DNA ligase and the pyridine nucleotide cycle in Salmonella typhimurium.

Bacterial DNA ligases use NAD as an energy source. In this study we addressed two questions about these enzymes. First, what is the physiological consequence of completely removing the NAD-dependent enzyme and replacing it with an ATP-dependent DNA ligase? We constructed Salmonella typhimurium strains in which the endogenous NAD-dependent DNA ligase activity was inactivated by an insertion mutation and the ATP-dependent enzyme from bacteriophage T4 was provided by a cloned phage gene. Such strains were physiologically indistinguishable from the wild type, even under conditions of UV irradiation or treatment with alkylating agents. These results suggest that specific functional interactions between DNA ligase and other replication and repair enzymes may be unimportant under the conditions tested. Second, the importance of DNA ligation as the initiating event of the bacterial pyridine nucleotide cycle was critically assessed in these mutant strains. Surprisingly, our results indicate that DNA ligation makes a minimal contribution to the pyridine nucleotide cycle; the Salmonella strains with only an ATP-dependent ligase had the same NAD turnover rates as the wild-type strain with an NAD-dependent ligase. However, we found that NAD turnover was significantly decreased under anaerobic conditions. We suggest that most intracellular pyridine nucleotide breakdown occurs in a process that protects the cell against oxygen damage but involves a biochemical mechanism other than DNA ligation.     

Hypoxanthine guanine phosphoribosyltransferase deficiency: nucleotide substitution causing Lesch-Nyhan syndrome identified for the first time among Japanese

Summary A previously undescribed nucleotide substitution at codon 51 (CGA to TGA) has been identified using the polymerase chain reaction technique in hypoxanthine guanine phosphoribosyltransferase (HPRT) cDNA; this is the first molecular evidence for a point mutation in a Japanese patient with Lesch-Nyhan syndrome. The present mutation is the 19th nucleotide substitution identified as a germ-line mutation at this locus and the second mutation generating a stop codon. The position of the nucelotide substitution is exactly the same as a previously described mutation HPRT_Toronto, indicating for the first time that nucleotide substitutions at the same position in the sequence of HPRT can generate different mutant alleles, one causing a partial deficiency and the other a complete deficiency. Although the type of nucleotide substitution is different between the two cases, a single base position has twice become the target of a mutation. However, the calculation of the probability of finding substitution mutations at the same base position in the coding region of hprt indicates that there is no evidence for the presence of a hot spot for substitution mutations in the human hprt germ line.     

Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products

The genes, rpoA, rpoB and rpoC of Escherichia coli, which encode the RNA polymerase alpha-, beta- and beta'-subunits, respectively, have been individually placed on expression plasmids under the control of the bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in host cells harboring each of the three plasmids resulted in the extensive overproduction of the three polypeptides. The overproduced subunits were purified and assembled into a functional enzyme, whose specific activity and dependence on the sigma-factor were indistinguishable from native RNA polymerase purified by conventional methods.     

A partial map of the barley genome incorporating restriction fragment length polymorphism, polymerase chain reaction, isozyme, and morphological marker loci

Nine low copy number genomic DNA clones, a ribosomal sequence, and seven cDNA clones were found to identify polymorphisms in cultivated barley (Hordeum vulgare L.). An F2 population consisting of 100 plants was produced from a cross between a high-yielding two-rowed feed barley cultivar and a genetic marker stock homozygous for nine recessive and one dominant morphological marker genes. Through the use of these 10 well-distributed marker genes, five previously mapped isozyme loci, and two storage-protein loci, the approximate recombinational location for each of 17 restriction fragment length polymorphism loci was estimated. One clone, pMSU21, identified variation that appeared to be the result of a small insertion-deletion event that differentiated two-rowed and six-rowed genotypes. This difference was characterized, and one allele was sequenced. Oligonucleotide primers that flanked the insertion-deletion event were synthesized, and DNA samples from the F2 population were subjected to polymerase chain reaction sequence amplification. The variation identified by this technique was determined to be allelic to the variation identified using pMSU21 in Southern blot analysis. Maps of previously undescribed informative clones are included.     

Immunocytochemical studies on the pituitary pars distalis of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary Immunocytochemical studies were performed to describe the characteristics of cell types and their distribution in the pars distalis of Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, collected at various stages of the reproductive cycle. Six distinct cell types have been identified in the pars distalis by the unlabeled immunoperoxidase technique and by the ABC method. Growth hormone (GH) and prolactin (PRL) cells were immunostained with antisera against chicken GH and ovine PRL. The GH-immunoreactive cells were round or oval orangeophilic cells distributed throughout the pars distalis with prominent aggregation in the posterolateral region. The PRL cells were pleomorphic carminophilic cells that occurred in small groups within the central and dorsocaudal regions of the pars distalis. They were sparsely distributed in the central region of the pars distalis in the hibernating bats, but increased significantly in the pregnant and lactating bats. The adrenocorticotropic (ACTH) cells were large round or polygonal amphophilic cells in the rostroventral and ventrolateral regions of the pars distalis. The thyrotropic (TSH) cells were small rounded or polygonal and distributed mainly in the ventrolateral region of the pars distalis. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) cells were identified immunocytochemically with antisera against the specific beta subunits of ovine LH and rat FSH. There were two populations of LH and FSH cells, one aggregated in the zona tuberalis and the other scattered singly throughout the rest of the pars distalis. The aggregated cells were immunoreactive with both antisera directed to LH and FSH, while scattered cells were reactive solely with antiserum to either LH or FSH and exhibited seasonal variations. In females, the proportional volume of the pars distalis occupied by LH cells was significantly reduced during pregnancy and lactation. No evidence of involution was observed in pars distalis cells except for PRL cells in males or females during hibernation.     

