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PubMed2005

24-hour changes in ACTH, corticosterone, growth hormone, and leptin levels in young male rats subjected to calorie restriction.

Calorie restriction of young male rats increases plasma prolactin, decreases luteinizing hormone (LH) and testosterone, and disrupts their 24 h secretory pattern. To study whether this could be the consequence of stress, we examined the 24 h variations of plasma adrenocorticotropic hormone (ACTH) corticosterone, growth hormone (GH), leptin, and adrenal corticosterone. Rats were submitted to a calorie restriction equivalent to a 66% of usual intake for 4 weeks, starting on day 35 of life. Controls were kept in individual cages and allowed to eat a normal calorie regimen. Significantly lower ACTH levels were detected in calorie-restricted rats. Plasma corticosterone levels during the light phase of the daily cycle were significantly higher in calorie-restricted rats. Time-of-day variation in plasma ACTH and corticosterone levels attained significance in calorie-restricted rats only, with a maximum toward the end of the resting phase. The daily pattern of adrenal gland corticosterone mirrored that of circulating corticosterone; however, calorie restriction reduced its levels. Plasma ACTH and corticosterone correlated significantly in controls only. Calorie restriction decreased plasma GH and leptin, and it distorted 24h rhythmicity. In a second study, plasma ACTH and corticosterone levels were measured in group-caged rats, isolated control rats, and calorie-restricted rats during the light phase of the daily cycle. Plasma ACTH of calorie-restricted rats was lower, and plasma corticosterone was higher, compared with isolated or group-caged controls. The changes in the secretory pattern of hormones hereby reported may be part of the neuroendocrine and metabolic mechanisms evolved to maximize survival during periods of food shortage.

Adrenal Glands↗

PubMed1996

Mitochondrial DNA restriction site map of Cochliomyia macellaria (Diptera:Calliphoridae).

The mitochondrial DNA of the secondary screwworm, Cochliomyia macellaria (F.), was cleaved with 15 restriction endonucleases to produce 54 restriction fragments. Forty-three restriction sites recognized by 12 enzymes were assigned positions on a restriction site map. Both the restriction fragments and the restriction site map were compared with published data from the screwworm, Cochliomyia hominivorax (Coquerel). The restriction site maps were aligned using a combination of highly conserved restriction sites. Estimated nucleotide divergence between the 2 species based on the fragments was 0.052. The divergence based on the restriction site map was 0.093. The data provide a baseline for future population or phylogenetic investigations.

Animals↗

PubMed2000

Dietary restriction of single essential amino acids reduces plasma insulin-like growth factor-I (IGF-I) but does not affect plasma IGF-binding protein-1 in rats.

The effects of dietary restriction of a single essential amino acid (EAA) on insulin-like growth factor-I (IGF-I) and IGF-binding protein (IGFBP)-1 were investigated in rats. Rats were fed experimental diets containing amino acid (AA) mixtures in which the concentrations of all EAA were at levels recommended by the National Research Council (control), in which a single EAA was restricted to 20% of that of the control diets (Leu(-), Lys(-), Met(-) or Thr(-)), or in which the diet was devoid of amino acids (AA(-)). To eliminate the effect of differences in energy intake, rats were fed the mean amount of food as consumed by the AA(-) group on the previous day. Growth was significantly retarded in rats fed diets restricted in just one EAA compared with that of rats fed the control diet, and further growth retardation was observed in rats fed the AA(-) diet. On the other hand, the plasma IGF-I concentrations in the groups with a single EAA restriction or in the AA(-) group were 66% (P: < 0. 05) and 50% (P: < 0.05) of that of the control group, respectively. The effect of any single EAA restriction was not significantly different from that of total AA deprivation. The plasma IGFBP-1 concentration in the control group did not differ from that of rats fed diets with the single EAA restrictions except for methionine restriction, but it was approximately 6-fold greater in the AA(-) group. Differences in plasma IGFBP-1 concentration under these conditions could be explained by differences in hepatic IGFBP-1 mRNA contents. Based on these results, we conclude that restriction of single EAA does not affect IGFBP-1 synthesis in vivo, although the deprivation of a single EAA has been reported to increase IGFBP-1 production in hepatocyte cultures. Our results also indicated that a single EAA restriction decreased IGF-I production but did not affect IGFBP-1 production. The present study suggests that not only plasma IGF-I, but also IGFBP-1, affects the magnitude of growth retardation in vivo.

