Sulfur metabolism in Beggiatoa alba

The metabolism of sulfide, sulfur, and acetate by Beggiatoa alba was investigated under oxic and anoxic conditions. B. alba oxidized acetate to carbon dioxide with the stoichiometric reduction of oxygen to water. In vivo acetate oxidation was suppressed by sulfide and by several classic respiratory inhibitors, including dibromothymoquinone, an inhibitor specific for ubiquinones. B. alba also carried out an oxygen-dependent conversion of sulfide to sulfur, a reaction that was inhibited by several electron transport inhibitors but not by dibromothymoquinone, indicating that the electrons released from sulfide oxidation were shuttled to oxygen without the involvement of ubiquinones. Intracellular sulfur stored by B. alba was not oxidized to sulfate or converted to an external soluble form under aerobic conditions. On the other hand, sulfur stored by filaments of Thiothrix nivea was oxidized to extracellular soluble oxidation products, including sulfate. Sulfur stored by filaments of B. alba, however, was reduced to sulfide under short-term anoxic conditions. This anaerobic reduction of sulfur was linked to the endogenous oxidation of stored carbon and to hydrogen oxidation.     

Interaction of small dermatan sulfate proteoglycan from fibroblasts with fibronectin

Immunogold labeling was used to localize the core protein of small dermatan sulfate proteoglycan (DS-PG) on the surface of cultured human fibroblasts. At 4 degrees C, DS-PG core protein was uniformly distributed over the cell surface. At 37 degrees C, gold particles either became rearranged in form of clusters or remained associated with fibrils. Double-label immunocytochemistry indicated the co-distribution of DS-PG core protein and fibronectin in the fibrils. In an enzyme-linked immunosorbent assay, binding of DS-PG from fibroblast secretions and of its core protein to fibronectin occurred at pH 7.4 and at physiological ionic strength. Larger amounts of core protein than of intact proteoglycan could be bound. Fibronectin peptides containing either the heparin-binding domain near the COOH-terminal end or the heparin-binding NH2 terminus were the only fragments interacting with DS-PG and core protein. Competition and replacement experiments with heparin and dermatan sulfate suggested the existence of adjacent binding sites for heparin and DS-PG core protein. It is hypothesized that heparan sulfate proteoglycans and DS-PG may competitively interact with fibronectin.     

Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters

We present a statistical-mechanical selection theory for the sequence analysis of a set of specific DNA regulatory sites that makes it possible to predict the relationship between individual base-pair choices in the site and specific activity (affinity). The theory is based on the assumption that specific DNA sequences have been selected to conform to some requirement for protein binding (or activity), and that all sequences that can fulfil this requirement are equally likely to occur. In most cases, the number of specific DNA sequences that are known for a certain DNA-binding protein is very small, and we discuss in detail the small-sample uncertainties that this leads to. When applied to the binding sites for cro repressor in phage lambda, the theory can predict, from the sequence statistics alone, their rank order binding affinities in reasonable agreement with measured values. However, the statistical uncertainty generated by such a small sample (only 6 sites known) limits the result to order-of-magnitude comparisons. When applied to the much larger sample of Escherichia coli promoter sequences, the theory predicts the correlation between in vitro activity (k2KB values) and homology score (closeness to the consensus sequence) observed by Mulligan et al. (1984). The analysis of base-pair frequencies in the promoter sample is consistent with the assumption that base-pairs at different positions in the sites contribute independently to the specific activity, except in a few marginal cases that are discussed. When the promoter sites are ordered according to predicted activities, they seem to conform to the Gaussian distribution that results from a requirement for maximal sequence variability within the constraint of providing a certain average activity. The theory allows us to compare the number of specific sites with a certain activity to the number that would be expected from random occurrence in the genome. While strong promoters are "overspecified", in the sense that their probability of random occurrence is very low, random sequences with weak promoter-like properties are expected to occur in very large numbers. This leads to the conclusion that functional specificity is based on other properties in addition to primary sequence recognition; some possibilities are discussed. Finally, we show that the sequence information, as defined by Schneider et al. (1986), can be used directly (at least in the case of equilibrium binding sites) to estimate the number of protein molecules that are specifically bound at random "pseudosites" in the genome.(ABSTRACT TRUNCATED AT 400 WORDS)     

Measurement of Acetylcholine Release in Freely Moving Rats by Means of Automated Intracerebral Dialysis

The present study demonstrates the feasibility of measuring acetylcholine in perfusion samples collected by means of in vivo brain dialysis in the striata of freely moving rats. The output of the dialysis device was directly connected to an automated sample valve of a HPLC-assay system that comprises a cation exchanger, a post-column enzyme reactor, and an electrochemical detector. The presence of an acetylcholinesterase inhibitor (neostigmine) in the perfusion fluid was required for the detection of acetylcholine in the perfusate. Increasing concentrations of neostigmine induced increasing amounts of acetylcholine. Continuous perfusion with a fixed concentration (2 microM) of neostigmine resulted in gradually increasing amounts of collected acetylcholine over time although a considerable variation between successive samples exists. The brain dialysis technique was further validated by studying the effect of various drugs. Systemically administered atropine increased the output of acetylcholine, whereas the addition of tetrodotoxin to the perfusion fluid resulted in a complete disappearance of the neurotransmitter.     

