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Literature Supporting The Use Of Dietary Nucleotides

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Title: Literature Supporting The Use Of Dietary Nucleotides


1
Literature Supporting The Use Of Dietary
Nucleotides
  • Dr. Garry Gordon
  • MD, DO, MD(H)

2
DIETARY NUCLEOTIDES
  • Dietary nucleotides have been shown to have
    important effects on several components of the
    immune system they may enhance intestinal
    absorption of iron they affect lipoprotein and
    long-chain polyunsaturated fatty acid metabolism
    they may alter intestinal flora and they have
    been demonstrated to have trophic effects on the
    intestinal mucosa and liver in several
    experimental situations. Clinical studies have
    shown nucleotide supplementation of infant
    formula reduces the incidence of diarrheal
    episodes
  • Cosgrove M. Nucleotides. Nutrition
    19981474851.

3
RNA CONTAINING FOODS CURRENTLY ON THE MARKET
  • IMPACT (novartis/sandoz)
  • Primary indications
  • Trauma
  • Major Surgery
  • Infections of pneumonia
  • Cancer
  • Ventilator dependent
  • Burn Injury

4
RNA CONTAINING FOODS CURRENTLY ON THE MARKET
  • There have been numerous studies of the
    clinical, immunological, nutritional and
    biochemical effects of IMPACT.
  • benefits were clinically confirmed by a
    significant reduction in infection rates and a
    reduction in wound healing complications, while
    average length of admission fell (Senkal et al.
    1997)
  • Clinically, this group had a significantly
    shorter length of hospital stay, a lower sepsis
    score and a trend to decreased infection. (Braga
    et al. 1996)
  • Complication rates and length of hospital stay
    were reduced (Daly et al. 1995)

5
RNA CONTAINING FOODS CURRENTLY ON THE MARKET
  • SMA Baby Food
  • contains added nucleotides which have been
    shown to be involved in the development of an
    infants immune system and may also reduce the
    incidence of diarrhoeal disease in babies.
  • Advanced Formula ENFAMIL
  • (Mead Johnson)
  • Nucleotide levels in ENFAMIL are patterned after
    the actual average free nucleotide levels found
    in breast milk.

6
RNA CONTAINING FOODS CURRENTLY ON THE MARKET
  • Breast milk has been reported to contain 1-12
    mg/dL of DNA and 10-60 mg/dL of RNA.
  • Sanguansermsri J, 1974 Am. J. Clin. Nutr. 2 859

7
  • Humans typically eat several grams of nucleotides
    in their diet each day.

8
(No Transcript)
9
LONGEVITY
  • research indicates that nucleotides such as
    RNA may help to reverse the effects of aging.
  • One Swedish study found that supplemental RNA
    enhances the effects of antioxidants
  • many patients followed a RNA supplement
    regime for many years without exhibiting harmful
    side effects. Instead, they demonstrate many
    benefits, including diminished skin wrinkling,
    improved circulation and peripheral nerve
    function.
  • rats injected weekly with RNA and DNA lived
    far longer than rats that did not receive the
    supplements.
  • LE Magazine August 1997

10
LONGEVITY
  • efficacy of RNA was its ability to increase
    cellular energy levels so as to facilitate the
    movement of young cells to the surface where they
    could replace unsightly senescent cells.
  • The nucleic acid RNA seemed to restore this
    repair mechanism.
  • LE Magazine January 2003

11
MEMORY
  • Other studies indicate that RNA may play a
    role in helping elderly people to avoid illness
    and remember things more clearly
  • LE Magazine August 1997

12
ENERGY
  • exogenous nucleosides increased the
    intercellular concentrations of UTP, UDP-glucose,
    CDP-choline and NAD
  • Arnaud A, Exogenous nucleosides alter the
    intracellular nucleotide pool in hepatic cell
    cultures. Implications in cell proliferation and
    function. Clin Nutr, 2003 Aug Vol. 22 (4), pp.
    391-9

13
ENERGY
  • Nucleotide food deprivation significantly
    decreased the ATP pool
  • Ortega MM, Nunez MC, Gil A and Sanchez-Pozo A,
    (1994) Dietary nucleotides accelerate intestinal
    recovery after food deprivation in old
    rats.Symposium Nucleotides and Nutrition
    Supplement in J. Nutr. (WA Walker, ed.), vol.
    124, pp1413-1418

14
BLOOD FLOW
  • the difference in blood flow velocities
    between baseline and 90 min was significantly
    greater with the formula with added nucleotides
    versus the formula with no added nucleotides
    These data suggest that orally administered
    nucleotides are associated with affects on the
    intestinal vasculature.
  • The higher blood flow velocity measured after
    nucleotide-supplementation may reflect dilation
    of the intestinal vascular bed.
  • Carver JD et al The Effects of Dietary
    Nucleotides on Intestinal Blood Flow Pediatric
    Research 52 (3) (2002)

15
BLOOD FLOW
  • Nucleotides may decrease the inflammatory
    response to ischemia-reperfusion. the number
    of leukocytes accumulated was reduced in the
    presence of nucleotides. Furthermore,
    nucleotides may have decreased protein leak and
    the production of nitric oxide during ischemia.
  • Bastamante, S. A., N. Sanches, J. Crosier, D.
    Miranda, G. Colombo, and M. J. S. Miller. 1994.
    Dietary nucleotideseffects on the
    gastrointestinal system in swine. J. Nutr.
    124149S-156S.

16
BLOOD CELLS
  • Studies on the rat everted gut have found that
    the nucleotides inosine, hypoxanthine and uric
    acid all significantly increased iron
    absorption.
  • Faelli A, Esposito G. Effect of inosine and its
    metabolites on intestinal iron absorption in the
    rat. Biochem Pharmacol 1970 19 2551-4.

17
DETOXIFICATION
  • Mycotoxin levels in the liver of pigs and
    poultry fed with nucleotide supplemented feed
    were significantly lower than of animals fed a
    standard diet, whereas the mycotoxin levels in
    the faeces of nucleotide fed animals were higher
    than of standard fed animals.
  • Koppel P, Physiological and nutritional
    functions of nucleotides. Chemoforma LTD
    Switzerland. Glasgow Veterinary School February
    (2003.)

