Investigations on microbial sulfur respiration

The reduction of elemental or sulfane sulfur to hydrogen sulfide by eubacteria was investigated. Spirillum 5175 had the most active sulfur oxidoreductase. It could be cultivated with fumarate (F), elemental sulfur (S) or nitrate (N) as electron acceptor. Maximum activity was found for Spirillum 5175_S but activity was also present in Spirillum 5175_F and Spirillum 5175_N, i.e. the sulfur oxidoreductase is a constitutive enzyme. It was localized in the membrane, and no activity was found in the cytoplasm in contrast to Desulfovibrio baculatus. Different procedures were applied for the measurement of the sulfur oxidoreductase activity. In the manometric assay hydrogenase was coupled to the sulfur oxidoreductase, and the uptake of dihydrogen was measured in the presence of elemental sulfur. Alternatively, H₂S was assayed directly or was trapped in 12% NaOH and determined by the methylene blue procedure. Using ³⁵S sulfur and ³⁵S-labelled compounds both the substrate and H₂S could be measured. A further increase in sensitivity was achieved using phenosafranin. It was reduced photochemically, and served as the electron donor to the sulfur oxidoreductase, i.e. no hydrogenase was required. This was an important result in view of the fact that not all sulfur-reducing bacteria contain hydrogenase. However, in those cases the hydrogenase isolated from Clostridium pasteurianum could be coupled to the sulfur oxidoreductase. Among the different forms of elemental sulfur Janek sulfur gave the best results in terms of activity and reproducibility. The reduction of elemental sulfur to hydrogen sulfide had a pH optimum at pH 8.7–8.9. There was always a lag-phase which was pH-dependent. During this period the turbidity of the solution changed. Addition of thiols, such as GSH, shortened the lag-phase and caused an increase in activity of the sulfur oxidoreductase. In the presence of p-chloromercuribenzenesulfonic acid the reaction rate decreased significantly. Comparable reaction rates and activity values of the sulfur oxidoreductase in Spirillum 5175_F were obtained with organic trisulfides, RS-S-SR. In contrast to elemental sulfur RS-S-SR are well-defined chemical compounds suitable for quantitative and mechanistic investigations. Labelling the central sulfur of RS-S-SR with ³⁵S gave a satisfactory recovery of the total radioactivity in form of (³⁵S) H₂S in our assay. Trisulfides were shown to be formed as reactive intermediates in bacteria. This process required the sulfur transferase rhodanese which was present in Spirillum 5175, or other sulfur-reducing eubacteria.Abbreviations EPR Electron Paramagnetic Resonance - A Absorbance - PCMS p-chloromercuribenzenesulfonic acid - Sp. 5175_F Splrillum 5175 grown with fumarate - Sp. 5175_S with sulfur - Sp. 5175_N with nitrate - SCE Standard Calomel Electrode     

Effects of analogues of prostaglandin E2 and F2 alpha on the decidual cell reaction in the rat

The identity of the prostaglandins (PGs) involved in the decidual cell reaction is uncertain. In the present study we investigated the ability of analogues of PGE2 and PGF2 alpha, 16,16-dimethyl-prostaglandin E2, methyl ester (16,16Me2PGE2) and 15(S)-15-methyl-prostaglandin F2 alpha (15MePGF2 alpha) respectively, to bring about decidualization when infused into the uterine lumen of rats sensitized for the decidual cell reaction. As indicated by uterine weights 5 days after the commencement of the infusions into rats in which endogenous PG production had been inhibited by treatment with indomethacin, 16,16Me2PGE2 produced decidualization which was equivalent to that produced by PGE2. By contrast, the infusion of 15MePGF2 alpha inhibited decidualization, even when PGE2 was infused concomitantly. As indicated by uterine radioactivity concentrations after i.v. administration of 125I-labeled bovine serum albumin, the PGF2 alpha analogue also inhibited the endometrial vascular permeability increase which precedes decidualization. Compared to PGE2, 16,16Me2PGE2 was slightly less effective at displacing 3H-PGE2 from an endometrial membrane preparation; by contrast 15MePGF2 alpha was considerably less effective. These data suggest that PGE2 mediates the decidual cell reaction, and that the decidualization obtained in response to PGF2 alpha may involve its conversion within the uterus to PGE2.     

A rapid and sensitive method for measuring the conditional emotional response. II: On-the-baseline excitatory conditioning and extinction

The present study outlines a rapid and sensitive on-the-baseline conditional emotional response (CER) procedure. Using rats as the experimental subject, the method detects delay conditioning, incubation, extinction and spontaneous recovery. In addition, the method detects conditional responding using electric shock ranging from 0.23 to 0.50 mA as the unconditional stimulus. Because of its speed and sensitivity, the method shelters the subject from unnecessary long-term deprivation and pain.     

A quantitative analysis of 2-D gels identifies proteins in which labeling is increased following long-term sensitization in Aplysia

Long-term memory for sensitization of the gill- and siphon-withdrawal reflex in Aplysia, produced by 4 days of training, is associated with increased synaptic efficacy of the connection between the sensory and motor neurons. This training is also accompanied by neuronal growth; there is an increase in the number of synaptic varicosities per sensory neuron and in the number of active zones. Such structural changes may be due to changes in the rates of synthesis of certain proteins. We have searched for proteins in which the rates of [35S]methionine labeling are altered during the maintenance phase of long-term memory for sensitization by using computer-assisted quantitative 2-D gel analysis. This method has allowed us to detect 4 proteins in which labeling is altered after 4 days of sensitization training.     

