Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Viability of an ectomycorrhizal fungus during cross-flow filtration

Factors affecting the viability and infectivity of an ectomycorrhizal fungus during moderate concentration by cross-flow filtration were determined. Mycelial suspensions were concentrated with three commercial membrane filters (Prostak Millipore Co., M14 Tech-Sep Co. and Ceraflo Norton Co.) under aseptic conditions. Medium components may reduce the filtration rate due to their low solubility. An antifoam agent did not reduce the average flux rates as much as did the malt extract. Clear unobstructed channels (I.D. 6mm) of the tubular modules (Tech-Sep) gave the best results both in terms of performance (filtration rate) and cell viability. Shear stresses caused by pumping and flow through narrow retentate channels were probably responsible for lowering viability and infectivity. There was no linear relationship between permeate fluxes and cell concentration. There is an optimum pore size both in terms of performance (filtration rate) and cell viability. Physical blockage of large pores by hyphae could explain lower permeate flux rates than those obtained with lower pore sizes membranes.     

Insulin binding in the hypothalamus of lean and genetically obese Zucker rats

Recent reports have suggested that the obesity and hyperphagia of the genetically obese Zucker rat may be related to defective insulin action or binding in the hypothalamus. We used quantitative autoradiography to determine if insulin binding is altered in specific hypothalamic nuclei associated with food intake. Insulin binding was measured in the arcuate (ARC), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei of 3–4-month-old lean (Fa/Fa) and genetically obese (fa/fa) Zucker rats. A consistently reproducible 15% increase in the total specific binding of 0.1 nM [¹²⁵I]-insulin was found in the ARC of the obese genotype. A slight increase in insulin binding in the DMN was also found. No difference in specific insulin binding was found between genotypes in the VMN. Nonlinear least squares analysis of competitive binding studies showed that the K_d of the ARC insulin binding site was 33% higher in the lean rats than in the obese rats, indicating an increased affinity for insulin. No difference in site number (B_max) was found in the ARC, DMN or VMN, and no evidence was found for reduced insulin binding in the hypothalamus of the obese (fa/fa) genotype. The results suggest that hyperphagia and obesity of the obese (fa/fa) Zucker rat genotype may be associated with increased insulin binding in the arcuate nucleus.     

Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) upon membrane ionic exchanges in sea urchin eggs

The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive ⁸⁶Rb uptake and amiloride-sensitive ²⁴Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na⁺/H⁺ and Na⁺/K⁺ exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na⁺/H⁺ activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na⁺/H⁺ exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA-pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 min. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na⁺ pump, Na⁺/H⁺ exchange) without eliciting corresponding regulatory mechanisms (Na⁺ stat, pH stat).     

Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius Rs approximately 7.5 nm, expected value for heteromeric complexes containing a dimer of the Mr approximately 90,000 heat shock protein, hsp90 (Rs approximately 7.0 nm). The oxysterol receptor represented a much smaller entity (Rs approximately 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at approximately 0.30 M and 0.26-0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11-0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10-0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.     

Scanning motion,ocellar morphology and orientation mechanisms in the phototaxis of the nematode Mermis nigrescens

Summary The putative ocellus of Mermis females consists of a hollow cylinder of dense hemoglobin pigmentation located in the anterior tip. The exact location of the photoreceptive nerve endings, however, is unknown. During phototaxis a continual bending or scanning motion of the head (anterior 2 mm) causes the orientation of the tip to swing about the direction of the source. By turning off (shuttering) the light source whenever the tip orientation was to one side of the source direction, the average orientation of the base of the head, and eventually the body orientation, was caused to be biased about 28° to the opposite side. Because the shuttering was synchronized with the scanning motion, the scanning motion must be involved in the maintenance of orientation to light. The direction of the bias rules out a two-signal comparison mechanism of orientation and demonstrates that a deviation of the tip from the source direction must decrease, rather than increase, the illumination of the photoreceptors. These findings, and the ocellar morphology, require that the photoreceptors be located inside the hollow tube of pigmentation where they can be shadowed by the pigment during deviations of the tip. Focusing by the curved anterior end should cause a similar modulation of the illumination at this location. The occasional episodes of transverse phototaxis can be explained by the leakiness of the pigment walls to transverse illumination. Analysis of the motion of the anterior in the presence and absence of shuttering indicates that the orientation of the base of the head, due to the motion of the neck, is controlled by the signals generated during one or more cycles of the scanning motion of the head. The orientation may be regulated by the phase relationship between the photoreceptor signal and putative proprioceptive signals that indicate the bending in the head.     

