Specificity of the collagenolytic enzyme from the fungus Entomophthora coronata: comparison with the bacterial collagenase from Achromobacter iophagus.

The specificity of the collagenolytic enzyme from the fungus Entomophthora coronata toward some inhibitors and the B chain of oxidized insulin was investigated and compared to that of the bacterial collagenase from Achromobacter iophagus. The fungal enzyme was completely inhibited by diisopropylfluorophosphate, tosyl-l-lysine chloromethyl ketone, and tosyl-amino-2-phenylethyl chloromethyl ketone but not at all by ethylenediaminetetraacetate. This indicates that it is not a metalloenzyme like the bacterial Achromobacter collagenase. The B chain of insulin was not hydrolysed at all by the bacterial enzyme under conditions where extensive digestion was observed with the Entomophthora enzyme. The fungal enzyme cleaves preferentially the bonds $\text{Leu}\text{15}\text{-}\text{Tyr}\text{16}\text{and}\text{Leu}\text{11}\text{Val}\text{12}$ as determined by automatic sequencing; the secondary cleavages were identified by a systematic analysis of the digestion mixture; thus, the fungal collagenolytic enzyme from Entomophthora coronata differs both structurally and functionally from the bacterial Achromobacter collagenase.     

Structure of isolated sensilla and sensory neurons in vitro: Observations on developing silkmoth antennae

Summary By combined enzymatic and mechanical treatment, it was possible to dissociate the sensory epithelium of developing antennae of male Antheraea polyphemus and A. pernyi silkmoths from the stage of separation of the antennal branches up to the early stages of cuticle deposition. Large numbers of entire developing trichoid sensilla were isolated. These are characterized by a large trichogen cell with a long apical, hair-forming process and a large nucleus. A cluster of 2–3 sensory neurons, enclosed by the thecogen cell, is situated in the basal region. The dendrites run past the nucleus of the trichogen cell into the apical process from which they protrude laterally. The nuclei of the tormogen and a 4^th enveloping cell can be distinguished near the base of the prospective hair. After further dissociation, only the neuron clusters remain, still enclosed by their thecogen cell and often attached to the antennal branch nerve via their axons. It is finally possible to disrupt the thecogen cells and the axons, leaving the sensory neurons with inner dendritic segments and axon stumps. The majority of these neurons can be expected to be olfactory.     

Both wound-inducible and tuber-specific expression are mediated by the promoter of a single member of the potato proteinase inhibitor II gene family

A chimeric gene consisting of 1.3 kb of the 5' regulatory region of a member of the potato proteinase inhibitor II gene family, the coding region of the bacterial β-glucuronidase (GUS) gene and 260 bp of the proteinase inhibitor II 3'-untranslated region containing the poly(A) addition site was introduced into potato and tobacco by Agrobacterium tumefaciens mediated transformation. Analysis of transgenic plants demonstrates systemic, wound-inducible expression of this gene in stem and leaves of potato and tobacco. Constitutive expression was found in stolons and tubers of non-wounded potato plants. Histochemical experiments based on the enzymatic activity of the GUS protein indicate an association of the proteinase inhibitor II promoter activity with vascular tissue in wounded as well as in systemically induced non-wounded leaves, petioles, potato stems and in developing tubers. These data prove that one single member of the proteinase inhibitor II gene family contains cis-active elements, which are able to respond to both developmental and environmental signals. Furthermore they support the hypothesis of an inducing signal (previously called proteinase inhibitor inducing factor), which is released at the wound site and subsequently transported to non-wounded parts of the plant via the vascular system from where it is released to the surrounding tissue.     

Morphogenesis of the antenna of the male silkmoth. Antheraea polyphemus, III. Development of olfactory sensilla and the properties of hair-forming cells

During adult development of the male silkmoth Antheraea polyphemus, the anlagen of olfactory sensilla arise within the first 2 days post-apolysis in the antennal epidermis (stage 1-3). Approximately on the second day, the primary dendrites as well as the axons grow out from the sensory neurons (stage 4). The trichogen cells start to grow apical processes approximately on the third day, and these hair-forming 'sprouts' reach their definite length around the ninth day (stages 5-6). Then the secretion of cuticle begins, the cuticulin layer having formed on day 10 (stage 7a). The primary dendrites are shed, the inner dendritic segments as well as the thecogen cells retract from the prospective hair bases, and the inner tormogen cells degenerate around days 10/11 (stage 7b). The hair shafts of the basiconic sensilla are completed around days 12/13 (stage 7c), and those of the trichoid sensilla around days 14/15 (stage 7d). The trichogen sprouts retract from the hairs after having finished cuticle formation, and the outer dendritic segments grow out into the hairs: in the basiconic sensilla directly through, and in the trichoid sensilla alongside, the sprouts. The trichogen sprouts contain numerous parallel-running microtubules. Besides their cytoskeletal function, these are most probably involved in the transport of membrane vesicles. During the phase of cuticle deposition, large numbers of vesicles are transported anterogradely from the cell bodies into the sprouts, where they fuse with the apical cell membrane and release their electron-dense contents (most probably cuticle precursors) to the outside. As the cuticle grows in thickness, the surface area of the sprouts is reduced by endocytosis of coated vesicles. When finally the sprouts retract from the completed hairs, the number of endocytotic vesicles is further increased and numerous membrane cisterns seem to be transported retrogradely along the microtubules to the cell bodies. Here the membrane material will most probably be used again in the formation of the sensillum lymph cavities. Thus, the trichogen cells are characterized by an intensive membrane recycling. The sensillum lymph cavities develop between days 16-20 (stage 8), mainly via apical invaginations of the trichogen cells. The imago emerges on day 21.     

