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PubMed1985

HLA-DR-, MB- and novel DC-related determinants restrict purified protein derivative of tuberculin (PPD)-stimulated human T cell proliferation.

Class II major histocompatibility complex determinants restricting recognition of tuberculin antigens (purified protein derivative; PPD) were studied by using monoclonal antibodies (mAb) to block lymphoproliferative responses. Anti-class II mAb were shown to exert inhibitory effects at the level of the antigen-presenting cells, without inducing suppressive lymphocytes or macrophages. Using panels of HLA-typed antigen-presenting cells and nonalloreactive proliferative T cell lines, derived by limiting dilution, restriction elements for PPD responses appeared to correlate with the donor's HLA-DRw6 specificity (one clone), MB1 (one clone), MB3 (one clone), or no established class II (or class I) specificity (three clones). mAb TU22, reacting with nonpolymorphic DC-like determinants, strongly inhibited stimulation of all clones except that restricted by DR antigens, suggesting the DC-like character not only of the MB1- and MB3-associated, but also of the unassigned, restriction elements of these cloned lines. In contrast, stimulation of the DR-restricted line was strongly inhibited by DR/SB-specific mAb which only weakly inhibited the stimulation of clones restricted by DC-like determinants. These results suggest that clonally distributed PPD-reactive proliferative lymphocytes from a single donor may be restricted by at least three different class II determinants (HLA-DR, MB, or a second, novel, DC-related molecule).

Antibodies, Monoclonal↗

PubMed1995

Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.

Type III restriction/modification systems recognize short non-palindromic sequences, only one strand of which can be methylated. Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation-specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site-specific, but EcoP15I-modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme-to-site ratio, in that site-saturating enzyme levels elicit cleavage exclusively between the closest pair of head-to-head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I-DNA complexes.

Adenosine Triphosphatases↗

PubMed1996

Regulation of the activity of the type IC EcoR124I restriction enzyme.

Restriction-modification (R-M) systems must regulate the expression of their genes so that the chromosomal genome is modified at all times by the methyltransferase to protect the host cell from the potential lethal action of the cognate restriction endonuclease. Since type I R-M systems can be transferred to non-modified Escherichia coli cells by conjugation or transformation without killing the recipient, they must have some means to regulate their restriction activity upon entering a new host cell to avoid restriction of unprotected host DNA and cell death. This is especially true for EcoR124I, a type IC family member, which is coded for by a conjugative plasmid. Control of EcoR124I restriction activity is most likely at the post-translational level as the transfer of the EcoR124I system into a recipient cell that already expressed the HsdR subunit of this system was not a lethal event. Additionally, the kinetics of restriction activity upon transfer of the genes coding for the EcoR124I RM system to a recipient cell are the same, irrespective of the modification state of the recipient cell or the presence or absence of the EcoR124I HsdR subunit in the new host cells. The mechanism controlling the restriction activity of a type IC R-M system upon transfer to a new host cell is different from that controlling the chromosomally coded type IA and IB R-M systems. The previously discovered hsdC mutant, which affects the establishment of the type IA system EcoKI, was shown to affect the establishment of the type IB system EcoAI, but to have no influence on EcoR124I.

Base Sequence↗

PubMed1996

Positive selection, cloning vectors for gram-positive bacteria based on a restriction endonuclease cassette.

