Haplotypic diversity in human CEACAM genes: effects on susceptibility to meningococcal disease.

Adhesion between the opacity-associated adhesin (Opa) proteins of Neisseria meningitidis and human carcino-embryonic antigen cell adhesion molecule (CEACAM) proteins is an important stage in the pathogenesis of meningococcal disease, a globally important bacterial infection. Most disease is caused by a small number of meningococcal genotypes known as hyperinvasive lineages. As these are also carried asymptomatically, acquisition of them alone cannot explain why only some hosts develop meningococcal disease. Our aim was to determine whether genetic diversity in CEACAM is associated with susceptibility to meningococcal disease. Frequency distributions of alleles, genotypes and haplotypes were compared in four CEACAM genes in 384 case samples and 190 controls. Linkage disequilibrium among polymorphic sites, haplotype structures and relationships were also analysed. A number of polymorphisms were observed in CEACAM genes but the diversity of CEACAM1, to which most Opa proteins bind, was lower, and a small number of high-frequency haplotypes were detected. Dose-dependent associations of three CEACAM haplotypes with meningococcal disease were observed, with the effect of carrying these haplotypes amplified in homozygous individuals. Two haplotypes were protective while one haplotype in CEACAM6 was associated with a twofold increase in disease susceptibility. These data imply that human CEACAM may be one determinant of human susceptibility to meningococcal disease.

Deletion analysis of a complex promoter for a developmentally regulated gene from Bacillus subtilis.

SpoVG is a developmentally regulated gene from the spore-forming bacterium Bacillus subtilis. The transcription initiation region for spoVG consists of two overlapping promoters whose startpoints of RNA synthesis are ten base pairs apart (Moran et al., 1981a). These startpoints are separately utilized by two forms of RNA polymerase holoenzyme containing different species of B. subtilis sigma factor. We have constructed a series of deletion mutations that extend into the spoVG promoter region from the downstream and from the upstream directions. Transcription studies with these mutated promoters showed that the functional boundaries of the spoVG promoters extended from the region of the transcription startpoints into an upstream A + T-rich box, which was located 76 to 51 base pairs preceding the downstream startsite. We have unexpectedly discovered that propagation of the spoVG promoter region on a high copy number plasmid in B. subtilis interferes with the process of sporulation by impairing development at an early stage. This was not a general effect of promoter amplification, since the propagation on plasmids of two other strong Bacillus promoters had little or no effect on spore formation. Deletion analysis established that the region of spoVG causing sporulation inhibition closely correlated with DNA sequences required for efficient promoter utilization in vitro. We propose that amplification of spoVG titrates a sporulation-specific regulatory protein that binds at or near the region of transcription initiation.

Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro.

We studied the regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486 [11 beta-(4-dimethylaminophenyl)17 beta-hydroxy-17 alpha-(prop-1-ynyl)estra-4,9-dien-3-one], a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Increased enzyme activity was specific for glucocorticoids; other steroid hormones were essentially without effect. The induction of glutamine synthetase was selective, in that glutaminase activity was not induced by dexamethasone treatment of L6 cells. Thus, glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

Neurosteroids: 3 alpha-hydroxy-5 alpha-pregnan-20-one and its precursors in the brain, plasma, and steroidogenic glands of male and female rats.

A RIA procedure for measuring progesterone (PROG), 5 alpha-pregnane-3,20-dione (5 alpha-DH PROG), and 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-TH PROG) has been developed and validated by GLC/mass spectrometry. Measurements were made in intact and adrenalectomized (ADX) male rats, in cyclic, pregnant, spayed, and spayed-ADX females, and in both males and spayed females injected with PROG. The predominant contribution of the ovary to the concentrations of 3 alpha,5 alpha-TH PROG in plasma and brain, was indicated by its larger levels in females, in particular during pregnancy, and by its presence in ovarian tissue and disappearance after ovariectomy. An additional adrenal origin in both males and females was shown. Neither PROG nor 5 alpha-DH PROG disappeared from brain, contrary to plasma, after combined adrenalectomy and gonadectomy, thus suggesting that PROG might be synthetized de novo in brain. However, the concentrations of 3 alpha,5 alpha-TH PROG in plasma and brain of female rats were positively correlated with the concentrations of PROG in plasma, indicating that plasma PROG was the major precursor of 3 alpha,5 alpha-TH PROG. The direct formation of 3 alpha,5 alpha-TH PROG from PROG in brain was strongly suggested by the increased 3 alpha,5 alpha-TH PROG/PROG ratios in brain vs. plasma, when measured in control females, and after injection of PROG to both males and OVX females. It was previously reported that 3 alpha,5 alpha-TH PROG is a sedative/anxiolytic steroid, as a result of its binding to gamma-aminobutyric acid (GABA)A receptors and allosteric potentiation of GABAcergic neurotransmission. Its concentrations in brain reach indeed the neuroactive range in cyclic and pregnant females, and are compatible with a physiological role of this neurosteroid.

