Studies on the phospholipid composition of pathogenic cell membranes of Mycoplasma hyopneumoniae

The membrane phospholipid and fatty acid compositions of Mycoplasma hyopneumoniae, a pathogen of porcine enzootic pneumoniae isolated in China, was studied by thin-layer chromatography and gas chromatography. The results showed that membrane phospholipids consisted predominantly of diphosphatidylglycerol. The percentage of C16 - C18 fatty acids comprised 79% of the total fatty acids, of which oleic acid as well as palmitic acid are the major fatty acids. Some differences were shown in fatty acid composition as compared with membranes of other species of Mycoplasma.     

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.     

Morphological studies of polycystic mouse ovaries induced by dehydroepiandrosterone

Summary Morphological alterations induced by dehydroepiandrosterone (DHA) were studied in polycystic mouse ovaries (PCO). Treated mice showed ovulatory failure and cystic changes; cysts and follicles in various stages of growth and atresia were present although corpora lutea were absent. The levels of testosterone, dihydrotestosterone, 3- and 3-androstanediol, estrone and androstenedione increased, whereas estradiol was not detectable.The ultrastructure of granulosa cells in healthy and atretic follicles was similar to that of control animals, although the membrana granulosa in cysts was reduced to a monolayer of flattened cells. The theca interna of healthy and atretic follicles and ovarian cysts showed ultrastructural signs of abnormal steroidogenic stimulation.No significant differences (0.7<P<0.8) were found between the extensive surface area of gap junctions of healthy follicles of control and DHA-treated animals. On the P-face of granulosa cells of large healthy follicles, meandering strands of tight junctional particles were observed; their average length was significantly longer than those in healthy follicles of control animals (P<0.001). This increase was probably related to the large amounts of androgens present in the treated animals.Theca interna cells possessed small gap junctions; no significant differences (P>0.9) in gap-junction surface area were observed between DHA-treated and control animals. These results suggest that the size of gap junctions is probably unrelated to the steroidogenic activities of theca cells.The following trivial names have been used: Dihydrotestosterone: 5-androstan 17 ol-13 one; 3-androstanediol: 5-androstan 3,17 diol; 3-androstanediol: 5-androstan 3,17 diol     

Correlation of 3,4-dihydroxybutyl 1-phosphonate resistance with a defect in cardiolipin synthesis in Escherichia coli.

Escherichia coli treated for 1 h with 100 microM rac-3,4-dihydroxybutyl 1-phosphonate (DBP), a glycerol-3-phosphate analog, die when sorted at 5 degrees C, whereas the viability of untreated cells is relatively unaffected. This observation formed the basis of a selection procedure that was used to isolate mutants that are partially resistant to DBP. One such mutant, strain 6204, is constitutive for DBP transport, exhibits a particularly high degree of cold resistance, has the same doubling time as the parent, and is similar to the parent strain in terms of incorporation of DBP into the lipid fraction. Glycerol-3-phosphate and phosphatidylglycerol phosphate synthetases obtained from strain 6204 and its parent were identical in terms of DBP recognition. The parent strain is killed when incubated in the presence of a combination of 70 microM rac-DBP and 0.25% deoxycholate, whereas strain 6204 continues to grow, albeit more slowly, in the presence of this combination. Strain 6204 can be distinguished from the parent strain on agar plates (low phosphate minimal medium with glucuronate as the sole carbon source) containing 15 microM rac-DBP. The insertion of Tn10 near the 6204 mutation has facilitated genetic manipulations. All phenotypic effects attributed to strain 6204 appear to be due to a single mutation. Genetic analysis indicates that Tn10, inserted near the gene responsible for DBP resistance, maps in the vicinity of 27 min. Three-factor crosses reveal a gene order of hemA-Dbpr-Tn10(zch)-trp. The only gene for phosphoglyceride metabolism known to map in this region is the gene associated with cardiolipin synthetase, cls. Genetic results suggest that the mutation responsible for DBP resistance maps in or very near cls. Analysis of the lipids isolated from untreated strain 6204 (and from each of the transductants prepared by P1 vir-mediated transfer of DBP resistance of wild-type strains) reveals that cardiolipin synthesis is defective. These results strongly suggest that the mutation responsible for DBP resistance has its primary effect on cardiolipin synthesis. To further test this hypothesis, strains with an authentic cls mutation were constructed and examined for resistance to DBP. These strains had growth properties that were identical with those of strain 6204. Wild-type strains and mutants defective in cardiolipin synthesis were treated with DBP and 20 mM magnesium or calcium chloride. Simultaneous treatment of either cell type with DBP and divalent cation not only failed to stimulate growth but, quite the contrary, had a marked synergistic growth inhibitory effect.     

