Bombyx acid cysteine protease (BCP): hormonal regulation of biosynthesis and accumulation in the ovary

We previously reported purification of the cysteine protease from Bombyx eggs (BCP) and the occurrence of the enzyme in various tissues of this insect. In the present paper, we present a detailed analysis of stage-specific changes in activity of BCP between the fourth larval instar and pupal-adult development. A synthetic fluorescent peptide, carbobenzoxy-L-Phenylalanyl-L-Arginine4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA), was used to assay proteolytic activity. When tissue extracts were treated with anti-BCP serum before assay of enzyme activity, most activity towards Z-Phe-Arg-MCA was removed from the extracts. Therefore proteolytic activity in the present experiments is due mainly to BCP. We used Western blot and Northern blot analyses to determine tissue and stage specific expression of the enzyme. In the 5th larval fat body and hemolymph, BCP activity dramatically increased at the time of spinning, returning to the basal level before ecdysis. Northern blot analysis showed that a 1.5 kilobase mRNA which hybridizes to BCPcDNA suddenly appears during this period. Similar results were obtained in 4th instar fat body. In pupal hemolymph and fat body, low basal activity of BCP was detected early (day 0 to day 3 after pupal ecdysis), followed by a pronounced increase to a maximum six days after ecdysis, before returning to the basal level. In ovariectomized female pupae, a significant amount of proteolytic activity accumulated in hemolymph, suggesting that the enzyme is synthesized in the fat body and transferred into the ovary along with vitellogenin. BCP activity increased three days after injection of 20-hydroxyecdysone into ligated pupae. Furthermore, putative BCPmRNA appeared in the fat body within 24 hours after injection. This increase was completely blocked by the administration of cycloheximide. The results suggest that, BCP is synthesized in extraovarian tissues such as fat body and ovarian follicle cells and accumulates in the ovary, thus representing a new class of yolk protein.     

Dimorphic expression of cerebrosides in the mycopathogen Sporothrix schenckii

Major neutral glycosphingolipid components were extracted from Sporothrix schenckii, a dimorphic fungus exhibiting a hyphal saprophytic phase and a yeast parasitic phase responsible for chronic mycotic infections in mammalian hosts. These components, one from the mycelial form and two from the yeast form, were purified and their structures were elucidated by (1)H nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and tandem ESI-MS/MS. All three were characterized as cerebrosides (monohexosylceramides) containing (4E, 8E)-9-methyl-4,8-sphingadienine as the long-chain base attached to N-2'-hydroxyoctadecanoate and N-2'-hydroxy-(E)-Delta(3)-octadecenoate as the fatty acyl components. However, while the mycelial form expressed only beta-glucopyranosylceramide, the yeast form expressed both beta-gluco- and beta-galactopyranosylceramides in approximately equal amounts. In addition, while the glucosylceramides of both mycelial and yeast forms had similar proportions of saturated and (E)-Delta(3) unsaturated 2-hydroxy fatty acid, the galactocerebroside of the yeast form had significantly higher levels of (E)-Delta(3) unsaturation.The differences in cerebroside hexose structure represent a novel type of glycosphingolipid dimorphism not previously reported in fungi. Possible implications of these findings with respect to regulation of morphological transitions in S. schenckii and other dimorphic fungi are discussed.     

Structural identification of phosphatidylcholines having an oxidatively shortened linoleate residue generated through its oxygenation with soybean or rabbit reticulocyte lipoxygenase

Phosphatidylcholines (PCs) with platelet-activating factor (PAF)-like biological activities are known to be generated by fragmentation of the sn-2-esterified polyunsaturated fatty acyl group. The reaction is free radical-mediated and triggered by oxidants such as metal ions, oxyhemoglobin, and organic hydroperoxides. In this study, we characterized the PAF-like phospholipids produced on reaction of PC having a linoleate group with lipoxygenase enzymes at low oxygen concentrations. When the oxidized PCs were analyzed by gas chromatography-mass spectrometry, two types of oxidatively fragmented PC were detected. One PC had an sn-2-short chain saturated or unsaturated acyl group (C(8)-C(13)) with an aldehydic terminal; the abundant species were PCs with C(9) and C(13). The other PC had a short chain saturated acyl group (C(6)-C(9)) with a methyl terminal, and the most predominant species was PC with C(8). When the extracts of oxidation products were subjected to catalytic hydrogenation, PCs having saturated acyl groups (C(6)-C(14)) were detected; the most abundant was C(12) species. The less regiospecific formation of PAF-like lipids suggests that they were generated by oxidative fragmentation of PC hydroperoxides formed by non-stereoselective oxygenation of the alkyl radical of esterified linoleate that escaped from the active centers of lipoxygenases. One of the PAF-like PC with an aldehydic terminal was found to be bioactive; it inhibited the production of nitric oxide induced by lipopolysaccharide and interferon-gamma in vascular smooth muscle cells from rat aorta.     