The cleavage site for the restriction endonuclease EcoRV is 5'-GAT/ATC-3'.

The cleavage site for the restriction endonuclease EcoRV has been found to be 5'-GAT/ATC-3', rather than 5'- GATAT /C-3' as reported earlier by Kholmina et al. [ Dokl . Akad . Nauk . 253 (1980) 495-497].

Developmental induction of glutaminase in primary cultures of cerebellar granule cells.

Glutaminase mRNA levels increased over 3-fold relative to total RNA, poly(A)+ RNA, and beta-actin mRNA in neonatal rat cerebellar granule cells as the cells differentiated between days 3 and 8 in culture. In contrast, mRNA levels of another glutamate cycle enzyme, glutamine synthetase, remained constant. Glutaminase protein levels increased per cell more than 2-fold between days 3 and 8, and at least 3-fold by day 10 in these cells. The total amount of glutamate per cell increased about 40% during this period. Glutaminase induction paralleled the development of Ca2+-dependent glutamate release, and the formation of neurites, synaptic vesicles, and synapses. The induction of glutaminase in developing granule cells is consistent with a special role for glutaminase in the synthesis of neurotransmitter glutamate.

Characterization of human cDNA and genomic clones for glial fibrillary acidic protein.

Both a partial cDNA clone and a complete genomic clone have been isolated for human gfa, the gene encoding the major component of astrocyte intermediate filaments, glial fibrillary acidic protein (GFAP). The nucleotide sequence of the entire coding region and 102 bp of the 5' flanking DNA was determined. The mRNA start site was identified by primer extension and probe protection experiments, and a novel in vitro transcription and translation procedure was then used to establish that the first ATG in the mRNA initiates GFAP synthesis. The predicted amino-terminal sequence for human GFAP differs greatly from that previously deduced for mouse GFAP from its gene sequence, despite otherwise high homology. This discrepancy was resolved by determining that the published mouse genomic sequence has an incorrect additional base. The corrected sequence produces strong homology between human and mouse GFAP in their predicted amino acid sequences, and suggests that human and mouse GFAP initiate at homologous positions. The beginning sequence deduced here for both proteins is matched closely by that previously obtained for porcine GFAP by direct sequencing of its amino-terminal end. This supports the protein initiation sites proposed, and also indicates that GFAP is not processed at its amino-terminal end.

Fatty acid activation of peroxisome proliferator-activated receptor (PPAR).

Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate peroxisome proliferator-activated receptor (PPAR), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from rat that is homologous to that from mouse, which encodes a 97% similar protein. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activated the receptor chimera. In addition, saturated fatty acids induced the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. To test whether a common PPAR binding metabolite might be formed from free fatty acids we tested the effects of differentially beta-oxidizable fatty acids and inhibitors of fatty acid metabolism. The peroxisomal proliferation-inducing, non-beta-oxidizable, tetradecylthioacetic acid activated PPAR to the same extent as the strong peroxisomal proliferator WY-14,643, whereas the homologous beta-oxidizable tetradecylthiopropionic acid was only as potent as a non-substituted fatty acid. Cyclooxygenase inhibitors, radical scavengers or cytochrome P450 inhibitors did not affect activation of PPAR. In conclusion, beta-oxidation is apparently not required for the formation of the PPAR-activating molecule and this moiety might be a fatty acid, its ester with CoA, or a further derivative of the activated fatty acid prior to beta-oxidation of the acyl-CoA ester.

A new value for mol percent guanine + cytosine of DNA for the salmonid fish pathogen Renibacterium salmoninarum.

The mol% G + C of DNA extracted from seven different isolates of Renibacterium salmoninarum was determined. Organisms studied were from selected geographical areas (U.S.A., Canada, England and France) and were isolated from five different species of salmonid fish. The mol% G + C was determined to be 55.5, higher than the currently reported value of 53.

Glutamic acid decarboxylase mRNA in rat brain: regional distribution and effects of intrastriatal kainic acid.