Characterization of the subunit structure of gonadotropin receptor in luteinized rat ovary

Gonadotropin receptors with specificity, high affinity and low capacity for luteinizing hormone and human chorionic gonadotropin (hCG) have been identified in rat luteal cells. To investigate the nature of the receptor, we have employed disuccinimidyl suberate, a cross-linker noncleavable by reducing agents, and dithiobis(succinimidyl propionate), a cleavable cross-linker, to covalently cross-link the 125I-hCG . receptor complex. The molecular weight of 125I-hCG-linked receptor complex and the receptor subunit structure were determined by electrophoresis in either 10 or 4.5% acrylamide in the presence of 0.1% sodium dodecyl sulfate with or without reducing agents. Autoradiographic analysis of the 125I-hCG-linked receptor separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing condition revealed a single labeled band corresponding to Mr = 305,000 +/- 15,000. However, electrophoresis performed in the presence of 50 mM dithiothreitol and 2% beta-mercaptoethanol resulted in the appearance of four labeled bands corresponding to Mr = 105,000 +/- 4,000, 96,000 +/- 5,000, 74,000 +/- 4,000, and 62,000 +/- 4,000 concomitant with the loss of the labeled band in the Mr = 305,000 region. Further experiments demonstrated that these four labeled bands were derived from the same molecular species. In addition, the 125I-hCG-linked receptor in the absence of reducing agent was not dissociated into subunits even by treatment with strong denaturing agent (8 M urea). The appearance of the cross-linked 125I-hCG . receptor was effectively inhibited by the unlabeled beta-subunit of hCG, intact hCG, and luteinizing hormone and partially inhibited by the alpha-subunit of hCG but not by choleratoxin, gonadotropin-releasing hormone, insulin or bovine serum albumin. These data suggest that 1) the hCG/luteinizing hormone receptor is an oligomeric complex linked by disulfide bonds and 2) that under reducing conditions, the oligomeric receptor dissociates into four nonidentical subunits.     

Influence of pulmonary inflations on discharge patterns of phrenic motoneurons

The purpose of this study was to assess the influence of pulmonary inflations on activities of single phrenic motoneurons. Studies were performed in decerebrate and paralyzed cats; activities of phrenic nerve and single phrenic motoneurons were recorded. Animals were ventilated with a servo-respirator which produced alterations in tracheal pressure in parallel with changes in integrated activity of the phrenic nerve. At end-tidal fractional concentrations of CO2 of 0.05, phrenic motoneurons were distributed into "early" and "late" populations, depending on time of onset of activity. During the late stages of neural inspiration, differences in levels of integrated activity of the phrenic nerve became evident between cycles with and without lung inflations. At a time approximating 90% of the inspiratory duration during inflations, integrated phrenic activity was higher for cycles with inflation. Concomitantly, with lung inflations, the discharge frequencies of early phrenic motoneurons were lower, and late motoneurons began to discharge sooner than when inflations were withheld. Similar results were obtained in hypercapnia. We conclude that reflexes activated by pulmonary inflations may produce augmentation, as well as inhibition of phrenic motoneuronal activities. Factors responsible for eliciting these reflex augmentations and inhibitions are discussed.     