Electron-microscopic studies on the threshold value of calcium concentration for the release of storage granules and the acceleration of their degradation in the rat parathyroid gland

Summary To determine both a threshold value of calcium concentration (CC) for the release of storage granules and that for the acceleration of degradation of these granules, the rat parathyroid glands were perfused in situ with HEPES-Ringer solutions containing different concentration of Ca²⁺ for 10 min. With perfusates containing 0.83–1.21 mM Ca²⁺ (equivalent to 8–11 mg/dl serum calcium), the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged. With perfusates containing 0.83 mM Ca²⁺ (7.5 mg/dl) or less, however, both NSG-I and NSG-II decreased remarkably and the former was larger than the latter. On the contrary, with perfusates containing 1.27 mM Ca²⁺ (11.5 mg/dl) or more, NSG-II increased and the ratio of NSG-I to NSG-II was changed reversely. We concluded that a thereshold value of CC required for the release of storage granules may be present between 0.88 and 0.83 mM Ca²⁺ (8 and 7.5 mg/dl) and that a threshold value of CC for accelerating the transformation of type-I granules into type-II, the degradation of storage granules, may be situated at about 1.27 mM Ca²⁺ (11.5 mg/dl). Additionally, it was suggested that both prosecretory and storage granules are not only formed at the innermost Golgi cisterna but also at the trans-Golgi network.     

Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRTAnn Arbor)

Summary HPRT_{Ann Arbor} is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in two brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased K_m for both substrates, a normal V_max, a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRT_{Ann Arbor} and determined its complete nucleotide sequence. A single nucleotide change (TG) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.     

Embryonic loss in pony mares induced by intrauterine infusion of Candida parapsilosis

Pony mares which were detected pregnant by transrectal ultrasonography received a single intrauterine infusion of either sterile saline (control, n = 12 mares) or 10(6)Candida parapsilosis (treated, n = 12 mares) between Days 11 to 14 postovulation. Subsequent embryonic loss was studied by daily ultrasonography of the mare's uterus, by serum progesterone levels, by endometrial swabs for cytologic and microbiologic examination and by endometrial biopsies that were taken after embryonic loss was detected. Significantly fewer (P<0.01) embryonic losses occurred in control than in treated mares (4 12 vs 12 12 ). The mean interval from intrauterine infusion until embryonic loss was 5.8 +/- 2.8 d for control mares (n = 4) and 2.1 +/- 0.2 d for treated mares (n = 12). Prior to embryonic loss, moderate to marked edema of the endometrial folds in 12 of 12 treated mares and free fluid in the uterine lumen of 5 of 12 treated mares were detected by ultrasonography. After embryonic loss, Candida parapsilosis was cultured from the uteri of 8 of 12 treated mares, and E . coli was cultured from the uteri of 2 of 4 control mares. Postloss endometrial smears had cytologic evidence of inflammation in 10 of 12 treated mares and 3 of 4 control mares. Intrauterine inoculation of C. parapsilosis consistently induced embryonic loss and may provide a basis to further study the relationship between endometritis and embryonic loss in mares.     

Purification and Characterization of Thermostable Pullulanase from Bacillus stearothermophilus and Molecular Cloning and Expression of the Gene in Bacillus subtilis

A thermostable pullulanase (alpha-dextrin 6-glucanohydrolase [EC 3.2.1.41]) from a newly isolated Bacillus stearothermophilus strain (TRS128) was purified and characterized. The enzyme hydrolyzed (1-->6)-alpha-d-glucosidic linkages of pullulan to produce maltotriose, and the optimum temperature was 65 degrees C. About 90% of the enzyme activity was retained after treatment at 65 degrees C for 60 min. By using pTB522 as a vector plasmid, the pullulanase gene was cloned and expressed in Bacillus subtilis.     

Hydrolysis of myelin basic protein in human myelin by terminal complement complexes

The participation of terminal complement complexes (TCC) in demyelination has been shown in rodent cerebellar cultures. Since TCC modulates activities of various membrane-associated enzymes and increases the level of cellular Ca2+ we investigated whether TCC could activate Ca2+-dependent neutral proteases in myelin that would lead to hydrolysis of myelin basic protein (BP). Addition of antibody and C7-deficient serum plus C7 to sealed myelin vesicles of two to six bilayers caused significant BP hydrolysis compared to the hydrolysis caused by antibody and C7-deficient serum. Significant hydrolysis occurred at the stage of C5b6,7 assembly, which increased in magnitude at the C5b6-8 stage. C5b6-9 formation did not enhance the effect of C5b6-8. BP hydrolysis by C5b6,7 did not require Ca2+ whereas the effect of C5b6-8/C5b6-9 was, in part, Ca2+-dependent. We postulated that TCC formation in myelin membranes causes activation of myelin-associated neutral proteases with subsequent hydrolysis of BP as a consequence of complement peptide insertion and channel formation. Such processes may alter the structure of myelin and augment the action of other inflammatory cells and their products in demyelinating diseases that could ultimately lead to the loss of myelin.