Glutamic acid decarboxylase (GAD) mRNA was quantified in different regions of rat brain using an antisense RNA probe (ribo-probe) prepared from a cloned feline cDNA. In all brain regions studied a single band of GAD mRNA of approximately 3.7 kb was detected. The level of GAD mRNA was highest in the cerebellum, followed by the hypothalamus greater than thalamus greater than striatum greater than hippocampus greater than frontal cortex = parietal cortex greater than or equal to medulla = pons. Since GAD has been previously localized to intrinsic neurons of the striatum, we examined the effects of intrastriatal kainic acid administration on striatal GAD mRNA. The level of GAD mRNA in the kainic acid-lesioned striatum was reduced by 70-75% when compared to the contralateral (unlesioned) striatum. In contrast, the level of glutamine synthetase (an enzyme localized to glia) mRNA was increased approximately 290% in the kainic acid-lesioned striatum. There were no significant differences in GAD mRNA levels between the ipsilateral and contralateral cerebral cortices and hippocampi of rats injected with intrastriatal kainic acid.

Isolation of a cDNA for rat brain glutaminase.

A single phage was isolated from a lambda gt11 rat brain cDNA library by screening with antibodies prepared against rat renal glutaminase. Partial proteolysis of the fusion protein produced by a lysogen of the isolated phage generated a series of immunoreactive peptides that co-migrated with those derived from the purified brain glutaminase. The cDNA has a single open reading frame which encodes 326 amino acids that are in frame with beta-galactosidase. A 72-kDa protein, corresponding in size to the precursor of mitochondrial glutaminase, was immunoprecipitated from the translation products of rat renal mRNA that selectively hybridized to the cDNA. A probe made from the glutaminase cDNA detected an mRNA about 6 kb in length. This mRNA was present in rat brain and normal kidney RNA, increased 6-fold in acidotic kidney RNA, but was not detectable in liver RNA.

Examination of the effectiveness of various treatment techniques for reducing tension.

The effectiveness of various tension-reducing techniques was investigated. Sixty-three subjects were randomly assigned to four treatment groups (EMG feedback, finger temperature feedback, EMG plus finger temperature feedback, autogenic training), a placebo control group, and a waiting-list control group. Physiological as well as a variety of cognitive measures were used to monitor changes in tension. The results showed no significant difference in effectiveness between the six groups. On virtually all cognitive measures there were significant decreases in tension, but not on the physiological measures. The decreases in tension on the cognitive measures were interpreted to be the results of non-specific (placebo) effects. These non-specific effects were not short-term, but did persist for at least three months after completion of treatment.

Seroprotection against serogroup C meningococcal disease in adolescents in the United Kingdom: observational study.

OBJECTIVE: To determine the persistence of bactericidal antibody titres following immunisation with serogroup C meningococcal glycoconjugate vaccine at age 6-15 years in order to examine changes in persistence of antibodies with age. DESIGN: Observational study. SETTING: Secondary and tertiary educational institutions in the United Kingdom. PARTICIPANTS: Healthy adolescents aged 11-20 years previously immunised between 6 and 15 years of age with one of the three serogroup C meningococcal vaccines. INTERVENTION: Serum obtained by venepuncture. MAIN OUTCOME MEASURES: Percentage of participants with (rabbit complement) serum bactericidal antibody titres of at least 1:8; geometric mean titres of serogroup C meningococcal serum bactericidal antibody. RESULTS: Five years after immunisation, 84.1% (95% confidence interval 81.6% to 86.3%) of 987 participants had a bactericidal antibody titre of at least 1:8. Geometric mean titres of bactericidal antibody were significantly lower in 11-13 year olds (147, 95% confidence interval 115 to 188) than in 14-16 year olds (300, 237 to 380) and 17-20 year olds (360, 252 to 515) (P<0.0001 for both comparisons). Within these age bands, no significant difference in geometric mean titres of bactericidal antibody between recipients of the different serogroup C meningococcal vaccines was seen. More than 70% of participants had received a vaccine from one manufacturer; in this cohort, geometric mean titres were higher in those immunised at aged 10 years or above than in those immunised before the age of 10. CONCLUSIONS: Higher concentrations of bactericidal antibody are seen five years after immunisation with serogroup C meningococcal vaccine at age 10 years or above than in younger age groups, possibly owing to immunological maturation. This provides support for adolescent immunisation programmes to generate sustained protection against serogroup C meningococcal disease not only for the vaccine recipients but also, through the maintenance of herd immunity, for younger children.

Toward a molecular etiology of Alzheimer's disease.

Recent progress in the biochemical characterization of Alzheimer's disease (AD) neuropathology has led to the proposal of three hypotheses for the molecular etiology of AD. One focuses on calcium-activated neutral proteases or calpains (Nixon, 1989). Another focuses on protein phosphorylation (Saitoh & Iimoto, 1989). A third is centered on altered phospholipid metabolism (Pettegrew, 1989). Interestingly, all three hypotheses are mutually compatible, involving closely interlocking biochemical systems. Disturbances in any one of these systems might result in the same type of neuropathology, consistent with suggestions that AD could have multiple etiologies. Future investigations of the function and interrelation of these systems in the central nervous system in general and at the synaptic junction in particular are likely to have significant bearing on our understanding of AD.