18
INFLAMMATION
  • peri-operative IMPACT administration reduced
    C-reactive protein release in the IMPACT group.
  • enteral formula containing nucleotides
  • O'Flaherty L, Immunonutrition and surgical
    practice. Proc Nutr Soc, 1999 Nov Vol. 58 (4),
    pp. 831-7

19
INFLAMMATION
  • enhancement of IL-12 production is responsible
    for the augmentation of the Th1 immune response
    by dietary nucleotides feeding of dietary
    nucleotides was found to decrease the
    antigen-specific IgE response significantly.
  • Nagafuchi et al, Dietary nucleotides can
    up-regulate antigen specific TH1 immune responses
    in mice International Archives of Allergy and
    Immunology 122, 33-41 (2000)

20
ALLERGIC INFLAMMATION
  • report that mice fed nucleotide-supplemeted
    diets had lower serum IgE levels, serum
    IgGIIgG2a ratios and IL-4
  • dietary nucleotides may upregulate the Th1
    immune response in systemic immunity.
  • Nagafuchi S, Katayanag T, Nakagawa E, Takahashi
    T, Yajima T, Yonekubo A, et al. Effects of
    dietary nucleotides on serum antibody and splenic
    cytokine production in mice. Nutr Res 1997 17
    1163-74

21
IMMUNE SURVELLIENCE SYSTEM
  • To date no side effects of nucleotides have
    been shown in clinical trials.
  • Lerner A and R. Shamir. Nucleotides in Infant
    Nutrition A Must or an Option
  • IMA. Vol 2, October 2000

22
IMMUNE SURVELLIENCE SYSTEM
  • Mice maintained on a nucleotide-free diet have
    been found to exhibit deficient macrophage
    activity.
  • Kulkarni AD, Fanslow WH, Drath DB, Rudolph FB,
    Van Buren Ct Influence of dietary nucleotide
    restriction on bacterial sepsis and phagocytic
    cell function in mice. Arch Surg 1986 121
    169-172.

23
HUMORAL IMMUNE SYSTEM
  • Infants fed the nucleotide-supplemented formula
    had significantly higher serum antibody
    concentrations
  • the nucleotide-supplemented formula was
    associated with higher antibody responses to two
    vaccines, Hib conjugate vaccine and DPT
  • Pickering L, Granoff D, Erickson JR, Masor M,
    Cordle CT, Scheller JP, Winship TR, Paule CL,
    Hilty MD. Modulation of the immune system by
    human milk and infant formula containing
    nucleotides. Pediatrics 19981012429.
  • Carver, JD. Dietary nucleotides effects on the
    immune and gastrointestinal systems. Acta
    Paediatrica, 09/21/99, Vol. 88 Issue 9

24
HUMORAL IMMUNE SYSTEM
  • using murine spleen cells showed that
    polynucleotides significantly increase in vitro
    antibody production in response to
    T-cell-dependent antigen. They seem to exert
    actions on T-helper cells at antigen
    presentation, perhaps during cognitive cell-cell
    interactions. Polynucleotides increase in vitro
    human immunoglobulin production in response to
    T-cell-dependent stimuli and antigen. Humoral
    immune responses to T-cell-dependent antigen were
    depressed in mice fed a nucleotide-free diet, but
    were restored by a mononucleotide-nucleoside
    mixture.
  • Jyonouchi H, (1994) Nucleotide Actions on the
    Humoral Immune Response
  • J. Nutr. 124, Suppl. January 1994 138S -143S

25
HUMORAL IMMUNE SYSTEM
  • higher plasma immunoglobulin M and A
    concentrations were found in preterm infants fed
    nucleotide-supplemented formula.
  • Martinez-Augustin O, Boza J, Navarro J,
    Martinez-Valverde A, Araya M, Gil A. Dietary
    nucleotides may influence the humoral immunity in
    immunocompromised children Nutrition 1997 13
    465-9.

26
HUMORAL IMMUNE SYSTEM
  • In mice and in humans, a nucleotide free diet
    significantly depressed IgM and IgG antibody
    production from spleen cells.
  • Jyonouchi, H. (1994). Nucleotide actions on
    humoral immune responses. The journal of
    Nutrition. Nucleotides and nutrition. Vol 124. No
    1S. 138S-143S.

27
HUMORAL IMMUNE SYSTEM
  • the specific immunoglobulin A (IgA) level
    significantly increased in mice fed a
    nucleotide-supplemented diet compared with those
    fed a nucleotide-free control diet.
  • Shinya N et al Dietary nucleotides increase
    mucosal IgA response and secretion of TGF
    Cytotechnology 4049-58(2002 ).

28
CELLULAR IMMUNE SYSTEM
  • oral nucleotides enhanced the response to
    some T dependent protein antigens.
  • Pickering L, Granoff D, Erickson JR, Masor M,
    Cordle CT, Scheller JP, Winship TR, Paule CL,
    Hilty MS. Modulation of the immune system by
    human milk and infant formula containing
    nucleotides. Pediatrics 1998 101 242-9.

29
CELLULAR IMMUNE SYSTEM
  • In animal models, nucleotides have been shown
    to improve delayed cutaneous hypersensitivity
    and alloantigen-induced lymphoproliferation
  • Pickering L, Granoff D, Erickson JR, Masor M,
    Cordle CT, Scheller JP, Winship TR, Paule CL,
    Hilty MD. Modulation of the immune system by
    human milk and infant formula containing
    nucleotides. Pediatrics 19981012429.

30
CELLULAR IMMUNE SYSTEM
  • the effect of a nucleotide-free diet on the
    immune function of mouse syngeneic bone marrow
    radiation chimeras. These results suggest that
    dietary nucleotides are important for the normal
    function of mouse T-lymphocytes.
  • Kulkarni, S. S., D. C. Bhately, A. R. Zander, C.
    T. Van Buren, F. B. Rudolph, K. A. Dicke, and A.
    D. Kulkarni. 1984. Functional impairment of
    T-lymphocytes in mouse radiation chimeras by a
    nucleotide-free diet. Exp. Hematol. 12694-699.

31
IMMUNE SYSTEM
  • Feeding nucleotide-supplemented rodents has
    been associated with
  • increases in the following immune responses
  • Graft-versus-host disease mortality
  • Rejection of allogenic grafts
  • Delayed cutaneous hypersensitivity
  • Alloantigen- and mitogen-induced
    lymphoproliferation
  • Reversal of malnutrition and starvation-induced
    immunosuppression
  • Natural killer cell activity and macrophage
    activation
  • Resistance to microbial challenge
  • Macrophage phagocytic capacity
  • Spleen cell production of interleukin-2 (IL-2)
    and expression of IL-2 receptors and lyt-1
    surface markers
  • The number of antibody-secreting spleen cells
    produced in cluture
  • Popliteal lymp-node cytokine secretion
  • Peripheral blood total leukocyte counts and
    neutrophil numbers following infection.
  • Carver, JD. Dietary nucleotides effects on the
    immune and gastrointestinal systems. Acta
    Paediatrica, 09/21/99, Vol. 88 Issue 9

32
IMMUNE SYSTEM
  • Dietary nucleotides are reported to play a role
    in the maintenance of the immune response.
  • Carver, JD. Dietary nucleotides effects on the
    immune and gastrointestinal systems. Acta
    Paediatrica, 09/21/99, Vol. 88 Issue 9

33
IMMUNE SYSTEM
  • Spleen cells of mice maintained on a
    nucleotide-free diet produce less interleukin-2
    (IL-2) and have lower natural killer (NK) cell
    cytotoxiicity and macrophage activiation compared
    with animals fed nucleotide-supplemented diet.
    In vivo lymphoproliferative response, macrophage
    phagocytic activity, and expression of IL-2
    receptor and Lyt1 surface marker are also lower
    in animals fed nucleotide-free diet.
  • Carver JD, Pimentel B, Cox WI, Barness LA.
    Dietary nucleotide effects upon immune function
    in infants. Pediatrics 19918835963.

