Removal of carbohydrate from influenza A virus and its hemagglutinin and the effect on biological activities.

Treatment of influenza virus and its purified hemagglutining with glycosidases from Diplococcus pneumoniae, which included beta-galactosidase, beta-N-acetylglucosminidase, and endoglycosidase D, released amino and neutral sugars from the virus and these as well as large oligosaccharides from the purified hemagglutinin. The released glucosamine-containing oligosaccharides were of one discrete size. Large oligosaccharides not removed by the glycosidases were found on the virus as well as the hemagglutinin. Some oligosaccharides on the virus were inaccessible to the enzymes, since they could be removed only from the purified hemagglutinin. Approximately 50% of the hemagglutinin carbohydrates could be removed without effect on hemagglutinating activity. Similarly, removal of 20 to 25% of the carbohydrates from intact virus particles did not alter infectivity.     

Bound Form Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of H₂O-soluble and NaOH-hydrolyzable bound forms of indole-3-acetic acid (IAA) in petiole slices of Nicotiana glauca, Nicotiana langsdorffii, and their tumorous and nontumorous hybrids in the presence of exogenous ¹⁴C-IAA was investigated. The synthesis of conjugates progressively increased during 6 hours of incubation in ¹⁴C-IAA. The results showed that the rate of synthesis of IAA conjugates was higher in tumorous hybrids supplied exogenous IAA than in the parental species similarly supplied, and the rate of synthesis was higher in amphidiploid tumor plants than in a nontumorous mutant. It was also found that after 10 to 12 hours of incubation, 45% of the IAA taken up by F1 hybrids was in conjugated form whereas only 10 to 25% of the IAA taken up by a nontumorous mutant, N. langsdorffii, or N. glauca was conjugated. An F1 hybrid and an amphidiploid hybrid were found equally efficient in conjugating exogenously supplied IAA. It is postulated on the basis of these and other findings that IAA conjugates play an important role in tumorigenesis in Nicotiana.     

Metronidazole in prevention and treatment of bacteroides infections in elective colonic surgery.

A double-blind randomised trial was carried out among 46 patients undergoing elective colonic surgery; 27 patients received prophylactic metronidazole and 19 received placebo. Anaerobic infections did not develop in any of the metronidazole-treated patients, but did develop in 11 (58%) of 19 controls who were subsequently successfully treated with metronidazole.     

A human 15-kDa IFN-induced protein induces the secretion of IFN-gamma.

A 15,000 molecular weight protein (15-kDa), induced and secreted by human PBMC after treatment with IFN-alpha or -beta, was assessed for its ability to modulate cellular function. Although it had no effect on growth or 2'5'-A synthetase activity in Daudi, U-937, or HL-60 cells, when incubated with fresh human PBMC, LPS-induced monocyte cytotoxicity against WEHI-164 target cells was augmented. This stimulation was inhibited by both an antibody against TNF-alpha and a rabbit polyclonal antiserum to the 15-kDa protein. Furthermore, when the 15-kDa protein was added to PBMC an increase in GTP cyclohydrolase I activity, as assessed by neopterin secretion, resulted. Neopterin secretion by PBMC in response to the 15-kDa was increased in a dose-responsive manner up to more than sixfold over baseline, with a 15-kDa concentration of less than 10 ng/ml effective. The 15-kDa protein also stimulated indoleamine 2,3-dioxygenase (IDO) activity in fresh, human PBMC. Induction of neopterin secretion and IDO activity was inhibited by a polyclonal antiserum to 15-kDa. LPS-induced cytotoxic activity was not augmented by 15-kDa pretreatment of purified monocytes, indicating the need for the presence of a second cell population and the indirect action of the 15-kDa on the induction of monocyte activities. When PBMC or purified CD3+ cells, but not purified CD14+ cells, were incubated with the 15-kDa protein, secretion of a factor was induced that resulted in the induction of IDO activity in PMA-differentiated THP-1 cells. An antibody to IFN-gamma, but not IFN-alpha, inhibited the induction of IDO activity by this secreted factor. In addition, antiserum to the 15-kDa blocked the secretion of IFN-gamma from the CD3+ cells. Thus, a 15-kDa product of IFN-alpha- and IFN-beta-treated monocytes and lymphocytes can stimulate secretion of IFN-gamma from CD3+ cells.     