Bidirectional effect of met-enkephalin on macrophage effector functions

Summary Met-enkephalin (ME) exerts a bimodal effect on functional activities of rat peritoneal macrophages (PM); in a range of low concentration (10^-9-10^-7 M) antibody dependent cellular cytotoxicity (ADCC)was markedly stimulated with a simultaneous decrease of Fc receptor (FcR) mediated phagocytosis while the opposite was observed at 10^-6-10^-5 M concentrations.Studying the possible underlying mechanism(s) the followings were recorded: (1) ME in all applied concentrations induced an early Na⁺ influx which was followed by a Ca²⁺ efflux in the range of low concentrations. In the range of high concentrations Na⁺ influx was accompanied by a Ca²⁺ influx. (2) ME at 10^-8 M concentration induced a rise in cGMP level with a plateau in the 60–120th min of incubation. This effect was prevented by 10^-5 M of naloxone. At 10^-6 M concentration a transient rise of cAMP level was recorded which was not affected by naloxone. (3) Verapamil in 10^-6 M abolished both the Ca²⁺ influx and the rise in cAMP level induced by 10^-6-10^-5 M ME but not the rise in cGMP level induced by lower ME concentrations. (4) cAMP elevation by high ME concentrations was abolished by enkephalinase inhibitory puromycin. (5) PM-enkephalinase as assessed by the cleavage of fluorogenic substrate L-alanine beta naphthylamide (ABNA), was inhibited by 10^-6-10^-5 M of ME. This inhibition was abolished by verapamil, but not affected by naloxone. In the range of low concentrations ME appears to act on specific delta opioid receptors and its action is positively coupled to guanylate cyclase. In relatively higher concentrations ME-action is not mediated by specific delta opioid receptors and it appears to involve Ca²⁺ influx, adenylate cyclase activation as well as the processing of hormone by PM-enkephalinase.     

Potentiation by the Tetraphenylboron Anion of the Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and Its Pyridinium Metabolite

The 1-methyl-4-phenylpyridinium species (MPP+) is the four-electron oxidation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is widely assumed to be the actual neurotoxic species responsible for the MPTP-induced destruction of dopaminergic neurons. MPTP is oxidized by the enzyme monoamine oxidase-B to a dihydropyridinium intermediate which is oxidized further to MPP+, an effective inhibitor of the oxidation of the Complex I substrates glutamate/malate in isolated mitochondrial preparations. In the present study, the tetraphenylboron anion (TPB) greatly potentiated the inhibitory effects of MPP+ and other selected pyridinium species on glutamate/malate respiration in isolated mouse liver mitochondria. At 10 microM TPB, the potentiation ranged from approximately 50-fold to greater than 1,000-fold for the several pyridinium species tested. In other experiments, TPB greatly enhanced the accumulation of [3H]MPP+ by isolated mitochondrial preparations. This facilitation by TPB of MPP+ accumulation into mitochondria explains, at least in part, the potentiation by TPB of the above-mentioned inhibition of mitochondrial respiration. Moreover, TPB addition increased the amount of lactate formed during the incubation of mouse neostriatal tissue slices with MPTP and other tetrahydropyridines. The administration of TPB also potentiated the dopaminergic neurotoxicity of MPTP in male Swiss-Webster mice. All of these observations, taken together, are consistent with the premise that the inhibitory effect of MPP+ on mitochondrial respiration within dopaminergic neurons is the ultimate mechanism to explain MPTP-induced neurotoxicity.     

Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition.

Eucaryotic, viral, and bacteriophage DNA polymerases of the alpha-like family share blocks of sequence similarity, the most conserved of which has been designated region I. Region I includes a YGDTDS motif that is almost invariant within the alpha-like family and that is similar to a motif conserved among RNA-directed polymerases and also includes adjacent amino acids that are more moderately conserved. To study the function of these conserved amino acids in vivo, site-specific mutagenesis was used to generate herpes simplex virus region I mutants. A recombinant virus constructed to contain a mutation within the nearly invariant YGDTDS motif was severely impaired for growth on Vero cells which do not contain a viral polymerase gene. However, three recombinants constructed to contain mutations altering more moderately conserved residues grew on Vero cells and exhibited altered sensitivities to nucleoside and PPi analogs and to aphidicolin. Marker rescue and DNA sequencing of one such recombinant demonstrated that the region I alteration confers the altered drug sensitivity phenotype. These results indicate that this region has an essential role in polymerase function in vivo and is involved directly or indirectly in drug and substrate recognition.