34
RNA therapy shows signs of enhancing the immune
system, improving transplanted organ viability
and even fighting off cancers.
supplemental ribonucleic acid (RNA) may help
boost our immune systems, increase energy
levels, neutralize toxins, repair cell
damage and improve skin elasticity.LE
Magazine August 1997
IMMUNE SYSTEM
35
studies suggesting potential benefits to
intestinal flora, immunity, iron absorption,
lipid metabolism and gut development.
Nucleotides in Infant Nutrition A Must or an
Option, Aaron Lerner and Raanan Shamir
IMMUNE SYSTEM
36
IMMUNE HEALTH
  • In human, parenteral solutions with nucleotides
    given to postoperative cancer patients improved
    immune function and infectious complications and
    length of hospital stay was reduced compared to a
    control group.
  • Van Buren, C.T., A.D. Kulkarni, F.B. Rudolph.
    (1994). The role of nucleotides in adult
    nutrition. The journal of Nutrition. Nucleotides
    and nutrition. Vol 124. No 1S. 160S-164S.

37
CANCER
  • Because dietary nucleotides are particularly
    essential for individuals under conditions of
    stress, oral administration of dietary
    nucleotides may result in tumor regression
    through enhancement of the production of IFN-?
    and IL-12.
  • Nagafuchi et al, Dietary nucleotides can
    up-regulate antigen specific TH1 immune responses
    in mice International Archives of Allergy and
    Immunology 122, 33-41 (2000)

38
CANCER
  • increased toxicity and tumor promotion
    observed in animals may in part be due to
    resulting imbalances in dNTP pools and
    interference with DNA replication.
  • Jackson C. et al Dietary NucleotidesEffects on
    Cell Proliferation in Rats Fed Folate/Methyl-Defic
    ient Diets. Journal of Nutrition 127(5)
    834S-837S.(1997)

39
INFECTION
  • Anil D. Kulkarni and his colleagues fed mice a
    diet supplemented with RNA and compared them with
    a control group of mice fed a nucleotide-free
    diet. Then they injected the mice with a strain
    of powerful bacteria. Half of the mice on the
    nucleotide-free diet died. The mice that had
    taken RNA, however, were able to fight off the
    infection.
  • LE Magazine August 1997

40
INFECTION
  • There was an inhibition of gram-negative enteric
    bacteria in mice fed a diet supplemented with
    a nucleoside-nucleotide mixture indicating that
    the nuclotide-nucleoside mixture blocked
    bacterial translocation by preventing
    endotoxin-induced mucosal or epithelial damage.
    These results suggest that the nucleoside-nucleoti
    de mixture could be used ot inhibit or reduce the
    incidence of bacterial translocation, and
    improve survival in a lethal model of
    endotoxemia.
  • Adjei AA, Yamamoto S. A dietary
    nucleoside-nucleotide mixture inhibits
    endotoxin-induced bacterial translocation in mice
    fed protein-free diet. J Nutr 1995125428.

41
INFECTION
  • Mice on the nucleotide-free diet suffered 100
    mortality following intravenous challenge with
    Staphylococcus aureus while RNA- or
    uracil-repleted mice demonstrated significantly
    greater resistance to this bacterial challenge.
    Macrophages from mice on the nucleotide-free diet
    had decreased phagocytic activity compared with
    mice maintained on the nucleotide-free diet
    supplemented with adenine, uracil, or RNA.
    provision of nucleotides to defined diets appears
    vital to maintain host resistance to bacterial
    challenge.
  • Kulkarni AD, Fanslow WC, Drath DB, Rudolph FB and
    Van Buren CT, (1986a)
  • Influence of dietary nucleotide restriction on
    bacterial sepsis and phagocytic cell function in
    mice Arch. Surg. 121 169-172

42
INFECTION
  • dietary nucleotide restriction increases the
    mortality from staphylococcal sepsis. Addition
    of certain dietary nucleotides appears to
    decrease this susceptibility to bacterial
    challenge.
  • Kulkarni AD et al Effect of Dietary nucleotides
    on response to bacterial infections. J Parenter
    Enteral Nutr. March 10(2)169-171 (1986)

43
INFECTION
  • mice maintained on a nucleotide-free diet
    exhibit a significantly decreased mean survival
    time and a significantly increased viable
    organism recovery in the spleen following
    intravenous injection of graded inocula of C.
    albicans compared to mice fed diets containing
    RNA or uracil as a nucleotide source.
  • Fanslow WC, Kulkarni AD, Van Buren CT and Rudolph
    FB (1988) Effect of nucleotide restriction and
    supplementation on resistance to experimental
    murine candidiasis
  • J. Parenter. Enteral Nutr. 12 49-52

44
HEART
  • An immune-boosting nutritional supplement can
    lower the risk of infection and death in patients
    undergoing heart surgery, researchers report.
    high-risk patients who took a supplement
    containing yeast RNA had lower rates of
    infection and organ dysfunction following
    coronary artery bypass than those who took an
    inactive placebo pill.
  • Tepaske R, et al. Effect of preoperative oral
    immune-enhancing nutritional supplements on
    patients at high risk of infection after cardiac
    surgery a randomised placebo-controlled trial.
    Lancet 358 696-701(2001)

45
ICU PATIENTS
  • Early enteral feeding of the formula containing
    dietary nucleotides was safe and well tolerated
    in ICU patients. In patients who received the
    nucleotide formula, particularly if they were
    septic on admission to the study, a substantial
    reduction in hospital length of stay was
    observed, along with a significant reduction in
    the frequency of acquired infections.
  • Bower RH, Cerra FB, Bershadsky B, Licari JJ, Hoyt
    DB, Jensen GL, Van Buren CT, Rothkopf MM, Daly
    JM, Adelsberg BR. Early enteral administration of
    a formula (Impact) supplemented with arginine,
    nucleotides, and fish oil in intensive care unit
    patients results of a multicenter, prospective,
    randomized, clinical trial.
  • Crit Care Med. 1995 Mar23(3)436-49.

