Complete enzymatic deglycosylation of native sex steroid-binding protein (SBP or SHBG) of human and rabbit plasma: effect on the steroid-binding activity.

An enzymatic procedure for the complete removal of the N-linked and O-linked oligosaccharide side chains of the sex steroid-binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N-glycanase, neuraminidase, and O-glycanase and was monitored by SDS-PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N-glycanase generated a major 39,777-Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar-free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066-19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino-terminus. The N- and O-linked side chains of human SBP were removed by sequential digestion with N-glycanase and neuraminidase/O-glycanase. A 38,771-Da protein was generated, which agrees well with the molecular weight of the sugar-free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584-7590). N-deglycosylation of human and rabbit SBP has no effect on the steroid-binding activity, but removal of the O-linked side chains of N-deglycosylated human SBP results in an apparent 50% loss of steroid-binding activity and an increase in the Kd for the binding of 5 alpha-dihydrotestosterone from 0.3 mM to 0.9 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

An antiserum to the C-terminus of the Alzheimer amyloid precursor recognizes a soluble 70 kDa protein

A rabbit antiserum to the C-terminus of the putative brain amyloid precursor was used to probe Western blots of tissue proteins separated by SDS-PAGE. The antiserum specifically labelled a protein of approx. 70 kDa in the Tris buffer-soluble fraction of brain samples from rat, Alzheimer subjects, cases of young and old Down's syndrome, and age-matched controls. The 70 kDa protein was present in low concentrations in human liver and kidney, and was undetectable in human skeletal muscle. The 70 kDa protein may be a metabolite of the amyloid precursor.     

Dynamic regulation of mean arterial pressure: special role of renal resistances

In this paper the general properties of homeostatic variables are discussed, and it is shown that mean state regulation must be defined over some stated epoch and that the variance associated with such regulation can permit maximum/minimum variations of 2:1. Dynamic regulation is then contrasted with (automatic) control, and mean systemic arterial pressure (MSAP) in mammals is shown to be under dynamic regulation in the long run, although it may be under control in the short run. The discussion is next developed around the branching rules for mammalian arterial trees. The heart and lymphatic system are introduced as separate, zero-back-pressure, sump pumps that "ground" central venous pressure and interstitial pressure, respectively. Hydraulic flow arguments, combined with arterial tree branching rules, are used to demonstrate the short-circuit character of the renal circulation, and the peculiar distribution of pressure drops within it. From that peculiar distribution it is proposed that there is a nonanatomic, functional resistance located approximately at the region of efferent arterioles, which adds 15 mm Hg of hydrostatic pressure, upstream, to the central arteries. The chief aim of the paper is to raise certain questions about inconsistencies in data about renal circulation, to suggest a resolution, and to show how MSAP is set at (approximately) 100 mm Hg.     

Effects of prior dynamic leg exercise on static effort of the elbow flexors

The isometric endurance of the elbow flexors was determined in a control condition and subsequent to a maximal effort exercise bout on a cycle ergometer in seven subjects. Maximum voluntary contraction (MVC), peak rate of tension development (+dP/dt), peak rate of tension relaxation (-dP/dt), one-half contraction time, and one-half relaxation time were also measured. Each subject was tested on four occasions: two control and two experimental sessions. During the control sessions each subject held 40% of MVC to exhaustion, whereas the experimental session included a 1-min maximal effort exercise bout on a cycle ergometer 6 min prior to the isometric endurance task. Arterialized blood samples were drawn and analyzed for lactate, pH, PCO2, and PO2. Plasma bicarbonate was calculated from the Henderson-Hasselbalch equation. Subsequent to the cycle ergometer bout, blood lactate concentration rose from 0.8 to 11 mM, pH decreased from 7.43 to 7.20, PCO2 decreased from 40 to 32 Torr, and plasma bicarbonate decreased from 26 to 12 mM. When compared with the control values, no significant changes were evident for any muscle contractile properties following the cycle ergometer bout. However, isometric endurance was significantly reduced from 115.0 +/- 7.2 to 86.3 +/- 7.3 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytological Detection of the c25H Deletion Involving the Albino (c) Locus on Chromosome 7 in the Mouse

A deletion of the albino (c) locus on mouse chromosome 7 has been demonstrated using Q- and G-banding methods in a mouse heterozygous for the radiation-induced lethal albino allele, c(25H). The deletion, which is thought to be 1-6 cM long, represents about 7.6% of the length of the metaphase chromosome.     

The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHIC STUDIES OF THE CAROTID BODY FOLLOWING INJECTIONS OF LABELED BIOGENIC AMINE PRECURSORS

Adult Syrian hamsters were given a subcutaneous injection of reserpine 3 days before an intraperitoneal injection of ³H-3,4 dihydroxyphenylalanine or ³H-5, hydroxytryptophan and the carotid bodies were subsequently prepared for electron microscopic radioautography. Other Syrian hamsters were given a subcutaneous injection of reserpine and the carotid bodies were subjected to a sensitive cytochemical test for the detection of unsubstituted amines. These studies were made to determine whether the labeled amine precursors were incorporated into the cells and to see whether the parenchymal cells were affected by reserpine treatment. Material from hamsters treated first with reserpine and subsequently injected with ³H-3,4 dihydroxyphenylalanine or ³H-5, hydroxytryptophan exhibited reduced grains of silver over the cells which were associated mainly with the dense cores of the cytoplasmic granules. These studies offer evidence that the granules of the carotid body incorporate catecholamine and indolamine precursors. Material from hamsters incubated for the presence of unsubstituted amines gave a positive reaction (opaque cytoplasmic granules) for catecholamines but not for indolamines. The latter substances may not be present in quantities sufficient to register a positive reaction in the cytochemical test. The opaque granules, indicative of the presence of catecholamines, decreased in density after reserpine treatment. 5 days after one reserpine injection the granules had regained opacity and were comparable to those seen in the control cells.     

Validation of signature polarlipid fatty acid biomarkers for alkane-utilizing bacteria in soils and subsurface aquifer materials

Abstract Extractable cell membrane-derived polarlipid ester-linked fatty acids (PLFA) obtained from aerated soils gassed with methane or propane and from methane- and propane-oxidizing bacteria isolated from the soils were analyzed by capillary gas chromatography/mass spectrometry. Exposure of aerated soils to methane resulted in the formation of a high proportion of an unusual 18-carbon mono-unsaturated PLFA, 18:lw8c. High proportions of this fatty acid biomarker are found in monocultures from this soil grown in minimal media with methane. This PLFA has been previously established as associated with authentic type II methane-oxidizing bacteria. The microbiota in aerated soils exposed to hydrocarbons containing propane, formed a suite of PLFA characterized by high proportions of a 16-carbon mono-unsaturated acid, 16:lw6c, and an 18-carbon saturated fatty acid with an additional methyl branch at the 10 position, 10 Me 18:0. This PLFA pattern has been detected in several monocultures enriched from the soil with propane-amended minimal media. The correspondence of high proportions of these unusual mono-unsaturated PLFA in the isolated monocultures and in situ in the soils after stimulation with the appropriate hydrocarbon is a strong validation of the utility of these biomarkers in defining the community structure of the surface soil microbial community.     

Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot

Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP arabinogalactan-protein - GlcY -glucosyl Yariv reagent - ELISA enzyme-linked immunosorbent assay We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society.