Lactococcus lactis contains numerous restriction and modification (R/M) systems of different specificities. A novel IIS type R/M system encoded by the LlaI operon has previously been characterized from the L. lactis conjugative plasmid pTR2030. The LlaI operon is composed of six genes: First, a small regulatory gene llaIC precedes the methylase gene llaIM. The following three genes, llaI.1, llaI.2, llaI.3, are all essential for restriction endonuclease activity and are designed as the restriction cassette llaIR. The forth open reading frame of unknown function follows the llaIR gene cassette. We have successfully subcloned the three llaIR genes, llaI.1, llaI.2, and llaI.3, without llaIM, as a suicide cassette into the three shuttle vectors pTRKL2, pTRKH2, and pBV5030. A promoter (P6) from Lactobacillus acidophilus ATCC4356, which is functional in E. coli, lactococci, and lactobacilli (Djordjevic and Topisirovic, unpublished) was cloned upstream of the three gene cassette. Restriction activity was evaluated in Escherichia coli and several gram-positive bacteria. The llaIR restriction cassette was not functional in E. coli, but its presence was lethal to L. lactis, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Carnobacterium pisicola, Enterococcus faecalis, Bacillus subtilis, and Leuconostoc gelidum. Several novel, positive selection cloning vectors were developed that can exploit unique cloning sites within the llaIR cassette. Insertions in llaI.1 resulted in complete inactivation of restriction activity and provided unconditional selection for recombinant plasmids in surviving transformants. These positive selection cloning vectors are the first for gram-positive bacteria that are based on a restriction endonuclease cassette. Functional activity of the llaIR genes in various gram-positive bacteria would also enable use of these cloning vectors for positive selection of promoters, terminators, and regulatory sequences across these genera.

Cloning, Molecular↗

PubMedDate not supplied

Household smoking restrictions and adolescents' exposure to environmental tobacco smoke.

BACKGROUND: Reducing adolescents' exposure to environmental tobacco smoke (ETS) is an important public health goal. This paper identifies the linkage between young people's exposure at home and household smoking restrictions, and suggests the promotion of such restrictions as a strategy to reduce health risks. METHODS: Data are from the 1993 Massachusetts Tobacco Survey, a telephone survey of 1,606 adolescents. RESULTS: Seventy-eight percent of adolescents reported exposure to ETS during the preceding week. Reported hours of exposure at home were associated with the number of adult smokers in the household (P < 0.001). Fifty-three percent of teens who lived with smokers reported no smoking restrictions for family members, 22% reported designated smoking areas, and 25% reported smoking bans. Among those households, smoking restrictions for family members were associated with significant reductions in mean hours of ETS exposure reported: no restrictions were associated with 33.2 hr of exposure during the prior week, designated smoking areas--12.7 hr, and household smoking bans--2.4 hr. Restrictions on visitor smoking were associated with significant reductions in home ETS exposure for teens who lived with nonsmokers, but had no independent effect among teens who lived with smokers. CONCLUSIONS: Health risks experienced by adolescents living with adult smokers can be substantially reduced by household smoking restrictions.

Adolescent↗

PubMed1992

Effects of voluntary exercise and/or food restriction on pancreatic tumorigenesis in male rats.

Studies were undertaken to evaluate the effects of caloric restriction on voluntary exercise in a rat model of pancreatic cancer. Suckling male Lewis rats were initiated with 3 doses (30 mg/kg body weight) of the pancreatic carcinogen azaserine. At 28 days of age they were weaned to one of four experimental protocols; namely, sedentary/ad libitum fed, voluntary exercise/ad libitum fed, sedentary/food restricted, and voluntary exercise/food restricted. Voluntary exercise was provided by free access to running wheels fitted with odometers. Food restriction was intended to be mild, at less than 10% reduction of ad libitum intake. Putative pre-neoplastic pancreatic foci were identified microscopically at 4 months post-azaserine treatment. As previously shown, food restriction led to increased wheel activity, but the increased activity could not be maintained beyond the first half of the post-initiation treatment phase. Additionally, the extensive wheel running occurred only when available food was restricted by greater than 10%. Exercise per se had a lesser effect compared to food restriction on pancreatic tumorigenesis.

Analysis of Variance↗

PubMed1983

Energy/protein interrelation in experimental food restriction.

