Antigenic structure and variation in an influenza virus N9 neuraminidase.

We previously determined, by X-ray crystallography, the three-dimensional structure of a complex between influenza virus N9 neuraminidase (NA) and the Fab fragments of monoclonal antibody NC-41 [P. M. Colman, W. G. Laver, J. N. Varghese, A. T. Baker, P. A. Tulloch, G. M. Air, and R. G. Webster, Nature (London) 326:358-363, 1987]. This antibody binds to an epitope on the upper surface of the NA which is made up of four polypeptide loops over an area of approximately 600 A2 (60 nm2). We now describe properties of NC-41 and other monoclonal antibodies to N9 NA and the properties of variants selected with these antibodies (escape mutants). All except one of the escape mutants had single amino acid sequence changes which affected the binding of NC-41 and which therefore are located within the NC-41 epitope. The other one had a change outside the epitope which did not affect the binding of any of the other antibodies. All the antibodies which selected variants inhibited enzyme activity with fetuin (molecular weight, 50,000) as the substrate, but only five, including NC-41, also inhibited enzyme activity with the small substrate N-acetylneuramin-lactose (molecular weight, 600). These five probably inhibited enzyme activity by distorting the catalytic site of the NA. Isolated, intact N9 NA molecules form rosettes in the absence of detergent, and these possess high levels of hemagglutinin activity (W.G. Laver, P.M. Colman, R.G. Webster, V.S. Hinshaw, and G.M. Air, Virology 137:314-323, 1984). The enzyme activity of N9 NA was inhibited efficiently by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, whereas hemagglutinin activity was unaffected. The NAs of several variants with sequence changes in the NC-41 epitope lost hemagglutinin activity without any loss of enzyme activity, suggesting that the two activities are associated with separate sites on the N9 NA head.     

GTP-mediated Ca2+ release in rough endoplasmic reticulum. Correlation with a GTP-sensitive increase in membrane permeability.

Guanine nucleotides have been reported to stimulate reticular Ca2+ release. By using the structure-linked latency of microsomal mannose-6-phosphate phosphatase as an index of microsomal permeability [Arion, Ballas, Lange & Wallin (1976) J. Biol. Chem. 251, 4901-4907], the effects of GTP on Ca2+ release and membrane permeability were compared in liver microsomes. In a stripped rough-microsome preparation, GTP caused a dose-dependent increase in mannose 6-phosphate permeability. Half-maximal and maximal effects were observed at 3 microM- and 10 microM-GTP respectively. The time course of the change in membrane permeability coincided with the time course of GTP-dependent Ca2+ release. This increase in microsomal permeability displayed positive to-operativity with respect to GTP (Hill coefficient = 1.8). By analogy to the GTP-dependent Ca2+ release process, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]-triphosphate inhibited the ability of GTP to alter microsomal permeability, but were without effect when added alone. In the presence of 50 microM-GTP, complete inhibition of the GTP-dependent increase in microsomal permeability was achieved with 10 microM-guanosine 5'-[gamma-thio]triphosphate, whereas a 25% inhibition was observed with 10 microM-guanosine 5'-[beta gamma-imido]triphosphate. In contrast with previous observations in crude microsomal preparations, GTP-dependent Ca2+ release in the stripped rough-microsome preparation did not require the addition of poly(ethylene glycol), although the latter did stimulate the rate of Ca2+ release. The ability of GTP to alter microsomal permeability was blocked by prior treatment with the thiol reagent p-hydroxymercuribenzoate; complete inhibition was observed after a 10 min exposure to 50 microM. Inhibition was reversed by subsequent treatment with dithiothreitol. The marked similarities between the two GTP-sensitive processes indicate that they may function via the same mechanism.     

Regulation of expression of glutamine synthetase in a symbiotic Nostoc strain associated with Anthoceros punctatus.

A characteristic of N2-fixing cyanobacteria in symbiotic associations appears to be release of N2-derived NH4+. The specific activity of the primary ammonium-assimilating enzyme, glutamine synthetase (GS), was found to be three- to fourfold lower in Nostoc sp. strain 7801 grown in symbiotic association with the bryophyte Anthoceros punctatus than in free-living Nostoc sp. strain 7801. Quantitative immunological assays with antisera against GS purified from Nostoc sp. strain 7801 and from Escherichia coli indicated that similar amounts of the GS protein were present in symbiotic (50 micrograms mg-1) and free-living (68 micrograms mg-1) cultures. The conclusion from these experiments is that GS is regulated by a posttranslational mechanism in Anthoceros-associated Nostoc sp. strain 7801. However, the results of comparative catalytic and immunological experiments between N2- and NH4+-grown free-living Nostoc sp. strain 7801 implied control of GS synthesis. A correlation was not observed between the level of GS expression and the extent of symbiotic heterocyst differentiation in Nostoc sp. strain 7801 associated with A. punctatus.     

Purification and characterization of trichosanthin. Homology to the ricin A chain and implications as to mechanism of abortifacient activity

Trichosanthin, a protein from the Chinese medicinal herb Trichosanthes kirilowii, was purified in two essentially quantitative steps involving CM-Sephadex chromatography and reverse-phase high performance liquid chromatography. The protein was found to have a molecular mass of 25-26 kDa, to contain no cysteine, and to contain no glycosidic linkages. Pure trichosanthin was found to have potent abortifacient activity in pregnant mice. In order to understand the molecular basis of this unique biological activity, we have examined the amino acid sequence of the protein. As purified, trichosanthin was found to contain two amino-terminal sequences which differed only in the absence or presence of a tyrosine at residue 1. Sequence analysis of trichosanthin has allowed for determination of the NH2-terminal 38-amino acid residues. Comparison of this sequence to those present in a data base revealed homology with the ricin A-chain. Consistent with this structural homology, we have found that trichosanthin is a potent inhibitor of protein synthesis in a reticulocyte lysate system.     