Demonstration of LH inhibitor(s) in sheep and human sera

In mature rat Leydig cells, the testosterone output (24 ng/10(6) Leydig cells/4hrs.) is increased 10 fold by LH; the addition of serum from either control or castrated or hypophysectomized rams inhibits (60%) the LH-stimulated testosterone production. Similarly, the incubation of immature rat Leydig cells with sera from hypophysectomized patients leads to a diminution (70 and 30% respectively) of both basal (0.98 ng) and LH stimulated (3.44 ng) testosterone biosynthesis. These data suggest the existence of an LH inhibitor (or inhibitors) in blood from ram and human; in addition, this substance is not only of testicular origin and is not an LH-related molecule.     

Soluble and particulate inositol 1,4,5-trisphosphate 5-phosphatases show common antigenic determinants

Inositol 1,4,5-trisphosphate 5-phosphatase catalyses the dephosphorylation of the phosphate in the 5-position from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. One particulate and two soluble enzymes were previously described in bovine brain. In this study, we have obtained a precipitating antiserum against soluble type I inositol 1,4,5-trisphosphate 5-phosphatase. The particulate, but not the soluble type II enzyme, was immunoprecipitated by the serum. Inositol 1,4,5-triphosphate 5-phosphatase activity from crude extracts of rat brain, human platelets and rat liver were immmunoprecipitated by the same antibodies, suggesting the existence of common antigenic determinant among inositol 1,4,5-trisphosphate 5-phosphatases of diverse sources.     

Comparison of incremental and steady state tests of endurance training

To compare the results obtained by incremental or constant work load exercises in the evaluation of endurance conditioning, a 20-week training programme was performed by 9 healthy human subjects on the bicycle ergometer for 1 h a day, 4 days a week, at 70-80% VO2max. Before and at the end of the training programme, (1) the blood lactate response to a progressive incremental exercise (18 W increments every 2nd min until exhaustion) was used to determine the aerobic and anaerobic thresholds (AeT and AnT respectively). On a different day, (2) blood lactate concentrations were measured during two sessions of constant work load exercises of 20 min duration corresponding to the relative intensities of AeT (1st session) and AnT (2nd session) levels obtained before training. A muscle biopsy was obtained from vastus lateralis at the end of these sessions to determine muscle lactate. AeT and AnT, when expressed as % VO2max, increased with training by 17% (p less than 0.01) and 9% (p less than 0.05) respectively. Constant workload exercise performed at AeT intensity was linked before training (60% VO2max) to a blood lactate steady state (4.8 +/- 1.4 mmol.l-1) whereas, after training, AeT intensity (73% VO2max) led to a blood lactate accumulation of up to 6.6 +/- 1.7 mmol.l-1 without significant modification of muscle lactate (7.6 +/- 3.1 and 8.2 +/- 2.8 mmol.kg-1 wet weight respectively). It is concluded that increase in AeT with training may reflect transient changes linked to lower early blood lactate accumulation during incremental exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship between RNA catalytic processes

Proposals that an RNA-based genetic system preceeded DNA, stem from the ability of RNA to store genetic information and to promote simple catalysis. However, to be a valid basis for the RNA world, RNA catalysis must demonstrate or be related to intrinsic chemical properties which could have existed in primordial times. We analyze this question by first classifying RNA catalysis and related processes according to their mechanism. We define: (A) thedisjunct nucleophile class which leads to 5-phosphates. These include Group I and II intron splicing, nuclear mRNA splicing and RNase P reactions. Although Group I introns and its excision mechanism is likely to have existed in primordial times, present-day examples have arisen independently in different phyla much more recently. Comparative methodology indicates that RNase P catalysis originated before the divergence of the major kingdoms. In addition, alldisjunct nucleophile reactions can be interrelated by a proposed mechanism involving a distant 2-OH nucleophile. (B) theconjunct nucleophile class leading to 3-phosphates. This class is composed of self-cleaving RNAs found in plant viruses and the newt. We propose that tRNA splicing is related to this mechanism rather than the previous one. The presence of introns in tRNA genes of eukaryotes and archaebacteria supports the idea that tRNA splicing predates the divergence of these cell types.