The effects of restriction of energy and/or protein intake were studied in rats during pregnancy and lactation and in adult mice. Three approaches were employed: sucrose stimulus-induced reduction in the intake of an adequate diet given simultaneously; restriction of intake of a complete food by 30% of ad libitum levels and selective protein or energy restriction of a high protein diet and a non-protein diet. Casein or lactalbumin was the protein source. During lactation the rat's natural food intake regulatory mechanism prevailed over the sucrose stimulus. Restricted intake of the complete food and selective restriction of protein or energy, variably influenced gestational and lactational performance and weight of young at weaning. A distinct regulation in the intake of protein and energy during pregnancy and lactation was observed on selective energy or protein restriction. Restricted feeding of the composite diet starting in the second year of life, to mice previously ad libitum fed a lab chow of constant composition, promoted the highest survival rate.

Age Factors↗

PubMed1988

Response of natural killer cells from dietary tyrosine- and phenylalanine-restricted mice to biological response modifiers.

The effect of dietary tyrosine and phenylalanine restriction on splenic natural killer (NK) cell activity was studied in tumor-free B6D2F1 and NIH nude mice and in B16 bladder-6 (BL6) melanoma-bearing B6D2F1 mice. This dietary restriction was found to suppress the naturally elevated NK-cell activity of nude mice and to induce a specific lymphocytopenia in B6D2F1 mice fed the restricted diet for a prolonged period. Baseline NK-cell activity was significantly lower in tumor-free B6D2F1 mice fed a diet restricted in tyrosine and phenylalanine (restricted diet) than in tumor-free mice fed a basal diet. Similar kinetics of activation after a single i.p. injection of 100 micrograms of polyinosinic:polycytidylic acid (poly I:C) were observed in mice fed both diets. NK-cell activity was not significantly augmented after i.v. inoculation of BL6 melanoma, irrespective of the diet fed; however, it was enhanced in tumor-bearing mice after poly I:C injection. This augmentation was similar to that observed in tumor-free mice. Spleen cells from mice fed either diet were responsive to stimulation of NK-cell activity after in vitro incubation with interleukin-2. These results indicate that dietary restriction of tyrosine and phenylalanine, a potentially useful therapeutic adjunct known to lower NK-cell activity, does not significantly interfere with poly I:C or interleukin-2 induction of NK cells. Our results also demonstrate that, while this dietary restriction causes lymphocytopenia, no effect of the diet could be found on total serum IgG or circulating immune complex levels.

Animals↗

PubMed1984

Kinetic impairment of restrictive streptomycin-resistant ribosomes.

Comparisons in vivo and in vitro of wild-type and otherwise isogenic bacteria with five different mutant alleles of the gene (rpsL) specifying ribosomal protein S12, all resistant to high levels of streptomycin, show that the streptomycin-resistant (Smr) phenotype can be subdivided into major groups: restrictive and non-restrictive. The restrictive bacteria have a characteristically lower frequency of nonsense suppression in vivo, and are also slower than the wild type in their rate of protein synthesis. Non-restrictive Smr bacteria on the other hand do not differ significantly from the wild type either in nonsense suppression frequencies or in the rate of translation. A complementary pattern is seen in vitro, where ribosomes from the restrictive Smr bacteria translate poly(U) with a significantly lower missense error frequency than wild-type ribosomes, and also show an increased Michaelis constant (KM) with respect to their substrate, i.e. ternary complexes. Both effects are correlated with the more aggressive proofreading function that is characteristic of these restrictive ribosomes. In contrast, ribosomes isolated from the non-restrictive Smr bacteria do not show any major difference in either proofreading or missense error in vitro when compared to the wild type.

Drug Resistance, Microbial↗

PubMed1979

Host-controlled modification and restriction in Bacillus subtilis: Bsu 168-system and BsuR-system in B. subtilis 168.