Amino Acids, Essential↗

PubMed2003

Prostate carcinogenesis in N-methyl-N-nitrosourea (NMU)-testosterone-treated rats fed tomato powder, lycopene, or energy-restricted diets.

BACKGROUND: Consumption of tomato products or lycopene and energy restriction have been hypothesized to reduce the risk of human prostate cancer. We investigated the effects of these dietary variables in a rat model of prostate carcinogenesis. METHODS: Male rats (n = 194) treated with N-methyl-N-nitrosourea and testosterone to induce prostate cancer were fed diets containing whole tomato powder (13 mg lycopene/kg diet), lycopene beadlets (161 mg lycopene/kg diet), or control beadlets. Rats in each group were randomly assigned to either ad libitum feeding or 20% diet restriction. Differences between Kaplan-Meier survival curves for diet composition or restriction were tested with the log-rank test. Cox proportional hazards models were developed to examine the combined effect of diet composition and restriction on survival. Statistical tests were two-sided. RESULTS: Risk of death with prostate cancer was lower for rats fed the tomato powder diet than for rats fed control beadlets (hazard ratio [HR] = 0.74, 95% confidence interval [CI] = 0.59 to 0.93; P =.009). In contrast, prostate cancer-specific mortality of the control and lycopene-fed rats was similar (P =.63). The proportions of rats dying with prostate cancer in the control, lycopene, and tomato powder groups were 80% (95% CI = 68% to 89%), 72% (95% CI = 60% to 83%), and 62% (95% CI = 48% to 75%), respectively. Rats in the diet-restricted group experienced longer prostate cancer-free survival than rats in the ad libitum-fed group (HR = 0.68, 95% CI = 0.49 to 0.96; P =.029). The proportion of rats that developed prostate cancer was 79% (95% CI = 69% to 86%) for ad libitum-fed rats and 65% (95% CI = 54% to 74%) for rats fed restricted diets. No interactions were observed between diet composition and dietary restriction. CONCLUSIONS: Consumption of tomato powder but not lycopene inhibited prostate carcinogenesis, suggesting that tomato products contain compounds in addition to lycopene that modify prostate carcinogenesis. Diet restriction also reduced the risk of prostate cancer. Tomato phytochemicals and diet restriction may act by independent mechanisms.

Animals↗

PubMed1991

Relative efficiencies of the maximum-parsimony and distance-matrix methods of phylogeny construction for restriction data.

The relative efficiencies of the maximum-parsimony (MP), UPGMA, and neighbor-joining (NJ) methods in obtaining the correct tree (topology) for restriction-site and restriction-fragment data were studied by computer simulation. In this simulation, six DNA sequences of 16,000 nucleotides were assumed to evolve following a given model tree. The recognition sequences of 20 different six-base restriction enzymes were used to identify the restriction sites of the DNA sequences generated. The restriction-site data and restriction-fragment data thus obtained were used to reconstruct a phylogenetic tree, and the tree obtained was compared with the model tree. This process was repeated 300 times. The results obtained indicate that when the rate of nucleotide substitution is constant the probability of obtaining the correct tree (Pc) is generally higher in the NJ method than in the MP method. However, if we use the average topological deviation from the model tree (dT) as the criterion of comparison, the NJ and MP methods are nearly equally efficient. When the rate of nucleotide substitution varies with evolutionary lineage, the NJ method is better than the MP method, whether Pc or dT is used as the criterion of comparison. With 500 nucleotides and when the number of nucleotide substitutions per site was very small, restriction-site data were, contrary to our expectation, more useful than sequence data. Restriction-fragment data were less useful than restriction-site data, except when the sequence divergence was very small. UPGMA seems to be useful only when the rate of nucleotide substitution is constant and sequence divergence is high.