46
KIDNEY
  • beneficial effects support use of an oral
    immune-enhancing nutritional supplement in
    patients who are undergoing surgery and are at
    high risk of infection. The study included 50
    patients who took the supplement or a placebo for
    at least 5 days before surgery. About 17 of
    patients who took the supplement developed an
    infection, compared with 55 of those who took
    the placebo. Patients who took the supplement
    also had better kidney function and remained in
    the hospital for fewer days than patients who
    were given the placebo.
  • Tepaske R, et al. Effect of preoperative oral
    immune-enhancing nutritional supplements on
    patients at high risk of infection after cardiac
    surgery a randomised placebo-controlled trial.
    Lancet 358 696-701(2001)

47
KIDNEY
  • Oral administration of nucleotides to fish daily
    resulted in enhanced activities in kidney
    phagocytic cells. This activation of kidney
    cells was observed for at least 10 days
    post-treatment.
  • Sakai, M Taniguchi, K Mamoto, K Ogawa, H
    Tabata, M. Immunostimulant effects of nucleotide
    isolated from yeast RNA on carp, Cyprinus carpio
    L. Journal of Fish Diseases, Oct2001, Vol. 24
    Issue 8, p433, 6p

48
LIPID BALANCE
  • It has been reported that early childhood levels
    of serum lipids and lipoproteins are good
    predictors of lipid levels in adulthood.
  • neonates fed formula supplemented with
    nucleotides from birth have significantly higher
    HDL-C and lower LDL-C serum levels than do
    neonates fed an unsupplemented formula.
  • the only known difference between groups was
    the supplementation of formula with nucleotides.
    This factor was identified as one of the
    independent variables influencing the HDL-C and
    LDL-C levels in the multiple regression analysis
    models. Therefore, it can be assumes that
    dietary nucleotides have an effect on
    lipoprotein-cholesterol serum concentrations
  • Siahanidou. Serum Lipids in Preterm Infants Fed
    a Formula Supplemented With Nucleotides. Journal
    of Pediatric Gastroenterology Nutrition,
    Jan2004, Vol. 38 Issue 1, p56, 5p
  • Webber LS, Srinivasan SR, Wattigney WA, et al.
    Tracking of serum lipids and lipoproteins from
    childhood to adulthood. Am J Epidemiol 1991 133
    884-9

49
LIPID BALANCE
  • previous studies all conclude that formula
    supplementation with nucleotides influences
    circulating lipid levels
  • Sanchez et al. found higher HDL and lower VLDL
    plasma levels in supplemented neonates.
  • The mechanism(s) by which dietary nucleotides
    affect HDL-C and LDL-C serum concentrations
    is/are not known.
  • It has been suggested that dietary nucleotides
    increase the synthesis of long-chain
    polyunsaturated fatty acids
  • Siahanidou. Serum Lipids in Preterm Infants Fed
    a Formula Supplemented With Nucleotides. Journal
    of Pediatric Gastroenterology Nutrition,
    Jan2004, Vol. 38 Issue 1, p56, 5p
  • Sanchez-Pozo A, Pita ML, Martinez A, et al.
    Effects of dietary nucleotides upon lipoprotein
    pattern of newborn infants. Nutr Res 1986 6
    763-71

50
LIPID BALANCE
  • It is well known that polyunsaturated fatty
    acids have a significant lowering effect on LDL-C
    circulating levels by increasing LDL receptor
    activity or reducing apoB secretion, and they
    also may increase the HDL lipoprotein levels by
    enhancing the apoA-1 secretion.
  • The combination of high HDL-C and low LDL-C
    circulating levels is considered to be a
    protective factor against the development of
    vascular disease.
  • Siahanidou. Serum Lipids in Preterm Infants Fed
    a Formula Supplemented With Nucleotides. Journal
    of Pediatric Gastroenterology Nutrition,
    Jan2004, Vol. 38 Issue 1, p56, 5p
  • .
  • American Academy of Pediatrics. Committee on
    Nutrition. Cholesterol in Childhood. Pediatrics
    1998 101 141-7.

51
LIPID METABOLISM
  • Dietary nucleotides supplementation had
    higher lecithin cholesterol acyl transferase
    (LCAT) activities and apoA-IV levels than those
    receiving the nucleotide-free formula. These
    findings indicate that nucleotide supplementation
    may improve dietary lipid tolerance by
    enhancing plasma LCAT activity, probably as a
    result of an increase in apoA-IV plasma
    concentrations
  • Sanchez-Pozo A, Ramirez M, Gil A, Maldonado J,
    van Biervliet JP, Rosseneu M. Dietary nucleotides
    enhance plasma lecithin cholesterol acyl
    transferase activity and apolipoprotein A-IV
    concentration in preterm newborn infants. Pediatr
    Res 19953732833.

52
LIPIDS
  • nucleotides are involved in the synthesis of
    proteins and other macromolecules such as
    phospholipids, and thereby facilitate lipoprotein
    synthesis. . . . These findings suggest that
    dietary nucleotides may enhance the synthesis of
    lipoproteins during the early neonatal period,
    especially in preterm infants.
  • Sanchez-Pozo, A., J. Morillas, I. Molto, R.
    Robles, A. Gils. (1994). Dietary nucleotides
    influence lipoprotein metabolism in newborn
    infants. Pediatr. Res. 35. 112-116.

53
STRESS
  • nucleotide-supplemented groups shoed
    significantly lowered corticosterone levels,
    resulting in a decreased stress response such a
    decrease in corticosterone levels may be one of
    the mechanisms of immune restoration. Thus it
    is possible that RNA and uracil may have
    suppressed the production of this stress hormone
    and enhanced certain cytokines that resulted in
    the significant increase in immune response.
  • Yamauchi K, Dietary nucleotides prevent decrease
    in cellular immunity in ground-based microgravity
    analog. J Appl Physiol, 2002 Jul Vol. 93 (1),
    pp. 161-6

54
STRESS
  • under certain stress conditions and extremely
    unusual environments including space flight, the
    endogenous supply of these compounds (nucleotides
    and nucleosides) may not be adequate for optimal
    functions
  • The results of our study have demonstrated that
    mice fed both CDR (RNA supplemented) and CDU
    (uracil supplemented) enhanced in vivo and ex
    vivo lymphocyte cell proliferation and IL-2 and
    INF-? production and decreased the stress level,
    indicated by corticosterone levels, compared with
    mice fed control CD (control diet).
  • Yamauchi K, Dietary nucleotides prevent decrease
    in cellular immunity in ground-based microgravity
    analog. J Appl Physiol, 2002 Jul Vol. 93 (1),
    pp. 161-6

55
STRESS
  • Roles for dietary sources of performed purines
    and pyrimidines such as RNA in various systems
    have been demonstrated in recent years. These
    compounds were previously thought to be
    non-essential but are now considered to be
    conditionally requires when various stresses
    are present.
  • Rudolph FB. The metabolic effects of enterally
    administered ribonucleic acids. Curr Opin Clin
    Nutr Metab Care, 1998 Nov Vol. 1 (6), pp. 527-30

56
STRESS
  • The effect of dietary nucleotide restriction was
    tested during stress These studies clearly
    indicate the nutritional role of nucleotides
  • Pizzini R, Kumar S, Kulkarni Ad, Rudolph FB and
    Van Buren CT, (1990)
  • Dietary nucleotides reverse malnutrition and
    starvation-induced immunosuppression
  • Arch. Surg. 125 86-90

57
ULCERS
  • We conclude that the yeast RNA accelerated
    ulcer healing, as indicated by decreased ulcer
    number and length. We postulate that the
    underlying mechanism(s) contributing to ulcer
    healing may be related, in part, to increased
    cell proliferation.
  • Sukumar P, Loo A, Magur E, Nandi J, Oler A,
    Levine RA. Dietary supplementation of nucleotides
    and arginine promotes healing of small bowel
    ulcers in experimental ulcerative ileitis. Dig
    Dis Sci. 1997 Jul42(7)1530-6.

