Conformational differences between linear alpha (2----8)-linked homosialooligosaccharides and the epitope of the group B meningococcal polysaccharide

The alpha-(2----8)-linked sialic acid oligosaccharides (NeuAc)n exhibit an unusual degree of heterogeneity in the conformation of their linkages. This was diagnosed by observation in their 13C NMR spectra of an equivalent and unique heterogeneity in the chemical shifts of their anomeric carbons and subsequently confirmed by more comprehensive 1H and 13C NMR studies. In these studies both one-dimensional and two-dimensional experiments were carried out on the trisaccharide (NeuAc)3 and colominic acid. In addition to the unambiguous assignment of the signals in the spectra, these experiments demonstrated that both linkages of (NeuAc)3 differed in conformation from each other and from the inner linkages of colominic acid. The NMR data indicate that these conformational differences extend to both terminal disaccharides of oligosaccharides larger than (NeuAc)5, a result that has considerable physical and biological significance. In the context of the group B meningococcal polysaccharide, it provides an explanation for the conformational epitope of the group B meningococcal polysaccharide, which was proposed on the evidence that (NeuAc)10, larger than the optimum size of an antibody site, was the smallest oligosaccharide able to bind to group B polysaccharide specific antibodies. Because the two terminal disaccharides of (NeuAc)10 differ in conformation to its inner residues, the immunologically functional part of (NeuAc)10 resides in its inner six residues. This number of residues is now consistent with the maximum size of an antibody site.     

ATP synthase from bovine mitochondria: sequences of imported precursors of oligomycin sensitivity conferral protein, factor 6, and adenosinetriphosphatase inhibitor protein

Oligomycin sensitivity conferral protein (OSCP), factor 6 (F6), and ATPase inhibitor protein are all components of the ATP synthase complex of bovine mitochondria. They are encoded in nuclear DNA. Complementary DNA clones encoding the precursors of these proteins have been isolated from a bovine library by using mixtures of synthetic oligonucleotides as hybridization probes, and their DNA sequences have been determined. The deduced protein sequences show that the OSCP, F6, and inhibitor proteins have N-terminal presequences of 23, 32, and 25 amino acids, respectively. These presequences are not present in the mature proteins. It is assumed that they serve to direct the proteins into the mitochondrial matrix. The cDNA clones have also been employed as hybridization probes to investigate the genetic complexity of the three proteins in cows and humans. These experiments indicate that the bovine and human inhibitor and bovine F6 proteins are encoded by single genes but suggest the possibility of the presence in both species of more than one gene (or pseudogenes) for the OSCP.     

Pulmonary vasodilatory response to neurohypophyseal peptides in the rat.

Experiments were performed on isolated salt-perfused rat lungs to determine the receptor type(s) responsible for the pulmonary vascular effects of the neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin. Bolus administration of AVP to lungs preconstricted with the thromboxane mimetic U-46619 resulted in a dose-dependent vasodilatory response (approximately 65% reversal of U-46619-induced vasoconstriction at the highest dose tested) that was blocked by pretreatment with a selective V1- but not by a selective V2-vasopressinergic receptor antagonist. Administration of a selective V1-agonist to the preconstricted pulmonary vasculature resulted in a vasodilatory response similar to that observed with AVP (approximately 55% reversal of U-46619 vasoconstriction), which was blocked by prior administration of the selective V1-receptor antagonist. Administration of the selective V2-receptor agonist desmopressin to the preconstricted pulmonary vasculature resulted in a small (approximately 8% reversal of U-46619 vasoconstriction) vasodilatory response that was, nevertheless, greater than that produced by addition of vehicle alone and was attenuated by pretreatment with a selective V2-receptor antagonist. Finally, oxytocin also caused vasodilation in the preconstricted pulmonary vasculature; however, the potency of oxytocin was approximately 1% of AVP, and the vasodilation produced by oxytocin was blocked by prior administration of a selective V1-receptor antagonist, suggesting that oxytocin acts via V1-vasopressinergic receptor stimulation. We conclude from these experiments that AVP and oxytocin dilate the preconstricted pulmonary vasculature primarily via stimulation of V1-vasopressinergic receptors. V2-receptor stimulation results in a minor vasodilatory response, although its physiological significance is unclear.     