Computer Simulation↗

PubMed2007

Is dietary restriction beneficial for human health, such as for immune function?

PURPOSE OF REVIEW: The impact of dietary restriction on physiologic function in humans is now beginning to be examined. The clinical trials are fueled by decades of animal experiments showing that dietary restriction delays the aging process and decreases the incidence of many age-associated diseases. The critical issue addressed in this article is whether or not dietary restriction long term is feasible or beneficial in humans. RECENT FINDINGS: Short-term dietary restriction in humans does appear to have beneficial effects at lowering metabolism, especially when examining carbohydrates and weight loss. Dietary restriction long term does, however, have detrimental psychological effects in humans, making its feasibility questionable. Even short-term dietary restriction can negatively impact physical activity and potentially some aspects of immunity. The best avenue for humans to benefit from dietary restriction would be for pharmacological or bioactive food ingredient mimetics to be developed which would be more applicable for long-term use. SUMMARY: Dietary restriction per se is unlikely to emerge as a feasible long-term strategy to improve human health. Developing dietary restriction mimetics targeting energy metabolism may prove beneficial, not only in aging, but also in diabetes and obesity.

Animals↗

PubMed1978

Genome organization of RNA tumor viruses II. Physical maps of in vitro-synthesized Moloney murine leukemia virus double-stranded DNA by restriction endonucleases.

Physical maps of the genome of Moloney murine leukemia virus (M-MLV) DNA were constructed by using bacterial restriction endonucleases. The in vitro-synthesized M-MLV double-stranded DNA was used as the source of the viral DNA. Restriction endonucleases Sal I and Hind III cleave viral DNA at only one site and, thus, generate two DNA fragments. The two DNA fragments generated by Sal I are Sal IA (molecular weight, 3.5 x 10(6)) and Sal IB (molecular weight, 2.4 x 10(6)) and by Hind III are Hind IIIA (molecular weight, 3.6 x 10(6) and Hind IIIB (molecular weight, 2.3 x 10(6)). Restriction endonuclease Bam I generates four fragments of molecular weights of 2.1 x 10(6) (Bam IA), 2 X 10(6) (Bam IB), 1.25 X 10(6) (Bam IC), and 0.24 x 10(6) (Bam ID), whereas restriction endonuclease Hpa I cleaves the M-MLV double-stranded DNA twice to give three fragments of molecular weights of 4.4 x 10(6) (Hpa IA), 0.84 X 10(6) (Hpa IB), and 0.74 x 10(6) (Hpa IC). Digestion of M-MLV double-stranded DNA with restriction endonuclease Sma I produces four fragments of molecular weights of 3.9 x 10(6) (Sma IA), 1.3 X 10(6) (Sma IB), 0.28 X 10(6) (Sma IC), and 0.21 x 10(6) (Sma ID). A mixture of restriction endonucleases Bgl I and Bgl II (Bgl I + II) cleaves the viral DNA at four sites generating five fragments of approximate molecular weights of 2 x 10(6) (Bgl + IIA), 1.75 X 10(6) (Bgl I + IIB), 1.25 X 10(6) (Bgl I + IIC), 0.40 X 10(6) (Bgl I + IID), and 0.31 x 10(6) (Bgl I + IIE). The order of the fragments in relation to the 5' end and 3' end of the genome was determined either by using fractional-length M-MLV double-stranded DNA for digestion by restriction endonucleases or by redigestion of Sal IA, Sal IB, Hind IIIA, and Hind IIIB fragments with other restriction endonucleases. In addition, a number of other restriction endonucleases that cleave in vitro-synthesized M-MLV double-stranded DNA have also been listed.

Cell-Free System↗

PubMed2002

Effect of maternal feed restriction during pregnancy on glucose tolerance in the adult guinea pig.