A Bsu168-specific restriction deficient (r168-) mutant of Bacillus subtilis Marburg 168 was transformed to be BsuR-specific restriction proficient (rR+) with B. subtilis R DNA as efficiently as the Bsu 168-specific restriction proficient (r168+) parental strain (hsrM+, hsdR-). We constructed rR+ mR+ r168+ m168+ strain (ISMR 4), rR+ mR+ r168- m168+ strain (ISR 11) and rR+ mR+ r168- m168- strain (ISR 6) from strain 101 (r168+ m168+), strain 1012 (r168- m168+) and strain RM125 (r168- m168-), respectively by transformation with B. subtilis R DNA, and tested their restriction and modification activities on phage phi 105C. The results show that the sites recognized by Bsu168-specific restriction and modification enzymes and the sites recognized by BsuR-specific ones are not overlapping. We conclude that the Bsu168-modification and restriction system and the BsuR-modification and restriction system are controlled independently by two distinct sets of genes in the rR+ mR+ transformant of r168+ m168+ strain B. subtilis 168.

Bacillus subtilis↗

PubMed1987

Highly transformable mutants of Streptomyces fradiae defective in several restriction systems.

Streptomyces fradiae JS85 is a mutant defective in tylosin production and an efficient recipient for conjugal transfer of tylosin genes. JS85 was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and derivatives defective in restriction were isolated by sequential selection for increased transformability by several plasmid DNAs. From the number of mutation and selection cycles required to eliminate most restriction, it was estimated that wild type S. fradiae expressed at least five restriction systems. From the patterns of restriction enzyme digestion of chromosomal DNA observed in the series of mutants that became progressively less restricting, it was suggested that wild type S. fradiae normally expresses modification (and presumably restriction) systems similar or analogous to PstI, XhoI, ScaI and EcoRI. The least restricting mutant of S. fradiae was readily transformable by many plasmids, including a bifunctional cosmid vector containing a large insert of Streptomyces DNA.

Bacteriophages↗

PubMed1985

Protection of nonmodified phage lambda against EcoK restriction mediated by recA protein.

A study was conducted to establish whether the EcoK-specific restriction, which is alleviated in E. coli cells after UV induction of the SOS response (Day 1977), is also alleviated under the influence of an increased level of recA protein without induction of other SOS functions. The host cells used were E. coli K-12, strain AB2497, and its derivatives; the nonmodified phage lambda was a mutant b2b5(vir). An increase of the recA protein level was induced using the plasmid pX02, which is a recombinant of pBR322 carrying the recA gene of E. coli. AB2497(pX02) cells were found to exhibit a lower level of restriction than those without plasmid. The results indicate that the recA protein protects phage DNA during the process of restriction. A further factor affecting restriction is the growth phase of the culture of the restricting host: cells in the late stationary phase exhibit lower restriction than those in the exponential phase of growth. By a combination of these two factors (presence of plasmid pX02 and stationary growth phase) one can reduce the restriction of nonmodified phage about 300 times.

Bacteriophage lambda↗

PubMed1989

Metabolic rate and aging: effects of food restriction and thyroid hormone on minimal oxygen consumption in rats.

Metabolic rate (MR) is widely regarded as an important component of aging processes. Decreased MR has been suggested as a possible mechanism of the life-prolonging action of food restriction in rodents and recent reports show lowered plasma levels of tri-iodothyronine (T3), a regulator of MR, in food restricted rodents. In order to study the relationship between MR, food restriction, thyroid status and aging we measured the Minimal Oxygen Consumption (MOC) of barrier-raised Fischer 344 male rats fed ad libitum (Group A) or fed a diet restricted to 60% of ad libitum intake (Group R). Oxygen consumption (VO2) was also measured over 24 hr to establish the relationship between MOC and VO2 under usual living conditions. Results show: (i) MOC declines with age in both groups of rats; (ii) there is no difference in MOC of Groups A and R rats; (iii) MOC of Group R rats is significantly more sensitive to doses of injected T3 than MOC of Groups A rats at all ages; and (iv) MOC of both groups of rats is significantly lower than the lowest VO2 recorded under usual living conditions. The results indicate that a decrease in MR is not the mechanism by which food restriction retards aging processes in rodents. The results also suggest no change in thyroid status due to restriction of food although there is increased sensitivity to T3 in food restricted rats.