Effect of antigravity suit inflation on cardiovascular, PRA, and PVP responses in humans

Blood pressure, pulse rate (PR), serum osmolality and electrolytes, as well as plasma vasopressin (PVP) and plasma renin activity (PRA), were measured in five men and two women [mean age 38.6 +/- 3.9 (SE) yr] before, during, and after inflation of an antigravity suit that covered the legs and abdomen. After 24 h of fluid deprivation the subjects stood quietly for 3 h: the 1st h without inflation, the 2nd with inflation to 60 Torr, and the 3rd without inflation. A similar control noninflation experiment was conducted 10 mo after the inflation experiment using five of the seven subjects except that the suit was not inflated during the 3-h period. Mean arterial pressure increased by 14 +/- 4 (SE) Torr (P less than 0.05) with inflation and decreased by 15 +/- 5 Torr (P less than 0.05) after deflation. Pulse pressure (PP) increased by 7 +/- 2 Torr (P less than 0.05) with inflation and PR decreased by 11 +/- 5 beats/min (P less than 0.05); PP and PR returned to preinflation levels after deflation. Plasma volume decreased by 6.1 +/- 1.5% and 5.3 +/- 1.6% (P less than 0.05) during hours 1 and 3, respectively, and returned to base line during inflation. Inflation decreased PVP from 6.8 +/- 1.1 to 5.6 +/- 1.4 pg/ml (P less than 0.05) and abolished the significant rise in PRA during hour 1. Both PVP and PRA increased significantly after deflation: delta = 18.0 +/- 5.1 pg/ml and 4.34 +/- 1.71 ng angiotensin I X ml-1 X h-1, respectively. Serum osmolality and Na+ and K+ concentrations were unchanged during the 3 h of standing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the major immediate-early polypeptides encoded by murine cytomegalovirus.

The immediate-early (IE) infected cell proteins induced by the murine cytomegalovirus (Smith strain) were studied. These polypeptides were identified as IE proteins by their synthesis in the presence of actinomycin D after removal from a protein synthesis block mediated by cycloheximide. By using a murine antiserum against murine cytomegalovirus, three abundant polypeptides of 89, 84, and 76 kilodaltons (kd) were immunoprecipitated. The three major proteins are phosphorylated but not glycosylated and share antigenic determinants recognized by monoclonal antibodies. The 84 and 76-kd polypeptides represent post-translational modification products of the 89-kd protein. Accordingly, in vitro translation of IE infected cell RNA revealed only the 89-kd polypeptide. The viral origin of the RNA species directing the synthesis of the major 89-kd IE polypeptide was verified by hybrid selection of IE RNA with DNA fragments representing the region from 0.769 to 0.815 map units of the murine cytomegalovirus genome. IE polypeptides were found to be located in the nuclei and the cytoplasm of infected cells. Studies on the kinetics of IE polypeptide synthesis revealed negative regulatory effects on IE gene expression correlated with the synthesis of early proteins.     

Carbohydrates of influenza virus. Structural elucidation of the individual glycans of the FPV hemagglutinin by two-dimensional 1H n.m.r. and methylation analysis.

The structures of the oligosaccharides of the hemagglutinin of fowl plague virus [influenza A/FPV/Rostock/34 (H7N1)] have been elucidated by one- and two-dimensional 1H n.m.r. spectroscopy at 500 MHz and by microscale methylation analysis. N-Glycosidic oligosaccharides of the oligomannosidic (OM) and of the N-acetyllactosaminic type have been found, the latter type comprising biantennary structures, without (A) or with (E) bisecting N-acetylglucosamine, and triantennary (C) structures. Analysis of the tryptic and thermolytic glycopeptides of the hemagglutinin allowed the allocation of these oligosaccharides to the individual glycosylation sites. Each attachment site contained a unique set of oligosaccharides. Asn12 contains predominantly structures C and E which are highly fucosylated. Asn28 contains OM and A structures that lack fucose and sulfate. Asn123 shows A that has incomplete antennae but is highly fucosylated and sulfated. Asn149 has fucosylated A and E. Asn231 shows fucosylated A and E with incomplete antennae. Asn406 has OM oligosaccharides. Asn478 has A and E with little fucose. Localization of the oligosaccharides on the three-dimensional structure of the hemagglutinin revealed that the oligomannosidic glycans are attached to glycosylation sites at which the enzymes responsible for carbohydrate processing do not have proper access. These observations demonstrate that an important structural determinant for the oligosaccharide side chains is the structure of the glycoprotein itself. In addition, evidence was obtained that the rate of glycoprotein synthesis also has an influence on carbohydrate structure.