Maternal nutrient restriction and impaired fetal growth are associated with postnatal insulin resistance, hyperinsulinemia, and glucose intolerance in humans but not consistently in other species, such as the rat or sheep. We therefore determined the effect of mild (85% ad libitum intake/kg body wt) or moderate (70% ad libitum intake/kg body wt) maternal feed restriction throughout pregnancy on glucose and insulin responses to an intravenous glucose tolerance test (IVGTT) in the young adult guinea pig. Maternal feed restriction reduced birth weight (mild and moderate: both P < 0.02) in male offspring. Moderate restriction increased plasma glucose area under the curve (P < 0.04) and decreased the glucose tolerance index (K(G)) (P < 0.02) during the IVGTT in male offspring compared with those of mildly restricted but not of ad libitum-fed mothers. Moderate restriction increased fasting plasma insulin (P < 0.04, adjusted for litter size) and the insulin response to IVGTT (P < 0.001), and both moderate and mild restriction increased the insulin-to-glucose ratio during the IVGTT (P < 0.003 and P < 0.02) in male offspring. When offspring were classed into tertiles according to birth weight, glucose tolerance was not altered, but fasting insulin concentrations were increased in low compared with medium birth weight males (P < 0.03). The insulin-to-glucose ratio throughout the IVGTT was increased in low compared with medium (P < 0.01) or high (P < 0.05) birth weight males. Thus maternal feed restriction in the guinea pig restricts fetal growth and causes hyperinsulinemia in young adult male offspring, suggestive of insulin resistance. These findings suggest that mild to moderate prenatal perturbation programs postnatal glucose homeostasis adversely in the guinea pig, as in the human.

Aging↗

PubMed2004

Effect of dietary energy restriction on vascular density during mammary carcinogenesis.

Inhibition of mammary carcinogenesis by dietary energy restriction is associated with a decrease in cell proliferation and the induction of apoptosis. Although changes in the metabolism of insulin-like growth factor I and glucocorticoids have been proposed to modulate these cellular processes, limitations in blood supply could induce similar effects. To investigate this possibility, female Sprague Dawley rats were given an injection of 1-methyl-1-nitrosourea and fed purified diets ad libitum or at 60% ad libitum intake, i.e., 40% dietary energy restriction. Premalignant mammary pathologies and mammary adenocarcinomas obtained from these rats were processed for vascular density analysis via CD-31 immunostaining. Vascular density, measured as vessels/unit area, of premalignant mammary pathologies and adenocarcinomas from dietary energy restriction rats was reduced 31 and 39%, respectively (P < 0.01). This effect, which was observed in a 50-microm wide band of tissue surrounding each pathology, was exerted on blood vessels > 25 microm2. Conversely, intratumoral vascular density was unaffected by dietary energy restriction. cDNA microarray and Western blot analyses of adenocarcinomas for evidence of dietary energy restriction-mediated effects on vascularization revealed that only the level of vascular endothelial growth factor receptor protein Flk-1 was significantly reduced (P < 0.001). It appears that dietary energy restriction imposes limitations in the supply of blood to developing pathologies, an effect that could directly inhibit the carcinogenic process. The vascular density data imply that dietary energy restriction inhibited the growth of endothelial cells but leave unresolved the question of whether dietary energy restriction had a specific effect on angiogenesis. The factors that account for the failure of dietary energy restriction to limit intratumoral vascularization are not obvious and merit additional investigation.

Animals↗

PubMed1997

Sodium restriction shifts circadian rhythm of blood pressure from nondipper to dipper in essential hypertension.