Responsiveness of a human parotid epithelial cell line (HSY) to autonomic stimulation: Muscarinic control of K+ transport

Summary Salivary electrolyte secretion is under the control of the autonomic nervous system. In this paper we report that HSY, an epithelial cell line derived from the acinar-intercalated duct region of the human parotid gland, responds to muscarinic-cholinergic (generation of Ca²⁺ signal) andβ-adrenergic (generation of cAMP signal), but not toα-adrenergic (lack of Ca²⁺ signal), receptor stimulation. The muscarinic response was studied in detail. Carbachol (10⁻⁴ M, muscarinic agonist) or A23187 (5 μM, calcium ionophore) stimulation of HSY cells increases both⁸⁶Rb (K⁺) influx and efflux, resulting in no change in net equilibrium⁸⁶Rb content. Atropine (10⁻⁵ M, muscarinic antagonist) blocks both the carbachol-generated Ca²⁺ signal and carbachol-stimulated⁸⁶Rb fluxes, but has no effect on either the A23187-generated Ca²⁺ signal or A23187-stimulated⁸⁶Rb fluxes. Carbachol- and A23187-stimulated⁸⁶Rb fluxes are substantially inhibited by two K⁺ channel blockers, quinine (0.3 mM) and scorpion venom containing charybdotoxin (33 μg/ml). The inhibition of these stimulated fluxes by another K⁺ channel blocker, tetraethylammonium chloride (5 mM), is less pronounced. Protein kinase C (PKC) seems to be involved in the regulation of the⁸⁶Rb fluxes as 10⁻⁷ M PMA (phorbol ester, phorbol-12-myristate-13-acetate) substantially inhibits the muscarinic-stimulated⁸⁶Rb efflux and influx. Because this concentration of PMA totally inhibits the carbachol-generated Ca²⁺ signal and only 80% of the muscarinic-stimulated⁸⁶Rb influx, it seems that a portion of the carbachol-stimulated⁸⁶Rb flux (i.e. that portion not inhibited by PMA) might occur independently of the Ca²⁺ signal. PMA fails to inhibit the A23187-stimulated⁸⁶Rb fluxes, however, suggesting that PKC regulates Ca²⁺-sensitive K⁺ channel activity by regulating the Ca²⁺ signal, and not steps distal to this event. 4-α-Phorbol-12,13-didecanoate, a phorbol ester which fails to activate PKC, fails to inhibit either the carbachol-stimulated increase in intracellular free Ca²⁺, or carbachol-stimulated⁸⁶Rb fluxes.     

Evidence that most of the radioimmunoassayable inhibin secreted by the corpus luteum of the common marmoset monkey is of a non-dimeric form.

Although recent data for several species of primate, including human and marmoset, indicate that the corpus luteum secretes high levels of radioimmunoassayable inhibin, the nature of the immunoreactive (ir) inhibin detected has not been established. In this study, plasma ir-inhibin levels during the ovarian cycle of the marmoset (n = 12 animals) were measured by alpha-subunit-directed inhibin RIA, and values were compared with those estimated by a recently developed two-site immunoradiometric assay (IRMA) specific for inhibin alpha-beta dimer. Consistent with earlier data, plasma levels of ir-inhibin measured by RIA (overall mean value 133 +/- 7 ng/ml; n = 171) reached values 4-fold higher (p less than 0.001) during the luteal phase (222 +/- 20 ng/ml) than during the follicular phase (58 +/- 8 ng/ml), being directly correlated with plasma progesterone levels (r = 0.65; p less than 0.001). In contrast, plasma ir-inhibin levels estimated by IRMA were substantially lower than those measured by RIA (overall mean value 9.62 +/- 1.08 ng/ml; n = 171) and did not vary significantly during the cycle. Administration of a luteolytic dose of cloprostenol during the late luteal phase/early pregnancy led to an abrupt fall in plasma concentrations of progesterone (95%) and alpha-inhibin measured by RIA (82%), whereas dimeric inhibin levels remained unchanged. Analysis of marmoset luteal extracts (n = 5) by RIA, IRMA, and inhibin bioassay yielded inhibin estimates of 102.6 +/- 21.0, 0.632 +/- 0.103, and less than 2.0 ng/mg, respectively, thus confirming that only a very small proportion of the inhibin produced was dimeric (i.e., bioactive) in nature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2′5′ ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (K_m = 12 micromolar) and coniferaldehyde (K_m = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.     