58
ULCERS
  • enteral and parenteral supplementation of
    nucleotides accelerates healing of ulcers in
    rats
  • Sukumar et al Dietary Nucleotides Augment Distal
    Colitis in Rats Journal of Nutrition
    1291377-1381 (1999).

59
STOMACH
  • dietary nucleotides are reported to play a role
    in growth and differentation of the
    gastrointestinal tract.
  • Uauy R. Dietary nucleotides and requirements in
    early life. In Lebenthal E, editor. Textbook of
    gastroenterology and nutrition. New York Raven
    Press, 1989 265-80

60
ENZYMES
  • Maltase activity was significantly greater the
    largest increase, 87, was seen in the proximal
    gut mucosa. The maltase/lactase ratio was also
    higher in this segment.
  • Uauy, R. G. Stringel, R. Thomas, and R. Quan.
    1990. Effect of dietary nucleotides on growth and
    maturation of the developing gut in the rat. J.
    Pediatr. Gastroenterol. Nutr. 10497-503.

61
ENZYMES
  • Nucleotide food deprivation significantly
    decreased maltase, sucrase, lactase and
    aminopeptidase activities.
  • Ortega MM, Nunez MC, Gil A and Sanchez-Pozo A,
    (1994) Dietary nucleotides accelerate intestinal
    recovery after food deprivation in old
    rats.Symposium Nucleotides and Nutrition
    Supplement in J. Nutr. (WA Walker, ed.), vol.
    124, pp1413-1418

62
INTESTINAL TRACT
  • investigators report decreased protein
    synthesis in the small intestine of rats fed
    nucleotide-free diets and accelerated intestinal
    recovery following food deprivation in rats fed
    nucleotide-supplemented diets. Dietary
    nucleotides may also be beneficial following
    intestinal injury.
  • intestinal histology and ultrastructure were
    improved by feeding nucleotide-supplemented
    versus nucleotide-free diets to rats following
    chronic diarrhea.
  • Dietary nucleotide supplementation promoted
    healing of small bowel ulcers in experimental
    ulcerative ileitis in rats
  • Carver, JD. Dietary nucleotides effects on the
    immune and gastrointestinal systems. Acta
    Paediatrica, 09/21/99, Vol. 88 Issue 9

63
INTESTINAL TRACT
  • Infants receiving the supplemented formula
    experienced fewer first episodes of diarrhea
  • infants fed nucleotide-supplemented formula
    had a significantly lower incidence of diarrhea
    compared with infants fed unsupplemented
    formula.
  • Carver, JD. Dietary nucleotides effects on the
    immune and gastrointestinal systems. Acta
    Paediatrica, 09/21/99, Vol. 88 Issue 9

64
INTESTINES
  • studies have shown that rapidly dividing cells
    of human intestinal tract are dependent on
    dietary nucleotides for their replication.
  • Sanderson, I.R., H. Youping. (1994). Nucleotide
    uptake and metabolism by intestinal epithelial
    cells. The journal of Nutrition. Nucleotides and
    nutrition. Vol 124. No 1S. 131S-137S.

65
DIARRHEA
  • The effects of a nucleotide-supplemented formula
    on diarrhoeal disease was studied The
    supplemented group experienced less episodes of
    diarrhoea including less first episodes and for a
    lesser number of days and a larger proportion
    never developed episodes of diarrhoea. The
    mechanisms through which nucleotides decrease the
    incidence of diarrhoeal disease in infants remain
    unclear.
  • Brunser O, Espinoza J, Araya M, Cruchet S, Gil A.
    Effect of dietary nucleotide supplementation on
    diarrhoeal disease in infants. Acta Paediatr
    19948318891.

66
DIARRHEA
  • the incidence and duration of acute diarrhoea
    is lower in infants fed supplemented-nucleotide
    formulas.
  • Gil A Modulation of the immune response mediated
    by dietary nucleotides. Eur J Clin Nutrition
    August 56(3)S1-4 (2002)

67
DIARRHEA
  • results suggest that dietary nucleotides
    promote earlier restoration of the ileal
    mitochondrial function after chronic diarrhea.
  • Arnaud, Journal of Pediatric Gastroenterology
    Nutrition Aug2003, Vol. 37 Issue 2, p124, 8p

68
LIVER
  • aspartate aminotransferase (AST), alanine
    aminotransferase (ALT), glutamic acid- oxalacetic
    transaminase (GOT) and glutamic acid- pyruvic
    transminase (GPT) levels were significantly lower
    in animals given a nucleotide-supplemented diet
    than those given the standard nutrition,
    indicating faster recovery from liver injury.
  • Ogoshi S, Iwasa M, Kitagawa S, Ohmori Y,
    Mizobuchi S, Iwasa Y and Tamiya T, (1988) Effects
    of total parenteral nutrition with nucleotide and
    nucleoside mixture on D-galacosamine-induced
    liver injury in rats.
  • J. Paranter. Enter. Nutr. 12 53-57

69
LIVER
  • Parenterally administered nucleotides promote
    recovery of liver injury and improve liver
    function and nitrogen balance after liver injury
    or partial hepatectomy.
  • Ogoshi, S., Iwasa, M., Kitagawa, S., Ohmori, Y.,
    Mizobuchi, S., Iwasa, Y. Tamiya, T. (1988)
    Effects of total parenteral nutrition with
    nucleosides and nucleotide mixture on
    D-galactosamine-induced live injury in rats. J.
    Parenter. Enteral Nutr. 18 62-66.

70
LIVER
  • In pre-term infants nucleotide supplement may
    improve lipid tolerance by enhancing plasma
    lecithin cholesterol acyltransferase activity.
  • Sanchez-Pozo A, Ramirez M, Gil A, Maldonado J,
    van Biervliet JP, Rosseneu M. Dietary nucleotides
    enhance plasma lecithin cholesterol
    acyltransferase activity and apolipoprotein A-IV
    concentration in preterm newborn infants. Pediatr
    Res 1995 37 328-33.

71
LIVER
  • The use of dietary nucleotides was found to
    decrease the percentage area of fibrous septae.
    In animals with liver cirrhosis fed the
    nucleotide-supplemented diet for two weeks, the
    total area of fibrosis was reduced. In
    conclusion, dietary nucleotides may be an
    important factor in the histological recovery of
    damaged liver in experimental cirrhosis.
  • Torres M. I. Effect of Dietary Nucleotides on
    Degree of Fibrosis and Steatosis Digestive
    Diseases and Sciences , 42 (6) 1322-132.(1997) .