BACKGROUND: Sodium restriction has been widely used for treatment of hypertension and renal diseases. Whether sodium restriction can transform the circadian rhythm of blood pressure from nondipper to dipper is examined. METHODS AND RESULTS: Patients (n=42) with essential hypertension were maintained on a high-sodium diet (12 to 15 g of NaCl per day) and a low-sodium diet (1 to 3 g/d) for 1 week each. On the last day of each diet, blood pressures were measured noninvasively every hour for 24 hours with an automatic oscillometric device. Twenty-one patients were classified as non-sodium sensitive whereas 21 were classified as sodium sensitive on the basis of a > or = 10% change in 24-hour mean arterial pressure caused by sodium restriction. Nocturnal blood pressure fall was significant in the non-sodium sensitive subjects but not in sodium-sensitive subjects. There was a significant interaction between sodium restriction and nocturnal fall in blood pressure only in the sodium-sensitive subjects, indicating that the degree of the nocturnal fall was affected by sodium restriction. Furthermore, changes in the nocturnal fall induced by sodium restriction had a positive relationship with sodium sensitivity (r=.38, P<.02) and a negative relationship with the nocturnal fall before sodium restriction (r=-.75, P<.0001). CONCLUSIONS: These findings show the difference in nocturnal fall in blood pressure between the non-sodium sensitive and sodium-sensitive types of essential hypertension. The diminished nocturnal fall, recognized in the sodium-sensitive type, is restored by sodium restriction, indicating that the circadian rhythm of blood pressure shifted from a nondipper to a dipper pattern. On the other hand, the nocturnal fall is not affected by sodium restriction in the non-sodium sensitive type, and the circadian rhythm remains of the dipper variety.

Adult↗

PubMed2006

Human TRIM5alpha mediated restriction of different HIV-1 subtypes and Lv2 sensitive and insensitive HIV-2 variants.

In order to characterize the antiviral activity of human TRIM5alpha in more detail human derived indicator cell lines over expressing wild type human TRIM5alpha were generated and challenged with HIV-1 and HIV-2 viruses pseudotyped with HIV envelope proteins in comparison to VSV-G pseudotyped particles. HIV envelope protein pseudotyped particles (HIV-1[NL4.3], HIV-1[BaL]) showed a similar restriction to infection (12 fold inhibition) compared to VSV-G pseudotyped viruses after challenging TZM-huTRIM5alpha cells. For HIV-2 a stronger restriction to infection was observed when the homologous envelope protein Env42S was pseudotyped onto these particles compared to VSV-G pseudotyped HIV-2 particles (8.6 fold inhibition versus 3.4 fold inhibition). It has been shown that HIV-2 is restricted by the restriction factor Lv2, acting on capsid like TRIM5alpha. A mutation of amino acid 73 (I73V) of HIV-2 capsid renders this virus Lv2-insensitive. Lv2-insensitive VSV-G pseudotyped HIV-2/I73V particles showed a similar restriction to infection as did HIV-2[VSV-G] particles (4 fold inhibition). HIV-2 envelope protein (Env42S)-pseudotyped HIV-2/I73V particles revealed a 9.3 fold increase in infection in TZM cells but remained restricted in TZM-huTRIM5alpha cells (80.6 fold inhibition) clearly indicating that at least two restriction factors, TRIM5alpha and Lv2, act on incoming HIV-2 particles. Further challenge experiments using primary isolates from different HIV-1 subtypes and from HIV-1 group O showed that wild type human TRIM5alpha restricted infection independent of coreceptor use of the infecting particle but to variable degrees (between 1.2 and 19.6 fold restriction).

Antiviral Restriction Factors↗

PubMed1999

The in vivo effects of adrenocorticotropin and sodium restriction on the formation of the different species of steroidogenic acute regulatory protein in rat adrenal.