Sources of plant sterol contaminants encountered in low level steroid analysis.

During development of an analytical method to characterize ligands to new members of the steroid hormone receptor superfamily, a persistant contaminant profile was observed during gas chromatographic analysis of reagent blanks. Mass spectrometric analysis identified three of the contaminant peaks as cholesterol and the plant sterols stigmasterol and sitosterol. Laboratory articles made of natural rubber, i.e. pipette fillers and latex gloves, were found to be the source of these and other compounds in the reagent blank profile.

Normal cerebellar glutamate dehydrogenase protein in spinocerebellar degeneration.

Immunochemical analyses (Western blots) of cerebellar homogenates for glutamate dehydrogenase (GDH) from patients with spinocerebellar degeneration and control subjects were conducted. Four patients with autosomal dominant Joseph disease type of spinocerebellar degeneration, one patient with autosomal dominant olivopontocerebellar degeneration and four control subjects were studied. GDH was of the same molecular weight and amount in all patients and control subjects. These data together with normal GDH activity from these same homogenates published previously support the view that GDH is not involved in the pathogenesis of these types of dominantly inherited spinocerebellar degeneration.

Cloning of multiple genes involved with cobalamin (Vitamin B12) biosynthesis in Bacillus megaterium.

An effective shotgun cloning procedure was developed for Bacillus megaterium by amplifying gene libraries in Bacillus subtilis. This technique was useful in isolating at least 11 genes from B. megaterium which are involved with cobalamin (vitamin B12) biosynthesis. Amplified plasmid banks were transformed into protoplasts of both a series of Cob mutants blocked before the biosynthesis of cobinamide and Cbl mutants blocked in the conversion of cobinamide into cobalamin. Amplification of gene libraries overcame the cloning barriers inherent in the relatively low protoplast transformation frequency of B. megaterium. A family of plasmids was isolated by complementation of seven different Cob and Cbl mutants. Each plasmid capable of complementing a Cob or Cbl mutant was transformed into each one of the series of Cob and Cbl mutants; many of the plasmids isolated by complementation of one mutation carried genetic activity for complementation of other mutations. By these criteria, four different complementation groups were resolved. At least six genes involved in the biosynthesis of cobinamide are carried on a fragment of DNA approximately 2.7 kilobase pairs in length; other genes involved in the biosynthesis of cobinamide were located in two other complementation groups. The physical and genetic data permitted an ordering of genes within several of the complementation groups. The presence of complementing plasmids in mutants blocked in cobalamin synthesis resulted in restoration of cobalamin biosynthesis.

Effect of diabetes on glutamine synthetase expression in rat skeletal muscles.

The regulation of glutamine synthetase expression in muscles from normal and diabetic (streptozotocin-treated) rats was studied. Muscle and body weights were markedly reduced in diabetic animals. Glutamine synthetase activity was significantly (2-fold) elevated 7 days after induction of diabetes. Increased enzyme activity persisted for at least 14 days after induction of diabetes, and it was apparent in both slow (soleus) and fast (plantaris) muscles. The diabetes-induced increase in enzyme activity was reflected in an increased steady-state level of glutamine synthetase mRNA. The increases in glutamine synthetase activity and mRNA level in muscle from diabetic rats were reversed by insulin administration. Increased expression of glutamine synthetase may be important for accelerated glutamine production by muscle from diabetic rats.

Changes in glutamate-cycle enzyme mRNA levels in a rat model of hepatic encephalopathy.

To detect possible changes in the regulation of glutamate/gamma-aminobutyric acid (GABA) enzymes at the level of gene expression in a thioacetamide-induced rat model of acute hepatic encephalopathy, we have examined changes in the mRNAs of four glutamate/GABA enzymes by quantitative RNA blot hybridization analysis. Such changes could reflect cell adaptation to excess ammonia or some other associated metabolic stress. The mRNA levels of glutamate dehydrogenase (GDH) decreased similarly in three different brain regions, whereas those of glutamine synthetase (GS) and glutaminase (GA) increased. The mRNA levels of glutamate decarboxylase (GAD) were unchanged. The results indicate that some effect of liver damage, presumably hyperammonemia, affected the expression of some, but not all, genes associated with ammonia and glutamate metabolism in the brain. This adaptation of gene expression to secondary effects of ammonia on brain amino acid neurotransmitter metabolism or brain energy metabolism could play a role in the physiological changes observed in hepatic encephalopathy.