Aging↗

PubMed1995

Effects of different types of anti-aging dietary restrictions on age-related atrial natriuretic factor changes: an immunochemical and ultrastructural study.

The effects of two types of anti-aging dietary restrictions restrictions-60% diet restriction (DR) and every-other-day feeding (intermittent feeding, IF)-on age-related changes in atrial natriuretic factor (ANF) metabolism were studied in male Sprague Dawley rats by standard radioimmunological procedures and transmission electron microscopy. Both diet restrictions decreased plasma ANP (atrial natriuretic peptide) levels significantly (12-month-old rats: ad libitum fed controls 96 +/- 17.3 pg/mL, IF 63 +/- 4.8 pg/mL, DR 74 +/- 14.4 pg/mL; 24-month-old rats: AL 109 +/- 6.3 pg/mL, IF 75 +/- 8.9 pg/mL, DR 65 +/- 9.1 pg/mL). Dietary restriction prevented the age-related increase in ANP concentration in both the right atrium (12-month-old rats: AL 0.25 +/- 0.033 microgram/mg, IF 0.22 +/- 0.023 microgram/mg, DR 0.30 +/- 0.044 microgram/mg; 24-month-old rats: AL 0.39 +/- 0.037 microgram/mg, IF 0.10 +/- 0.015 microgram/mg, DR 0.07 +/- 0.011 microgram/mg) and left atrium (12-month-old rats: AL 0.23 +/- 0.033 microgram/mg, IF 0.13 +/- 0.019 microgram/mg, DR 0.17 +/- 0.035 microgram/mg; 24-month-old rats: AL 0.44 +/- 0.037 microgram/mg; IF 0.07 +/- 0.009 microgram/mg, DR 0.07 +/- 0.011 microgram/mg). Endocrine cardiocytes of diet-restricted senescent rats could be readily distinguished ultrastructurally from cells of ad libitum fed controls due to a higher number of beta-atrial specific granules of lower electrondensity. In conclusion, anti-aging diet restriction regimens fully prevented the age-related increase in the hormone store in atrial tissue, and lowered plasma ANP levels.

Age Distribution↗

PubMed1998

Dietary Mg and/or K restriction enhances paraquat toxicity in rats.

Effect of mineral restriction was studied to clarify which mineral in the diet is most indispensable in preventing paraquat (PQ) toxicosis. ODS rats were chosen as the experimental animal owing to the inability to synthesize vitamin C similarly to humans. Rats were fed with either mineral-adequate or restricted diets dosed with 125 ppm PQ. The mineral-adequate diet was based on the American Institute of Nutrition-76, and the restricted diet was one-half the amounts. Measurements were made on the onset day of PQ toxicosis, body weight changes during the feeding experiment, and changes of two acute phase reactant proteins cysteine proteinase inhibitor and alpha1-proteinase inhibitor. The minerals tested were divided into three classes: I, largely needed, Ca, K, Na, and Mg; II, moderately needed, Mn, Fe, Zn, and Cu; and III, minutely needed, Cr and Se, respectively. Rats fed with a Mg-restricted diet showed a severe toxicosis but those with a K-restricted diet, a mild toxicosis. No appreciable effect was observed by restriction of other minerals. A synergistic effect was observed in the restriction of Mg and K.

Animals↗

PubMed1997

Changes in the plantaris muscle as an indicator of alterations in lean body mass of obese Zucker rats following prolonged energy restriction and subsequent partial recovery.