We have studied the in vivo expression of steroidogenic acute regulatory protein (StAR) in adrenals of control, ACTH-treated, and Na+-restricted rats. Indirect immunofluorescence by microscopy revealed the presence of StAR in the zonae glomerulosa (ZG) and fasciculata-reticularis (ZFR). An increased signal was observed in the ZG and zona fasciculata, 5 h after ACTH injection; a few cells of the medulla were also positive. Immunogold electron microscopy showed that StAR was mainly located over mitochondria (MT). By immunoblotting, a major 29-kDa and other minor StAR bands migrating between 30 and 39 kDa were increased 5 h after ACTH treatment but remained unchanged after 1 h. By two-dimensional-PAGE, four StAR species were revealed in homogenates of control ZG, and their intensity was increased 5 h after ACTH treatment but not after 1 h. Also, additional acidic species were seen 5 h after treatment. Other bands with basic isoelectric point were revealed between 29 and 37 kDa. Analyses on whole gland MT and supernatant (SN) revealed four bands in the control SN and five in ACTH SN; the intensity of one band was increased, and that of another one was decreased, in SN of treated rats. ACTH treatment resulted in the localization of many low-isoelectric point StARs in MT. After two-dimensional-PAGE, differences were found in the mobility of some StAR species in the ZG between controls and Na+-restricted rats. In MT, four bands were revealed in the ZG preparations of Na+-restricted and two bands in controls. Four bands were revealed in the ZG SNs of control and Na+-restricted rats; an additional band was observed only in the SN of treated animals, whereas the intensity of another band decreased. Na+ restriction did not affect StAR in the ZFR. In conclusion, StAR was present in the rat adrenal cortex ZG and ZFR and was mainly located in MT. StAR expression was inducible in the ZG and the ZF by ACTH, resulting in the formation of many StAR acidic species; interestingly, such changes were detectable 5 h, but not 1 h, after ACTH administration, suggesting that steroidogenesis stimulation by StAR might occur mainly outside MT. Although less spectacular than for ACTH, Na+ restriction also affected StAR expression in the ZG but not in the ZFR, by increasing two mitochondrial and one SN species, implying that StAR is involved in the mechanism of action of Na+ restriction in promoting aldosterone formation. These results suggest that differential processing and/or changes in phosphorylation may occur in vivo upon ACTH treatment and Na+ restriction. We hypothesize that modification of a relatively small quantity of StAR, mainly located outside MT, is necessary to increase adrenal steroidogenesis challenged either by ACTH or Na+ restriction.

Adrenal Glands↗

PubMed1995

Effects of caloric or protein restriction on insulin-like growth factor-I (IGF-I) and IGF-binding proteins in children and adults.

Serum concentrations of insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1), -2, and -3 are influenced by dietary intake in normal adults. These studies were undertaken to determine the influence of dietary factors on these proteins in children and to compare the responses in children to those in adults. Eight adults and eight pubertal children underwent energy restriction (50% reduction intake) for 6 days and were refed a normal diet for an additional 6 days. Basal energy intakes during the prerestriction periods were 70 Cal/kg in children and 35 Cal/kg in adults. A second group of 8 adults and 6 children underwent protein restriction (decreased from 1.0 to 0.66 g/kg in both groups) for 6 days and were refed a normal diet for 6 days. Calorie restriction resulted in a significant decrease in nitrogen balance in adults and children. Likewise, IGF-I concentrations declined significantly in both adults and children. In contrast, IGFBP-1 concentrations were significantly increased in adults (from 40 +/- 6 to 62 +/- 4 ng/mL; P < 0.05), but not in children. Serum concentrations of IGFBP-2 did not change in either group in response to energy restriction. IGFBP-3 declined significantly in the children (3189 +/- 90 to 2843 +/- 96 ng/mL; P < 0.05), but not in the adults. Protein restriction also caused negative nitrogen balance in both children and adults and a decline in the mean IGF-I concentration in the adults. IGFBP-2 concentrations rose significantly in both adults (131 +/- 15 to 164 +/- 15 ng/mL; P < 0.005) and children (126 +/- 13 to 158 +/- 15 ng/mL; P < 0.05) in response to protein restriction and returned to normal during refeeding. IGFBP-3 was slightly, but significantly, reduced in response to protein restriction in adults (3518 +/- 180 to 3328 +/- 151 ng/mL; P < 0.05), but not children. The findings indicate that protein or energy restriction in children leads to changes in IGF-I or specific IGFBPs. Changes in IGFBP-2 are sensitive to protein restriction, and measurement of IGFBP-2 may be useful in monitoring the response to refeeding in children who have been ingesting suboptimal amounts of protein.

Adult↗

PubMed1992

Dietary sodium restriction and pressor responsiveness to tyramine in spontaneously hypertensive rats.