Upper pleistocene fossil hominids from Sri Lanka

Between 1978 and 1983 hominid skeletal remains were collected from the cave sites of Batadomba lena and Beli lena Kitulgala in Sri Lanka (formerly Ceylon). These are the most ancient specimens of anatomically modern Homo sapiens found thus far in South Asia, radiocarbon dates placing them in the Upper Pleistocene. Morphometric analysis of the remains of some 38 individuals from the two sites indicates that these populations were characterized by medium stature, moderate to pronounced cranial and postcranial robusticity, medium-size permanent tooth crown measurements, prognathic alveolar facial proportions, and low incidence of osseous and dental pathological conditions. Comparisons of these ancient Sri Lankans with other prehistoric skeletal series from South Asia and elsewhere support the hypothesis that muscular-skeletal robusticity was a significant physical adaptation of earlier hunting-foraging populations. A trend towards reduction of sexual dimorphism and development of more gracile body form and smaller teeth appears to have accelerated with the socioeconomic transition to food-production strategies involving agriculture and pastoralism and refinement of technologies for food procurement and preparation, as documented by morphometric studies of later prehistoric inhabitants of South Asia.     

Microbial Hydroxylation of 1,4-Cineole

Microorganisms were examined for their potential to hydroxylate the oxygenated monoterpene 1,4-cineole. Using gas chromatography and thin-layer chromatography, screening experiments revealed that hydroxylation at position 2 was the most commonly observed microbial transformation reaction. In most microorganisms, the predominant alcohol metabolite was the 2-endo-alcohol isomer. Preparative-scale incubations were conducted in order to isolate and characterize microbial transformation products by comparison of proton nuclear magnetic resonance, mass spectrometry, and chromatography profiles with those of cineole standards. Streptomyces griseus yielded 8-hydroxy-1,4-cineole as the major hydroxylation product together with 2-exo- and 2-endo-hydroxy-1,4-cineoles.     

Chloromethane, a Novel Methyl Donor for Biosynthesis of Esters and Anisoles in Phellinus pomaceus

Chloromethane (CH₃Cl), a gaseous natural product released as a secondary metabolite by many woodrotting fungi of the family Hymenochaetaceae, has been shown to act as a methyl donor for biosynthesis of methyl esters of benzoic and furoic acid in the primary metabolism of Phellinus pomaceus. The broad-specificity methylating system could esterify a wide range of aromatic and aliphatic acids. In addition to CH₃Cl, both bromo- and iodomethanes acted as methyl donors. Methylation did not appear to proceed via methanol or a coenzyme A intermediate. The initial growth-related accumulation of methyl benzoate during culture of P. pomaceus was paralleled by an increase in activity of the methylating system in the mycelium. Changes in percent incorporation of C²H₃ from exogenous C²H₃Cl during growth indicated that although utilization of CH₃Cl was initially closely coupled to biosynthesis of the compound, the system became less tightly channeled later in growth. This phase coincided with release of gaseous CH₃Cl by the fungus. A biochemically distinct CH₃Cl-utilizing system capable of methylating phenols and thiophenol was also identified in the fungus, but in contrast with the carboxylic acid-methylating system, it attained maximum activity in the idiophase. Preliminary investigations of a non-CH₃Cl-releasing fungus, Fomitopsis pinicola, have shown the presence of a CH₃Cl-utilizing system capable of methylating benzoic acid, suggesting that CH₃Cl biosynthesis may occur in non-hymenochaetaceous fungi. Halogenated compounds hitherto found in nature are mainly stable end products of metabolism. The participation of CH₃Cl in primary fungal metabolism demonstrates that some halometabolites may have a previously unrecognized role as intermediates in the biosynthesis of nonhalogenated natural products.     

Genetic and other effects on antibody and cell mediated immune response in swine leucocyte antigen (SLA)-defined miniature pigs

Miniature pigs of eight swine leucocyte antigens (SLA) haplotypes were immunized with sheep erythrocytes (SRBC), hen egg white lysozyme (HEWL) and the synthetic peptide (T, G)-A--L to induce antibody. Bacillus Calmette Geurin (BCG) and dinitrochlorobenzene (DNCB) were used to induce cell mediated immune response (CMI). Analysis of variance by least squares was used to assess the effects of SLA haplotype, sire, dam, litter and sex of pig on the magnitude of the primary and secondary antibody response and on dermal delayed type hypersensitivity induced by purified protein derivative of tuberculin (PPD) and DNCB-induced contact hypersensitivity. The statistical model accounted for 43.50-77.30% of the observed variability in antibody and CMI at various times after immunization or challenge. While SLA had a significant effect on both antibody and CMI to some antigens at some, but not all times, sire, dam and litter were more frequently significant and to a greater degree. Haplotypes dd, dg and gg produced more antibody to SRBC and (T, G)-A--L while dg and gg had higher primary, but not secondary antibody response to HEWL. Delayed hypersensitivity to PPD was most marked in pigs of dd, dg and gg haplotypes while contact hypersensitivity to DNCB was expressed least in the dg and gg haplotype pigs. Heritability estimates were high for response to (T, G)-A--L and HEWL indicating feasibility of selective breeding for these traits.