Restriction sites containing CpG show a higher frequency of polymorphism in human DNA

Unique loci in the human genome were examined with restriction enzymes in order to detect restriction fragment length polymorphisms (RFLPs). Of 31 arbitrary loci, nine were detectably polymorphic, reflecting ten polymorphic restriction sites. Nine of the ten polymorphic sites were revealed with two restriction enzymes, Msp I and Taq I, whose recognition sequences have in common the dimer sequence CpG. The cytosines in the CpG sequence are known to be frequently methylated in mammals, and the occurrence of significant variation in Msp I and Taq I sites supports the view that methylated cytosine residues are hotspots for mutation in mammalian DNA.     

Molecular cloning of human mevalonate kinase and identification of a missense mutation in the genetic disease mevalonic aciduria.

Mevalonic aciduria is the first proposed inherited disorder of the cholesterol/isoprene biosynthetic pathway in humans, and it is presumed to be caused by a mutation in the gene coding for mevalonate kinase. To elucidate the molecular basis of this inherited disorder, a 2.0-kilobase human mevalonate kinase cDNA clone was isolated and sequenced. The 1188-base pair open reading frame coded for a 396-amino acid polypeptide with a deduced M(r) of 42,450. The predicted protein sequence displayed similarity to those of galactokinase and the yeast RAR1 protein, indicating that they may belong to a common gene family. Southern hybridization studies demonstrated that the mevalonate kinase gene is located on human chromosome 12 and is a single copy gene. No major rearrangements were detected in the mevalonic aciduria subject. The relative size (2 kilobases) and amounts of human mevalonate kinase mRNA were not changed in mevalonic aciduria fibroblasts. Approximately half of the mevalonic aciduria cDNA clones encoding mevalonate kinase contained a single base substitution (A to C) in the coding region at nucleotide 902 that changed an asparagine residue to a threonine residue. The presence of this missense mutation was confirmed by polymerase chain reaction amplification and allele-specific hybridization of the genomic DNAs from the proband and the proband's father and brother. Similar analysis failed to detect this mutation in the proband's mother, seven normal subjects, or four additional mevalonic aciduria subjects, indicating that the mutation does not represent a common gene polymorphism. Functional analysis of the defect by transient expression confirmed that the mutation produced an enzyme with diminished activity. Our data suggest that the index case is a compound heterozygote for a mutation in the mevalonate kinase gene.     

Induction of activated macrophages in C3H/HeJ mice by avirulent Salmonella

A single injection of viable Salmonella typhimurium SL3235, an avirulent organism blocked in the aromatic pathway, induced the generation of activated peritoneal macrophages in three different C3H mouse strains, including macrophage-defective C3H/HeJ mice. Macrophages obtained from immunized mice were cytotoxic for B16 melanoma cells, P815 mastocytoma cells, and TU-5 fibrosarcoma cells and microbicidal in vitro for the obligate, intracellular, protozoan parasite Leishmania major. The capacity of live SL3235 to activate C3H/HeJ macrophages contrasts with the failure of live Bacillus Calmette-Guérin to induce activated macrophages in this mouse strain. Although viable SL3235 were capable of fully activating cells of both normal and defective mice, a dose-dependent difference was observed in the number of organisms necessary for induction of tumoricidal macrophages in C3HeB/FeJ (normal) and C3H/HeJ (defective) animals. As few as 80 viable SL3235 were capable of activating C3HeB/FeJ macrophages whereas 5 X 10(4) organisms were required to activate C3H/HeJ macrophages. Maximal macrophage activation occurred 7 to 10 days after SL3235 inoculation in C3H/HeJ and C3HeB/FeJ mice. Acetone-killed cells of SL3235 had some but not all of the activity of the living Salmonella. A single in vivo injection of the nonviable preparation resulted in the induction of tumoricidal macrophages in C3HeB/FeJ but not in C3H/HeJ mice, even when tested over a wide dosage range. Injection of acetone-killed cells of SL3235 did, however, result in a population of primed macrophages in C3H/HeJ mice, as explanted cells could be induced to express activated macrophage effector activities after additional treatment in vitro with either LPS or IFN-gamma. Thus, in vivo administration of viable SL3235 is, by itself, capable of eliciting the full series of steps required for activation of C3H/HeJ macrophages, whereas killed SL3235 only provides signals sufficient to prime these defective macrophages for further activation in vitro. AI 15613     