OBJECTIVE: To determine in vivo whether in young spontaneously hypertensive rats (SHR) dietary sodium restriction decreases adrenergic transmitter release from the sympathetic nerve terminal. DESIGN: Dietary sodium restriction was initiated in young and mature SHR and Wistar-Kyoto (WKY) rats, and subsequently changes in pressor responsiveness to norepinephrine and to the indirectly acting sympathomimetic tyramine were determined in relation to their effects upon plasma catecholamines. RESULTS: In young SHR sodium restriction for 3-6 weeks prevented the development of hypertension, whereas in mature SHR sodium restriction did not affect blood pressure. Sodium restriction caused modest decreases in pressor responsiveness to the exogenous alpha-agonist, not different in young and mature SHR compared with WKY rats. In contrast, sodium restriction markedly inhibited pressor-responses to tyramine in young SHR and WKY rats, but not at all in mature rats. Tyramine increased plasma norepinephrine 5-10-fold. However, sodium restriction did not affect this response. The pressor response to tyramine was related to increases in total peripheral resistance, with minimal changes in cardiac output, and could be blocked by alpha 1-receptor blockade in rats on either control or low-sodium diets. CONCLUSIONS: These results show that sodium restriction causes only a small decrease in the pressor response to norepinephrine, but a more marked inhibition of the pressor response to tyramine in young SHR and WKY rats without affecting the plasma norepinephrine response to tyramine. These results suggest that dietary sodium can indeed affect presynaptic functions in vivo, but that plasma norepinephrine responses to tyramine may not reflect changes in arterial norepinephrine release, or that sodium restriction affects a co-transmitter rather than norepinephrine release per se.

Animals↗

PubMed2002

Restriction enzymes in the analysis of genetic alterations responsible for cancer progression.

BACKGROUND: Molecular approaches are increasingly being employed to dissect the genetic changes accompanying tumour formation. These methods can often be confusing to the non-specialist as they include complex molecular steps. This can reduce the usefulness of such molecular data to clinicians. The authors aim to aid interpretation of molecular studies in general by presenting a comprehensive review of one molecular approach, i.e. the use of restriction enzymes in molecular studies of tumour development. METHOD: A review was made of the molecular studies that have employed restriction enzymes in gastrointestinal cancer research. These studies have used restriction enzymes to analyse point mutation induction, gene methylation status and the deletion of chromosomal loci. In addition, emphasis is placed on some of the important considerations for the molecular analysis of tumours that can affect the molecular data obtained. RESULTS: Restriction enzyme digestion has played, and continues to play, a major role in analysing the genetic changes in cancer. Many adaptations of basic restriction enzyme methodologies have enhanced the application of this approach in cancer genetics. CONCLUSION: The availability of 200 different restriction enzymes, each recognizing different sequences in DNA, has been invaluable in studying cancer genetics. It is hoped that current advances in protein engineering will facilitate the creation of novel restriction enzymes with tailor-made sequence specificities. This will further improve the applicability of restriction enzymes in cancer genetics.

DNA Methylation↗

PubMed1984

Major histocompatibility complex restriction of tetanus toxoid-specific human T lymphocyte clones.

Peripheral blood mononuclear cells (PBMC) from an HLA DRw6/7 individual were stimulated with tetanus toxoid (TT). T cell blasts were cloned by the limiting dilution technique in the presence of TT and irradiated autologous PBMC (iPBMC). Twelve were propagated under interleukin 2 and restimulated weekly with TT and iPBMC. All proliferated specifically in response to TT or either the alpha or beta chain of the toxin molecule. HLA restriction of specific proliferative responses was analyzed using a panel of HLA-typed unrelated donors and selected families, and blocking experiments with anti-HLA class I and class II monoclonal antibodies (mAb). Three types of restriction were observed: (a) autologous restriction; the inhibition observed using anti-HLA DR mAb as well as family studies performed previously with a similarly restricted clone obtained from the same donor suggest an HLA DRw6-linked restriction; (b) an HLA DR7 restriction was found for 2 clones, specific for alpha or beta chain; the identical pattern of inhibition obtained with two different mAb belonging to the same cluster suggests that these clones may be restricted by the same (or a very close) epitope of the HLA DR7 molecule; (c) an unusual restriction pattern was found for one clone; PBMC from more than 80% of donors could present TT whatever their degree of HLA compatibility with the autologous donor. Family studies were unable to disclose any restriction with known class II (or class I) antigens. While no inhibition was observed with anti-DR or -DC reagents, a mAb that recognizes class I antigen when associated with beta 2-microglobulin did inhibit the proliferation of this clone.