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)     

Feeding trials ofAphelenchus avenae on soil bacteria and actinomycetes

Summary Aphelenchus avenae and various unidentified bacteria and actinomycetes were cultured from soil samples taken from a pine-forest and a wheat-field. The nematode failed to reproduce in significant numbers on cultures of these micro-organisms and was apparently unable to utilize them as food sources.     

The anal sac secretion of the red fox (Vulpes vulpes); its chemistry and microbiology. A comparison with the anal sac secretion of the lion (Panthera leo)

Phenylacetic, 3-phenylpropionic, $\text{p}$-hydroxyphenylacetic and 3 ($\text{p}$-hydroxyphenyl) propionic acids together with the series of C₂ to C₆ saturated fatty acids previously reported in the anal sac secretion of the red fox ($\text{Vulpes vulpes}$) are identified as constituents of the anal sac secretion of the lion (Panthera leo). All these compounds are also observed in the anal sac secretion of the red fox using gas chromatography. The aerobic microflora of red fox and domestic dog ($\text{Canis familiaris}$) anal sac secretion samples invariably consisted predominantly of $\text{Streptococcus faecium}$ and $\text{Streptococcus faecalis}$. The hypothesis that the secretion volatiles so far identified may be microbiologically produced is examined.     

Enhanced ability of skeletal muscle containing cyclocreatine phosphate to sustain ATP levels during ischemia following beta-adrenergic stimulation

Breast muscle of young chicks fed chow diets containing the creatine analog 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine) accumulated up to 40 mumol/g wet weight of the synthetic phosphagen 1-carboxymethyl-2-imino-3-phosphonoimidazolidine (cyclocreatine-P2-). ATP levels were sustained at high values substantially longer in breast muscle of cyclocreatine-fed chicks, compared to control-fed chicks, during total ischemia initiated 2 h after injection of both groups with the beta-adrenergic agonist isoproterenol (5 mg/kg subcutaneous). For example, in chicks fed 0.5% cyclocreatine for 10-19 days ATP levels in isoproterenol-stimulated breast muscles after 1 h of ischemia at 37 degrees C were 6.1 mumol/g, compared to 1.9 mumol/g for the control-fed group, and after 2 h of ischemia were 3.5 mumol/g compared to 0.6 mumol/g for controls. Creatine-P reserves in isoproterenol-stimulated breast muscles of all dietary groups were essentially exhausted within the first hour of ischemia. In contrast, breast muscle of chicks fed either 1 or 0.5% cyclocreatine still contained 28 and 19 mumol/g of cyclocreatine-P, respectively, after 1 h of ischemia; after 2 h of ischemia, the respective cyclocreatine-P values were 20 and 13 mumol/g. Isoproterenol-stimulated chick breast muscle provides the first skeletal muscle model system for studying the molecular mechanisms by which dietary cyclocreatine helps sustain ATP levels during ischemia. Although adaptive factors are also involved, it is suggested that a significant portion of the ATP-sustaining activity of dietary cyclocreatine in ischemic breast muscle can be attributed to the unique thermodynamic properties of the accumulated cyclocreatine-P. These properties enable cyclocreatine-P to continue to thermodynamically buffer the adenylate system and transport high energy phosphate throughout the long muscle fibers at cytosolic pH values and phosphorylation potentials well below the range where the creatine-P system can function effectively. Synergism between glycolysis and this long-acting synthetic phosphagen might well help delay depletion of ATP levels in skeletal muscles during ischemia. Cyclocreatine feeding provides a unique experimental tool for quantitative evaluation of the proposed protective role of ATP against irreversible cellular damage in skeletal and cardiac muscles during ischemic episodes.