To study the effects of a prolonged (80 day), severe (64% body mass loss) energy restriction and subsequent refeeding on skeletal muscle tissue, specifically the plantaris muscle, 21 genetically obese Zucker rats were selected for this study. Six rats were initially killed and served as baseline (BASE), then 15 rats underwent severe energy restriction for 80 days. Seven of these restricted rats (RESTRICT) were then killed and assessed while the other eight rats (REHAB) were fed a dry rehabilitation diet that provided 100% of the recommended energy and 1.5 times the recommended protein for growth. Once the REHAB rats had recovered approximately 45% of their original mass loss, these animals were then evaluated. Within 20 min after being killed, the plantaris muscles from each animal in each group had been removed, weighed, and frozen. Analyses included total plantaris mass, as well as differences in fiber diameters, esterase activity, and fiber type distributions between three groups (BASE, RESTRICT, and REHAB). The extreme body mass loss of 64% in genetically obese Zucker rats resulted in significant tissue weight loss and reduced fiber diameters in the plantaris muscle. Refeeding resulted in larger muscle fiber diameters that approached baseline values but an 11% difference in muscle weight remained and may be due to a decreased fiber number. Esterase activity seemed to indicate an initial fat utilization for the RESTRICT group, followed by suppressed esterase activity in the REHAB group, suggesting increased fat storage. No significant differences were found in fiber type distribution between BASE, RESTRICT, or REHAB animals.

Animals↗

PubMed1993

Sensitization to the psychomotor effects of amphetamine and morphine induced by food restriction depends on corticosterone secretion.

Food restriction has been shown to enhance the behavioral sensitivity to addictive drugs. The biological factors involved in this effect are largely unknown. Since food restriction, among other factors, increases corticosterone secretion, the role of this hormone in the effects of food restriction on the response to psychostimulants and opioids was investigated. The effects of food restriction on amphetamine- and morphine-induced locomotor activity were compared in: (i) animals with an intact hypothalamo-pituitary-adrenal axis; (ii) animals in which food restriction-induced corticosterone secretion was suppressed by adrenalectomy, but which received exogenous corticosterone from a subcutaneous implant, which slowly releases corticosterone, producing a stable plasma level within the normal physiological range over a period of 20 days. It was found that food restriction enhanced sensitivity to the psychomotor effects of amphetamine (1 mg/kg i.p.) and morphine (1 mg/kg i.p.) in animals with an intact hypothalamo-pituitary-adrenal axis, but not in animals in which endogenous corticosterone secretion was eliminated. These results suggest that corticosterone secretion may be one of the mechanisms by which food restriction amplifies the behavioral responses to amphetamine and morphine. Since an enhanced locomotor reactivity to addictive drugs has been found to be frequently associated with an enhanced vulnerability to drug self-administration, these findings point to a role for glucocorticoids in the susceptibility to drug abuse.

Adrenalectomy↗

PubMed1994

Effects of chronic food restriction on prodynorphin-derived peptides in rat brain regions.

Chronic food restriction produces a variety of physiological and behavioral adaptations including a potentiation of the reinforcing effect of food, drugs and lateral hypothalamic electrical stimulation. Previous work in this laboratory has revealed that the lowering of self-stimulation threshold by food restriction is reduced by mu- and kappa-selective opioid antagonists. In the present study, the effect of chronic food restriction on levels of three prodynorphin-derived peptides, namely dynorphin A1-17 (A1-17), dynorphin A1-8 (A1-8) and dynorphin B1-13 (B1-13) were measured in eleven brain regions known to be involved in appetite, taste and reward. Food restriction increased levels of A1-17 in dorsal medial (+19.6%), ventral medial (+24.2%) and medial preoptic (+82.9%) hypothalamic areas. Levels of A1-17 decreased in the central nucleus of the amygdala (-35.1%). Food restriction increased levels of A1-8 in nucleus accumbens (+34.4%), bed nucleus of the stria terminalis (+24.5%) and lateral hypothalamus (+41.9%). Food restriction had no effect on levels of B1-13. A1-17 is highly kappa-preferring and the brain regions in which levels increased all have a high ratio of kappa: mu and delta receptors. A1-8 is less discriminating among opioid receptor types and the brain regions in which levels increased have a low ratio of kappa: mu and delta receptors. The present results suggest that food restriction alters posttranslational processing within the dynorphin A domain of the prodynorphin precursor, possibly leading to a change in the balance between kappa and non-kappa opioid receptor stimulation in specific brain regions.

Animals↗