Antibodies, Monoclonal↗

PubMed1995

Food restriction stimulates conjugation of p-nitrophenol in perfused rat liver.

Rates of conjugation of p-nitrophenol were studied in livers from normal and food-restricted rats perfused with either p-nitroanisole or p-nitrophenol. Female Sprague-Dawley rats had ad libitum access to a Purina 5001 nonpurified diet (control) or were given 65% of the intake of controls for 3 weeks. Livers were perfused with oxygenated Krebs-Henseleit buffer using a nonrecirculating system. Maximal rates of conjugation of p-nitrophenol, generated either from the O-demethylation of p-nitroanisole (200 microM) or from the infusion of p-nitrophenol (70 microM), were elevated significantly nearly twofold by food restriction. Thus, food restriction stimulates conjugation in the intact liver cell. Specifically, rates of conjugation were increased from 2.1 +/- 0.2 to 3.7 +/- 0.4 and from 3.3 +/- 0.6 to 5.8 +/- 0.5 mumol/g/h when 200 microM p-nitroanisole or 70 microM p-nitrophenol were infused, respectively. On the other hand, rates of conjugation were not affected by food restriction when low concentrations of p-nitroanisole (50 microM) or p-nitrophenol (20 microM) were infused. Further, food restriction did not alter rates of conjugation in isolated microsomes supplemented with excess UDPGA. Interestingly, both UDP-glucose and UDP-glucuronic acid were increased significantly in liver extracts from food-restricted rats when livers were perfused with high but not low concentrations of p-nitrophenol. Under these conditions, the increase in UDP-glucuronic acid was threefold. Moreover, food restriction increased carbohydrate release from the liver about twofold. Glycogen content was also increased significantly in liver extracts from 8.4 +/- 1.9 to 60.4 +/- 13.8 mmol/kg wet weight by food restriction. Taken together, these data support the hypothesis that food restriction stimulates conjugation of p-nitrophenol concentrations by increasing the supply of the pivotal cofactor UDP-glucuronic acid from carbohydrate reserves (e.g., glycogen).

Animals↗

PubMed1997

The increase in the induction of IL-2 expression with caloric restriction is correlated to changes in the transcription factor NFAT.

The objective of this study was to determine if the increase in the induction of interleukin-2 (IL-2) expression with caloric restriction correlates with changes in binding activity of the IL-2-specific transcription factor NFAT (nuclear factor of activated T cells) and/or the ubiquitous transcription factor AP-1 in T cells from male Fischer 344 rats. Splenic T cells were isolated from young (6-month) and old (24-month) rats fed ad libitum and from old (24-month) rats fed a restricted diet (40% caloric restriction) that began at 6 weeks of age. T cells were stimulated with concanavalin A (Con A) and the expression of IL-2 and the DNA binding activity of the transcription factors NFAT and AP-1 were measured in these cells. We found that the induction of IL-2 activity and mRNA levels decreased with age and that caloric restriction significantly (P < 0.05) reduced the age-related decline in IL-2 expression. The ability of nuclear extracts from T cells isolated from old rats fed ad libitum and restricted old rats to bind to the NFAT oligonucleotide or AP-1 oligonucleotide decreased with age. Caloric restriction significantly (P < 0.05) reduced the age-related decline in NFAT but had no significant effect on AP-1 binding activity. We also measured the induction of c-fos and c-jun expression by Con A in T cells from young and old rats fed ad libitum or caloric-restricted diet. The induction of c-fos protein and mRNA levels but not c-jun protein or mRNA levels decreased significantly with age. Caloric restriction significantly (P < 0.05) reduced the age-related decline in c-fos expression but had no significant effect on c-jun expression. Therefore, the increase in IL-2 expression with caloric restriction correlates with an increase in binding activity of transcription factor NFAT and an increase in the expression of c-fos, which is a component of the NFAT-protein complex.

Aging↗