Determination of small quantities of sulfate (0-12 nmol) in serum, urine, and cartilage of the mouse

The colorimetric benzidine method of K. S. Dodgson and B. Spencer (1953, Biochem. J. 55, 436-440) for the measurement of inorganic sulfate can be scaled down about 100 times by using disposable 96-well microplates instead of individual cuvettes. Ten-microliter samples of serum and urine, derived from mice, can be analyzed in a simple, rapid, and reliable way without sacrificing the animals. Without prior isolation of sulfated glycosaminoglycans, ester sulfate in mouse patellar cartilage is liberated quantitatively as inorganic sulfate upon acid hydrolysis in 3 M HCl for 16 h at 80 degrees C. To this end the articular cartilage layer of the patella must be separated in toto from the underlying bone. Subsequent hydrolysis in polypropylene tubes gives accurate results. In contrast, hydrolysis in borosilicate glass vials is useless, since nanomoles of sulfate added cannot be recovered adequately. The thin patellar cartilage layer obtained from 10-week-old male mice contains about 5 nmol of sulfate, an amount easily measured with the developed microplate benzidine method.     

GC-MS identification of GA20-13-O-glucoside formed from GA20 in normal plants and dwarf-1 mutants ofZea mays L.

After feeding GA₂₀ to excised seedlings ofZea mays L. normals (N) and dwarf-1 mutants (d1), GA₂₀-13-O-glucoside (9) was identified by HPLC and by GC-MS of its permethylated derivative. The glucosylation rate of GA₂₀ was found to be higher in the dwarf-1 mutant (26%) than in the normal plant (3.6%). This article includes a GC-MS study in which diagnostic fragments from the spectra of permethylated synthetic GA glucosides have been selected that proved to be useful for the identification of permethylated GA glucosides.     

Internal motions in yeast phenylalanine transfer RNA from 13C NMR relaxation rates of modified base methyl groups: a model-free approach

Internal motions at specific locations through yeast phenylalanine tRNA were measured by using nucleic acid biosynthetically enriched in 13C at modified base methyl groups. Carbon NMR spectra of isotopically enriched tRNA(Phe) reveal 12 individual peaks for 13 of the 14 methyl groups known to be present. The two methyls of N2,N2-dimethylguanosine (m22G-26) have indistinguishable resonances, whereas the fourteenth methyl bound to ring carbon-11 of the hypermodified nucleoside 3' adjacent to the anticodon, wyosine (Y-37), does not come from the [methyl-13C]methionine substrate. Assignments to individual nucleosides within the tRNA were made on the basis of chemical shifts of the mononucleosides [Agris, P. F., Kovacs, S. A. H., Smith, C., Kopper, R. A., & Schmidt, P. G. (1983) Biochemistry 22, 1402-1408; Smith, C., Schmidt, P. G., Petsch, J., & Agris, P. F. (1985) Biochemistry 24, 1434-1440] and correlation of 13C resonances with proton NMR chemical shifts via two-dimensional heteronuclear proton-carbon correlation spectroscopy [Agris, P. F., Sierzputowska-Gracz, H., & Smith, C. (1986) Biochemistry 25, 5126-5131]. Values of 13C longitudinal relaxation (T1) and the nuclear Overhauser enhancements (NOE) were determined at 22.5, 75.5, and 118 MHz for tRNA(Phe) in a physiological buffer solution with 10 mM MgCl2, at 22 degrees C. These data were used to extract two physical parameters that define the system with regard to fast internal motion: the generalized order parameters (S2) and effective correlation times (tau e) for internal motion of the C-H internuclear vectors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The genotypic response of mouse embryos to multiple freezing variables

Four- and eight-cell embryos from 3 mouse genotypes were cryopreserved to study the relationship of genetics and freezing variables on post-thaw survival. Embryos from outbred N:NIH(S), N:NIH(S)-B and inbred (C57BL/6N) mice were exposed to 1 of 2 cryoprotectants (glycerol [GLYC] versus dimethyl sulfoxide [DMSO]) and stored in 1 of 2 containers (ampules [AMP] versus straws [STR]). Containerized embryos were cooled at a rate of 0.5 degrees C/min to a minimum of -35 degrees C, transferred into liquid nitrogen, and later thawed and cultured in vitro. Genotypic differences (p less than 0.05) were noted for 4 interrelated embryo characteristics including post-thaw survival (PTS), embryo degeneration rate (EDR), and quality grade (QG) and viability grade (VG) ratings. The PTS for outbred embryos was greater (p less than 0.05) in GLYC than DMSO, whereas inbred C57BL/6N embryos survived similarly (p greater than 0.05) in either cryoprotectant. Compared to DMSO counterparts, embryos from GLYC-treated outbred groups had improved QG and VG ratings and reduced EDR (p less than 0.05), but comparable results were observed between GLYC- AND DMSO-treated embryos in the C57BL/6N group. Between genotypes, type of container affected PTS and EDR (p less than 0.05) but not QG or VG ratings (p greater than 0.05). Within genotypes, PTS generally ranged 15 to 20% higher (p less than 0.01) in AMP than STR groups. Increased PTS was noted in outbred mouse x GLYC x AMP groups; however, based on the degrees of difference, the inbred C57BL/6N strain appeared less affected by this 3-way interaction.(ABSTRACT TRUNCATED AT 250 WORDS)