72
LIVER
  • The serum GOT and GPT concentrations were
    significantly lower in the group supplemented
    with nucleoside-nucleotide mixture than those in
    other groups.
  • the nucleoside/nucleotide mixture inhibited
    the liver injury. Thus infusion of a
    physiological and balanced mixture of nucleosides
    or nucleotides may improve liver function in rats
    with liver injury.
  • Ogoshi S, Iwasa M, Kitagawa S, Ohmori Y,
    Mizobuchi S, Iwasa Y and Tamiya T, (1988) Effects
    of total parenteral nutrition with nucleotide and
    nucleoside mixture on D-galacosamine-induced
    liver injury in rats.J. Paranter. Enter. Nutr.
    12 53-57

73
BRAIN
  • The learning ability of rats fed the
    nucleotides-supplemented diet, which was
    evaluated by the water-filled multiple T-maze
    test and passive avoidance test, was superior to
    the rats fed the nucleotides-free diet. The
    results presented here suggest that dietary
    nucleotides may influence lipid metabolism of the
    cerebral cortex and contribute to the rise in
    learning ability of rats.
  • Sato N, Effects of dietary nucleotides on lipid
    metabolism and learning ability of rats. Biosci
    Biotechnol Biochem, 1995 Jul Vol. 59 (7), pp.
    1267-71

74
FATTY ACIDS
  • dietary nucleotides play a role in the in vivo
    desaturation and elongation of essential fatty
    acids to long chain PUFA during early life for
    the human newborn.
  • DeLucchi C, Pita ML, Faus MJ, Molina JA, Uauy R,
    Gil A. Effects of dietary nucleotides on the
    fatty acid composition of erythrocyte membrane
    lipids in term infants. J Pediatr Gastroenterol
    Nutr 1987656874.

75
FATTY ACIDS
  • Plasma polyunsaturated fatty acids with more
    than 18 carbons of the omega 6 family were
    significantly increased These results suggest
    that dietary nucleotides may be involved in the
    conversion of linoleic acid to longer chain
    polyunsaturated fatty acids during early life.
  • Gil A, Pita M, Martinez J, Molina JA and Sanchez
    Medina F, (1985)
  • Effect of dietary nucleotides on the plasma fatty
    acids in at-term neonates.
  • Hum. Nutr. Clin. Nutr. 40 185-195

76
PROTEIN SYNTHESIS
  • These findings suggest that the
    nucleoside-nucleotide mixture was used for the
    syntheses of RNA and DNA through the salvage
    pathway and that this resulted in enhanced
    protein turnover with simultaneous increase in
    protein synthesis.
  • Ogoshi S, Iwasa M, Yonezawa T and Tamiya T,
    (1985) Effect of nucleotide and nucleoside
    mixture on rats given total parenteral nutrition
    after 70 hepatectomy.
  • J. Paranter. Enter. Nutr. 9 339-342

77
APOPTOSIS
  • in vitro modulation of cell proliferation and
    apoptosis has been described by ribonucleosides,
    in particular by modified components using human
    cell culture models.
  • Schlimme, E., D. Martin, and H. Meisel. 2000.
    Nucleosides and nucleotides natural bioactive
    substances in milk and colostrums. Br. J. Nutr.
    84S59

78
CYTOKINES
  • Dietary nucleotides have been shown to enhance
    the production and the genetic expression of IL-6
    and IL-8 Dietary nucleotides may affect the
    levels of intestinal cytokines.
  • Gil A Modulation of the immune response mediated
    by dietary nucleotides. Eur J Clin Nutrition
    August 56(3)S1-4 (2002)

79
NERVES
  • Guanine nucleotides regulate the affinity of
    melatonin receptors The effect of guanine
    nucleotides and related analogues on the binding
    of melatonin to membranes was studied. These
    results are consistent with a melatonin receptor
    existing in an equilibrium between high- and
    low-affinity states, with GTP and related
    analogues able to cause a shift in the
    equilibrium
  • Morgan P.J.et al . Guanine nucleotides regulate
    the affinity of melatonin receptors on the ovine
    pars tuberalis. Neuroendocrinology, 50, 359362.
    (1989)

80
SKIN
  • topical application of RNA improved cell
    energy metabolism and therefore the health and
    appearance of the skin.
  • Dietary nucleotides affect tissue repair.
  • LE Magazine January 2003
  • Ramírez M, Effect of dietary nucleotides and
    orotate on the blood levels of prostacyclin
    (PGI2) and thromboxane (TXA2) in the weanling
    rat. Prostaglandins Leukot Essent Fatty Acids,
    1991 May Vol. 43 (1), pp. 49-54

81
MICROBES
  • In animal models, nucleotides have been shown
    to enhance T-cell maturation and function.
  • Pickering L, Granoff D, Erickson JR, Masor M,
    Cordle CT, Scheller JP, Winship TR, Paule CL,
    Hilty MD. Modulation of the immune system by
    human milk and infant formula containing
    nucleotides. Pediatrics 19981012429.

82
MICROBES
  • One possible anti-infective mode of action of
    nucleotide may be that dietary nucleotide can
    inhibit endotoxin-induced bacterial translocation
    in protein-malnourished mice.
  • Adjei AA, Yamamoto S. A dietary
    nucleoside-nucleotide mixture inhibits
    endotoxin-induced bacterial translocation in mice
    fed protein-free diet. J Nutr 1995 125 42-8.

83
WEIGHT LOSS
  • Dietary RNA is required to restore lost immune
    function after protein deprivation. Adequate
    calories and protein alone do not return immune
    function to normal. Dietary nucleotides can
    restore lost immune function even during protein
    starvation and weight loss.
  • Van Buren C.T., Kulkarni A.D.. Rudolph F.B., The
    Role of Dietary Nucleotides in Adult Nutrition,
    The Journal of Nutrition, January
    124124S-160S.(1994)