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans

Dopamine-modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine-mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1-like dopamine receptor gene (dop-1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop-1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild-type copy of the gene. The dop-1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop-1 gene in mechanosensory neurons using the mec-7 promoter rescues the mechanosensory deficit in dop-1 mutant animals. The tyrosine hydroxylase-deficient C. elegans mutant (cat-2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.     

Desensitization of prostaglandin-activated platelet adenylate cyclase.

Prostaglandin D2 (PGD2) is one of several prostaglandins that can inhibit platelet aggregation and activate adenylate cyclase. Platelets were exposed to varying concentrations of PGD2 washed, and the adenylate cyclase response to prostaglandins, epinephrine, and sodium fluoride determined. Incubating platelets with 5 x 10(-5) M PGD2 for 2 hr resulted in a 45% decrease in PGD2 activation of adenylate cyclase and a 25% decrease in stimulation by PGE1. Fluoride activation (7-fold) epinephrine inhibition (30%) and basal enzyme activity were unchanged by exposure of the platelets to PGD2. Desensitization was concentration dependent, with loss of enzyme activity first noted when platelets were incubated with 10(-7) M PGD2. Enzyme sensitivity could be partially restored when desensitized platelets were washed free of PGD2 and incubated in buffer for 2 hr; complete resensitization required incubation for 24 hr in plasma. Regulation of prostaglandin sensitive platelet adenylate cyclase could be of importance in mediating the response of platelets to aggregating agents.     

Glycosaminoglycan composition of human amniotic fluid

The glycosaminoglycan composition of human amniotic fluid between 12–21 weeks gestation has been studied by Dowex column chromatography coupled with enzymatic analyses of the specific glycosaminoglycan in each column fraction. The total uronic acid recovered from the columns consisted of “glycopeptides” (7%), hyaluronic acid (34%), nonsulfated chondroitin (14%), chondroitin-4-sulfate (13%), chondroitin-6-sulfate (20%), dermatan sulfate (5%), and heparan sulfate (6%). Based on these studies a simple screening procedure was devised to detect increased quantities of heparan sulfate and dermatan sulfate in 5–10-ml samples of amniotic fluid and tested in the antenatal diagnosis of Hurler and Hunter's syndrome. A false negative result was recorded in a Hunter fluid obtained early gestation and a false positive result recorded in a normal fluid obtained at weeks. These data suggest that the time in gestation when amniotic fluid is sampled for chemical analysis is an important variable affecting glycosaminoglycan composition in both normal and pathological pregnancies.     

Comparing beta-carotene,vitamin E and nitric oxide as membrane antioxidants

Singlet oxygen initiates lipid peroxidation via a nonfree radical mechanism by reacting directly with unsaturated lipids to form lipid hydroperoxides (LOOHs). These LOOHs can initiate free radical chain reactions leading to membrane leakage and cell death. Here we compare the ability and mechanism by which three small-molecule membrane antioxidants (beta-carotene, alpha-tocopherol and nitric oxide) inhibit lipid peroxidation in membranes. We demonstrate that beta-carotene provides protection against singlet oxygen-mediated lipid peroxidation, but does not slow free radical-mediated lipid peroxidation. Alpha-Tocopherol does not protect cells from singlet oxygen, but does inhibit free radical formation in cell membranes. Nitric oxide provides no direct protection against singlet oxygen exposure, but is an exceptional chain-breaking antioxidant as evident from its ability to blunt oxygen consumption during free radical-mediated lipid peroxidation. These three small-molecule antioxidants appear to have complementary mechanisms for the protection of cell membranes from detrimental oxidations.