A Phorbol Ester-Sensitive Kinase Catalyzes the Phosphorylation of P0 Glycoprotein in Myelin

The proposed structural protein of peripheral nerve myelin, P0, has been shown to have several covalent modifications. In addition to being glycosylated, sulfated, and acylated, P0 is phosphorylated, with the intracellular site of this latter addition being in question. By employing nerve injury models that exhibit different levels of P0 biosynthesis in the absence and presence of myelin assembly, we have examined the cellular location of P0 phosphorylation. It is demonstrated that there is comparable P0 phosphorylation in both normal and crush-injured adult rat sciatic nerves, although the level of biosynthesis of P0 differs between these myelin maintaining and actively myelinating nerve models, respectively. The glycoprotein does not appear to be phosphorylated readily in the transected adult sciatic nerve, a preparation in which P0 biosynthesis is observed but that lacks myelin membrane. These observations suggest that the modification is not associated with the biosynthesis or maturation of P0 in the endoplasmic reticulum or Golgi, but that it instead occurs after myelin assembly. That P0 phosphorylation occurs in the normal nerve even when translation is inhibited by cycloheximide treatment lends further support to this conclusion. P0 is shown to be phosphorylated on one or more serine residues, with all or most of the phosphate group(s) being labile as evidenced by pulse-chase analysis. Addition of a biologically active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate or 4 beta-phorbol 12,13-dibutyrate, substantially increases the extent of [32P]orthophosphate incorporation into the glycoprotein of normal and crushed nerve but not transected nerve. Biologically inactive 4 alpha-phorbol 12,13-didecanoate has no effect on P0 phosphorylation. Similarly, the addition of the cyclic AMP analog 8-bromo-cyclic AMP causes no appreciable changes in P0 labeling. These findings indicate that the phorbol ester-sensitive enzyme, protein kinase C, may be responsible for the phosphorylation of P0 within the myelin membrane.     

Taste systems of the petrosal ganglion of the rat glossopharyngeal nerve

Single unit recordings were taken from sensory ganglion cellsin the petrosal ganglion (PG) of the glossopharyngeal nerveof the rat. These taste units were examined with respect tospontaneous and evoked discharge patterns and responsivenessto a wide variety of chemical compounds, most of natural occurrence.Spontaneous activity patterns, with few exceptions, tended tobe extremely irregular with both bursting (clusters of 2–3spikes) and grouping (large groups of spikes as in evoked discharges).Most interspike interval histograms of spontaneous activitywere multimodal, similar to rat geniculate ganglion (GG) units.Evoked discharges usually displayed grouping of spikes, andlong latencies of onset and persistence of discharge after rinsewere sometimes seen. Little response was shown to nucleotidesor salts. Units responsive to amino acids tended to show largedischarge to only one or two amino acids; and the most responsiveamino acid usually varied from cell to cell. Units responsiveto alkaloids only responded to a few alkaloids with atropineand quinine being the most stimulatory. Units responsive toacids only discharged to a few of the acids tested and oftenacids of low pH elicited no discharge. Saccharin activated unitsresponsive to both sugar and alkaloids. A few units highly responsiveto both sugar and alkaloids were seen. The units were placedinto four clusters on the basis of chemicals activating themand certain neurophysiological characteristics: PG salt units,PG acid units and, tentatively, amino acid (sugar) units andX (alkaloid and alkaloid plus) units. The PG salt units didnot show the exclusive sensitivity to sodium and lithium compoundsas did the GG salt units. The PG acid units could also be differentiatedfrom the GG acid units. The petrosal amino acid and X units,on the other hand, could not be differentiated from similarunits in the rat GG.     

Circumferential analysis of digitalized gamma angiocardiography by assessment of regional left ventricular contraction

After acquisition of a digital equilibrium gamma-angiocardiographie, circumferential analysis of end-diastolic and end-systolic frames gives 120 points diastolic and systolic curves. Their difference represents systolic volume and leads to regional left ventricular ejection fraction assessment at the considered radius level. The circumferential analysis evolute gives the regional left ventricular ejection fraction representative curves which allows especially differential diagnosis between left ventricular akinesia and dyskinesia.     

bcd: A mutation affecting the width of organelle domains in the cortex of Tetrahymena thermophila

Summary A single-gene recessive mutation, bcd (broadened cortical domains), of Tetrahymena thermophila is characterized by a variable broadening of the spatial domains within which cortical organelles, including both the contractile vacuole pores (CVP) and oral apparatus (OA), are formed. The phenotype is not temperature-sensitive. During the development of the organelles of the mutant prior to cell division, extra CVPs and extra oral primordia (OP) appear near ciliary rows adjacent to the rows at which these structures normally form. In the later stages of development, some, but not all, of these extra structures are resorbed, or in the case of the oral domain, multiple adjacent OPs may be completely or partially integrated into a single enlarged OA. When multiple OAs persist, one or more of these may display a reversed orientation reminiscent of those encountered in janus mutants. However, unlike janus, bcd cells do not express any sign of a mirror-image global organization.Our results can best be accounted for by postulating that the bcd mutation affects some common determinant of the widths of both CVP and OA domains. Studies are in progress which explore the relationship between this width-determining mechanism(s) and the mechanism(s) determining the location of cortical organelles around the cell circumference.