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86
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
RNAi What's All the Noise About Gene
Silencing? Posted 04/02/2004 Ernie
Hood Introduction It all began with petunias. In
the late 1980s, geneticist Richard Jorgensen,
then working at a California plant biotechnology
company, attempted to deepen the hue of purple
petunias by introducing more of the gene that
gives them their color, in the form of
double-stranded RNA (dsRNA). Instead, some of the
engineered flowers became variegated and others
turned white, indicating that expression of both
the introduced pigmentation gene and its
homologous endogenous gene had been knocked down
or knocked out altogether. Jorgensen had
serendipitously discovered an age-old natural
biologic process now recognized to be
evolutionarily conserved in most, if not all,
forms of life. Today, gene silencingor RNA
interference (RNAi), as it is now knownhas
revolutionized genetics and is on the verge of
spawning an entirelynew class of drugs to treat
human diseases with a genetic component. The
ability to selectively silence genes is one of
the hottest topics in biology today. Science
crowned RNAi as its "Breakthrough of the Year" in
2002. Nobel laureate and RNAi pioneer Phillip
Sharp, who is Salvador E. Luria Professor of
Biology and director of the McGovern Institute
for Brain Research at the Massachusetts Institute
of Technology (MIT), calls it "the most exciting
discovery in the last decade," adding that
"there's not an area of biological science this
will not touch." John Maraganore, who is
president, CEO, and director of Alnylam
Pharmaceuticals, touts RNAi as "presenting
perhaps the broadest new class of therapeutics
since recombinant proteins and monoclonal
antibodies." Can RNAi live up to the hype? That
remains to be seen, of course, but academic and
industrial researchers are optimistic that it can
and will, if the significant remaining barriers
to its progress can be overcome. Given the rapid
pace of discovery in the field, such optimism may
well be justified.
87
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. RNA Redefined It once seemed so simple, so
straightforward basically, DNA makes messenger
RNA (mRNA) mRNA makes proteins. But the
discoveries associated with RNAi have shown that
the real story is far more complex. RNA has been
unveiled as the "man behind the curtain" in the
cell, wielding previously unimagined control over
and influence upon cellular processes (including
gene expression and regulation) and organism
development. RNAi has been revealed to be an
ancient mechanism protecting cells from invading
viruses and from damage by transposable genetic
elements, performing a variety of cellular
housekeeping functions essential to survival,
health, and development. RNAi was first
described and so named by molecular biologists
Andrew Fire of the Carnegie Institute of
Washington and Craig Mello of the University of
Massachusetts, along with their colleagues, in a
landmark 19 February 1998 Nature paper that
electrified the biology community. The team found
that administering tiny amounts of dsRNA to
Caenorhabditis elegans resulted in potent
sequence-specific gene silencing. Tantalized by
the possibility of acquiring a powerful new tool
for genetic manipulation and analysis,
investigators around the world began
investigating RNAi. The flood of significant
discoveries that followed soon established the
basic outlines of the mechanisms involved in
RNAi. Researchers using Drosophila found in 2000
that long-strand dsRNA was processed in cells
into 21- to 23-nucleotide snippets of RNA, which
then cleaved to precisely matching homologous
mRNA sequences, degrading the mRNA and
effectively silencing the corresponding gene by
blocking its ability to encode for proteins. The
higher life forms, such as mammals, while
conserving this ability, use it in different
ways the response to dsRNA is more complicated,
triggering a cellular immune response involving
the release of interferon that ultimately kills
the cell.
88
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
89
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. RNA Redefined Then, in 2001, Thomas
Tuschl, then of the Max Planck Institute for
Biophysical Chemistry in Göttingen, Germany,
discovered with his colleagues that RNAi could be
prompted through the use of shorter pieces of RNA
known as small interfering RNAs (siRNAs). Soon
thereafter, they showed that duplexes of
21-nucleotide siRNAs mediated RNAi in cultured
mammalian cells and demonstrated that siRNAs
could be designed to silence specific genes
without activating the interferon response. In
other words, scientists could potentially silence
any gene of interest in a highly predictable,
reproducible, and accurate fashion. Research
scientist Gregory Hannon and his colleagues at
New York's Cold Spring Harbor Laboratory
contributed several key discoveries during the
same period. They identified, described, and
named the "Dicer" enzyme, which chops dsRNA into
siRNAs, as well as the RNA-induced silencing
complex (RISC), which mediates the silencing
process by degrading the homologous mRNA. In
2002, they described the use in mammalian cells
of so-called short hairpin RNAs (shRNAs), which
generate endogenous siRNAs within cells and thus
provide stable, heritable gene silencing (in
contrast, administered siRNAs are transient in
their silencing effect). They whimsically named
this effect "short hairpin-activated gene
silencing," or SHAGging. This discovery allowed
the development of cell lines and animal models
with permanently silenced genesa major step
forward for basic science in general, and
especially for functional genomics. Further
advances in the past few years have added the
ability to silence the expression of just the
mutant copy of a gene, leaving the normal copy
intact, as well as to modulate the level of
silencing in order to produce a range of
phenotypes. Plus, researchers can now induce
silencing in a controlled manner and target
multiple genes for silencing. These discoveries
alone are quite importantall of these
capabilities are crucial in a variety of critical
applications. But some proponents believe this is
only the beginning, and the best may be yet to
come.
90
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. Knock Down Genes, Drag Out Knowledge With
the continual refinement and improvement of
techniques to silence genes with exquisite
specificity, RNAi has already had a major impact
on molecular biology. For example, the pace of
discovery in functional genomics has accelerated
as a consequence of researchers' enhanced ability
to practice reverse genetics, in which a gene's
function can be inferred by silencing its
expression. With complete sequences of several
genomes now on hand, including those of C.
elegans, Drosophila, the mouse, and the human,
investigators can now quickly, easily, reliably,
and relatively inexpensively use siRNAs to
silence genes of interest and determine their
functions. Several companies are already selling
made-to-order siRNAs for use in functional
genomics work, as well as for drug target
identification and validation. The ability to
knock down genes either stably (that is, creating
heritable phenotypes through germline
transmission of permanently silenced genes) or
transiently (as opposed to knocking them out
altogether) has some important advantages in the
production of animal models and in vitro cell
lines. When a gene is considered silenced by
RNAi, expression is typically reduced by 70 or
more. This allows the method to be used in
so-called essential genes, which cannot be
knocked out in animal models without killing the
animal. Also, "turning down" a gene by a certain
amount can sometimes more closely resemble a
disease state, allowing the fashioning of more
useful, refined models of some diseases. Russell
Thomas, director of the functional genomics
research program at the CIIT Centers for Health
Research in Research Triangle Park, North
Carolina, points out another advance that RNAi
has brought to animal studies "When you knock
out a gene in mice, you have to live with the
consequences," he says. "The targeted gene is
knocked out for the remainder of the animal's
life span. In contrast, shRNAs with inducible
promoters allow an investigator to control the
timing for the knockdown of the targeted gene.
With this technology, you don't have residual
developmental effects, and you can have more
sophisticated experimental designs since you can
look at wild-type expression and knockdown in the
same animal and at multiple times throughout the
animal's life span."
91
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. Knock Down Genes, Drag Out Knowledge Sharp
observes that RNAi will in some cases obviate the
need for animal knockouts. "You can do gene
knockouts in mice, and then relate phenotype to
somatic cells in the human, but it's very
expensive, the gene has to be nonessential to get
an interesting phenotype, and you have to ask the
question in the context of the developing mouse,"
he says. By comparison, he explains, RNAi allows
researchers to inactivate the gene and observe in
real time the changes in the metabolic, cell
biologic, or other phenotype of the cell, and