Manganese-stimulated phosphorylation of a rat pancreatic protein: identity with elongation factor 2

To investigate the effect of Mn2+ on pancreatic protein phosphorylation, we incubated rat pancreatic cytosol in Tris buffer (pH 7.5) with [gamma-32P]ATP. Analysis using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography revealed a single protein (p98), with an Mr of 98,000 and a pI of 6.4-6.5, which was phosphorylated in a dose-dependent manner by Mn2+. A threshold effect was observed at 35 microM, and maximal effect at 1.1 mM Mn2+. Ca2+ and calmodulin (CaM) did not cause p98 phosphorylation, but Mg2+ (10 mM) caused faint non-specific phosphorylation of p98. Ca2+ (0.03-3 mM) and CaM (1-10 micrograms/ml) significantly enhanced, whereas trifluoperazine (TFP) and Mg2+ inhibited Mn(2+)-stimulated p98 phosphorylation. Under the above incubation conditions, Mn(2+)-stimulated protein phosphorylation of p98 was also observed in isolated pancreatic acini, but not in cytosols from liver or kidney. Partial purification of p98 and amino acid sequencing of the protein band corresponding to p98 indicated complete sequence homology with rat elongation factor 2 (EF-2). Furthermore, the combination of Ca2+, Mg2+ and CaM, which is known to induce the phosphorylation of EF-2, mimicked the actions of Mn2+. Inasmuch as EF-2 is the major substrate for CaM-dependent protein kinase III (CaM-PK III), these studies suggest that in the pancreatic acinar cell Mn2+/CaM protein kinase activity is mediated via CaM-PK III and the Mn2+ participates in the regulation of this enzyme in the pancreas.     

Ventilation-perfusion relationships in the lung during head-out water immersion.

Water immersion can cause airways closure during tidal breathing, and his may result in areas of low ventilation-perfusion (VA/Q) ratios (VA/Q less than or equal to 0.1) and/or shunt and, ultimately, hypoxemia. We studied this in 12 normal males: 6 young (Y; aged 20-29 yr) with closing volume (CV) less than expiratory reserve volume (ERV), and six older (O; aged 40-54 yr) with CV greater than ERV during seated head-out immersion. Arterial and expired inert gas concentrations and dye-dilution cardiac output (Q) were measured before and at 2, 5, 10, 15, and 20 min in 35 degrees C water. During immersion, Y showed increases in expired minute ventilation (VE; 8.3-10.3 l/min), Q (6.1-8.2 l/min), and arterial PO2 (PaO2; 91-98 Torr; P less than or equal to 0.05). However, O2 uptake (VO2), shunt, amount of low-VA/Q areas (% of Q), and the log standard deviation of the perfusion distribution (log SDQ) were unchanged. During immersion, O showed increases in shunt (0.6-1.8% of Q), VE (8.5-11.4 l/min), and VO2 (0.31-0.40 l/min) but showed no change in low-VA/Q areas, log SDQ, Q, or PaO2. Throughout, O showed more VA/Q inequality (greater log SDQ) than Y (O, 0.69 vs. Y, 0.47).(ABSTRACT TRUNCATED AT 250 WORDS)