characterize the role of the gene in that
particular situation. Although these applications
of RNAi are not yet perfected and are unlikely to
completely replace classic knockout studies, many
scientists are excited about adding these new
capabilities and efficiencies to their bag of
laboratory tricks. "It's fundamentally changing
how we do laboratory science," says Sharp. "It
will change how we do animal genetics, and we
have not even scratched the surface of all the
ways it will be used. RNAi and the Big
Picture The ability to reproducibly and robustly
silence every single gene in the genome is
expected to facilitate the acquisition of
profound new knowledge regarding function and
regulation at the cell and whole-organism levels.
Several organizations are in the process of
constructing large-scale RNAi libraries that
should be available for use very soon.
Genomewide screening using RNAi libraries will
help researchers learn more about global
questions in systems biology, elucidating the
nature and role of the complex, often
interrelated pathways and signaling networks at
work in organisms.
92
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. RNAi and the Big Picture Leona Samson,
director of the MIT Center for Environmental
Health Sciences, has already used such a library
for the 4,800 nonessential genes in Saccharomyces
cerevisiae. She says that higher-organism RNAi
construct libraries will aid toxicogenomics
research by allowing researchers to interrogate
each and every gene of an organismto examine a
specific gene to determine its function, or to
screen large amounts of genes in the context of a
specific function, to see which ones contribute
to the function, and possibly what roles they
might play and how they might interact. As a
result, Samson says, "you can start to identify
the important pathways for helping cells recover
from toxic insults. Samson says there are
important toxicogenomics treatment and prevention
end points to be achieved with this type of
large-scale screening. By interrogating every
gene, it's possible to compile a portrait of all
the pathways that are relevant to cellular
function and response, such that researchers
could look at which genes are being expressed in
members of a population and accurately predict
the effects of exposure to particular
environmental agents. "In the end," Samson says,
"we're going to get an integrated systems view,
and we need that to be able to make
predictions. There's More to the Machinery It
turns out that gene silencing through degradation
of mRNA by siRNAs is not the only cellular
mechanism regulated by small pieces of RNA.
Thanks to groundbreaking work in C. elegans,
researchers have discovered a class of natural
small RNA molecules called microRNAs that appear
to be crucial in regulating development. Although
they apparently use the same tools as siRNAs to
carry out their functionsDicer, RISC, and a
family of proteins known as ArgonautesmicroRNAs
differ in that their sequences do not precisely
match their mRNA targets. As such, they regulate
the expression of proteins by those targets,
rather than degrade them altogether, which leads
investigators to believe that they play an
important role in the timing and nature of
developmentperhaps to the point of controlling
differentiation in embryonic stem cells.
93
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. There's More to the Machinery Tuschl, who
is now group leader of the Laboratory of RNA
Molecular Biology at Rockefeller University,
says, "It's a very complicated regulatory
machinery that you have in your cells, with a
very complicated biology behind it. We know that
there are genes that express dsRNA and microRNAs,
and that this gene family in humans is about 250
genes, and many of these genes are conserved. . .
. The question is what all these genes are doing,
and how the RNAi machinery ties in to the gene
regulation mediated by the microRNA
genes." Recent studies further suggest there is
yet a third mechanism controlled by microRNAs, an
arm of the silencing machinery in the nucleus of
the cell that modifies heterochromatin. The
result is transcriptional repression of gene
expression. As Tuschl puts it, "This is one of
the real highlights of the discovery of RNAithat
it's a new cellular mechanism involved in
regulating gene expression, and it's as
complicated and as effective as transcriptional
gene regulation." More thorough understanding of
this mechanism could eventually lead to
beneficial insights into development and disease
processes, particularly carcinogenesis. Silencing
Disease Although biologists are excited about
the long-range additions to knowledge that could
emerge from studying the complexities of the RNAi
machinery, many are focusing major efforts on
exploiting what is already knownthat the
gene-silencing effect of RNAi holds tremendous
promise in treating human disease. RNAi
therapeutics will be judged on the same criteria
as any other prospective drug potency,
stability, and safety. Despite the great deal of
work yet to be done, many researchers believe
that RNAi-based agents will eventually pass
muster on those issues, and will actually have
inherent advantages over presently available
classes of drugs.
94
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. Silencing Disease For example, the fact
that RNAi is a natural cellular process may mean
that drugs based upon the phenomenon can be
expected to be quite efficacious. "The siRNA
process depends upon the endogenous pathway of
microRNA biology, a process that's present in all
cells," says Sharp. "Therefore, it's efficient,
it's a normal biological process, and we are
learning how to design siRNAs that are more
efficiently taken up in that process and more
efficiently used in silencing. Maraganore
agrees. "What's very unique about RNAi is that
it's the first natural mechanism that's ever been
discovered that would allow people to silence
genes," he says. "Because of that leveraging of
that natural mechanism, you have a high degree of
specificity and potency in the action of
siRNAs." According to Nassim Usman, chief
operating officer and senior vice president of
Sirna Therapeutics in Boulder, Colorado, there is
ample precedent for this opinion. "If you look at
the history of successful biotechnology drugs, a
lot of them that have been successful are the
ones that either use or replace a naturally
occurring mechanism," he says. "The two clearest
examples are recombinant proteins and
antibodies. The potential therapeutic value of
RNAi has been repeatedly demonstrated in a wide
variety of in vitro studies, and more and more in
vivo experiments are confirming that early
promise. Efficacy in gene silencing has been
shown in viral diseases (such as HIV/AIDS,
influenza, human papillomavirus infection,
various hepatitis strains, smallpox, and SARS),
neurodegenerative diseases (such as Parkinson
disease, amyotrophic lateral sclerosis, and
Alzheimer disease), cancer, inflammatory diseases
(such as rheumatoid arthritis), and autoimmune
diseases (such as type 1 diabetes mellitus). The
intense interest in RNAi therapeutics has come at
least in part because of its potential broad
applicability across such a wide spectrum of
disorders. Researchers also cite the specificity
of RNAi targeting siRNAs may be able to
effectively reach cellular targets that have
previously been inaccessible or highly resistant
to other forms of therapy. Success in reaching
such targets could lead to significant advances
in the treatment of several diseases with
currently unmet medical needs.
95
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. The Principle of the Thing Researchers in
academic and industrial labs around the world are
pursuing therapeutic applications of RNAi. One
good illustration is the work conducted by senior
investigator and associate pediatrics professor
Judy Lieberman and her group at the Institute for
Biomedical Research of the Harvard Medical School
Center for Blood Research. In 2003, the team
published two RNAi studies of landmark
importance. In one, an in vitro experiment
presented in the Journal of Virology in July 2003
(issue 13), the team achieved sustained
siRNA-mediated silencing of HIV-1 in primary
macrophages, in essence preventing infection from
taking hold. In previous studies conducted in
rapidly dividing cells, such as tumors, the RNAi
effect lasted only 3-5 days. In these
macrophages, however, as Lieberman explains, "the
silencing lasted very long, in fact for as long
as we could keep the cultures growing, for some
genes." The experiment co-targeted the viral p24
structural gene and CCR5, the major HIV-1
co-receptor in macrophages, cells that are known
to be reservoirs of HIV infection and that are
stubbornly resistant to current antiviral
therapies. The team found that they could
completely suppress HIV replication in
macrophages by using this co-targeting stratagem.
They also found that they could inhibit viral
replication in cells that were already infected,
in which HIV was integrated into the host cell.
As Lieberman summarized, those are "pretty
encouraging data for the possibility of using
siRNA against HIV." The experiment also sh
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