Crystal structure of tobacco necrosis virus at 2.25 A resolution

The crystal structure of tobacco necrosis virus (TNV) has been determined by real-space averaging with 5-fold non-crystallographic symmetry, and refined to R=25.3 % for diffraction data to 2.25 A resolution. A total of 180 subunits form a T=3 virus shell with a diameter of about 280 A and a small protrusion at the 5-fold axis. In 276 amino acid residues, the respective amino terminal 86, 87 and 56 residues of the A, B and C subunits are disordered. No density for the RNA was found. The subunits have a "jelly roll" beta-barrel structure, as have the structures of the subunits of other spherical viruses. The tertiary and quaternary structures of TNV are, in particular, similar to those of southern bean mosaic virus, although they are classified in different groups. Invisible residues 1 to 56 with a high level of basic residues are considered to be located inside the particle. Sequence comparison of the coat proteins of several TNV strains showed that the sequences of the disordered segment diverge considerably as compared with those of the ordered segment, consistent with a small tertiary structural constraint being imposed on the N-terminal segment. Basic residues are localized on the subunit interfaces or inner surface of the capsid. Positive charges of the basic residues facing the interior, as well as those of the N-terminal segment, may neutralize the negative charge of the RNA inside. Five calcium ions per icosahedral asymmetric unit are located at the subunit interfaces; three are close to the exterior surface, the other two away from it. The environments of the first three are similar, and those of the other two sites are similar. These calcium ions are assumed to be responsible for the stabilization/transition of the quaternary structure of the shell. Three peptide segments ordered only in the C subunits are clustered around each 3-fold (quasi-6-fold) axis forming a beta-annulus, and may lead to quasi-equivalent interactions for the organization of the T=3 shell.     

Fast folding of Escherichia coli cyclophilin A: a hypothesis of a unique hydrophobic core with a phenylalanine cluster

Escherichia coli cyclophilin A, a 164 residue globular protein, shows fast and slow phases of refolding kinetics from the urea-induced unfolded state at pH 7.0. Given that the slow phases are independent of the denaturant concentration and may be rate-limited by cis/trans isomerizations of prolyl peptide bonds, the fast phase represents the true folding reaction. The extrapolation of the fast-phase rate constant to 0 M urea indicates that the folding reaction of cyclophilin A is extraordinarily fast and has about 700 s(-1) of the rate constant. Interrupted refolding experiments showed that the protein molecules formed in the fast phase had already been fully folded to the native state. This finding overthrows the accepted view that the fast folding is observed only in small proteins of fewer than 100 amino acid residues. Examination of the X-ray structure of cyclophilin A has shown that this protein has only one unique hydrophobic core (phenylalanine cluster) formed by evolutionarily conserved phenylalanine residues, and suggests that this architecture of the molecule may be responsible for the fast folding behavior.     

Cerebral primitive neuroectodermal tumor in an adult male. A case report

BACKGROUND: Primitive neuroectodermal tumors (PNETs) are very rare. Malignant tumors of the cerebrum in young individuals are composed predominantly of undifferentiated cells, with moderate differentiation along either neuronal or glial lines. To our knowledge, cerebral PNETs in adults are extraordinarily rare and have been reported in only 11 cases, with little cytologic documentation in the literature. The cytopathologic, immunohistochemical and ultrastructural features of cerebral PNET arising in an adult male are presented. CASE: A cystic tumor, on computed tomography and magnetic resonance imaging, arose from the left frontal lobe in a 39-year-old man and contained histopathologic features of PNET. Specimens obtained from surgery revealed the presence of an undifferentiated type of PNET with moderate neuronal and glial differentiation and mild characteristic findings of peripheral PNET. The cytologic and histologic specimens showed evidence of a scattered pattern of blastic and undifferentiated tumor cells and a neural arrangement with Homer-Wright-like rosettes. Immunohistochemically, the tumor cells were glial fibrillary acidic protein, neuron-specific enolase, synaptophysin and CD-99 positive and epithelial membrane antigen, S-100 protein and vimentin negative. Ultrastructurally, neither microtubular structures nor intermediate filaments, except neurosecretory granules, were found in the tumor cells. CONCLUSION: Both immunohistochemical and ultrastructural studies on cytologic and histologic slides were important for the diagnosis of PNET because of establishing not only undifferentiated tumor cells but also neural and glial differentiation.     

Fine needle aspiration cytology of adenocarcinoma of the rectovaginal septum. A case report

BACKGROUND: Adenocarcinoma arising in the rectovaginal septum is exceedingly rare and is difficult to diagnose by pathologic examination prior to surgery because of the anatomic position of the tumor. CASE: A 42-year-old woman presumed to have adenocarcinoma of the rectovaginal septum underwent fine needle aspiration for diagnosis. Although a previously performed biopsy from the posterior vaginal fornix was unsuccessful, fine needle aspiration cytology via the posterior vaginal wall detected adenocarcinoma cells. The cell clusters were composed of cells with enlarged and hyperchromatic nuclei. The nuclei themselves demonstrated round and/or irregular morphologic patterns, with high nuclear/cytoplasmic ratios, and often contained an enlarged, round nucleolus and sometimes multiple ones in a single nucleus. Aniso-nucleosis was severe, and the chromatin patterns ranged from coarse to finely granular. The cytoplasm was narrow and lightly stained. Following fine needle aspiration, the patient underwent posterior exenteration on the basis of the cytologic diagnosis. CONCLUSION: Fine needle aspiration cytology was useful in establishing the preoperative diagnosis of adenocarcinoma of the rectovaginal septum, and curative exenterative surgery could be then performed. To our knowledge, this is the first report of fine needle aspiration cytology of adenocarcinoma at this location.     

Search for UV-responsive genes in human cells by differential mRNA display: involvement of human ras-related GTP-binding protein, Rheb, in UV susceptibility

We have previously demonstrated that glucose deprivation alters the glycosylation of the GLUT1 glucose transporter in 3T3-L1 adipocytes. Many aberrantly glycosylated proteins are retained in the endoplasmic reticulum by interaction with chaperones. Herein, we use three independent procedures to show that GLUT1 is targeted to the plasma membrane, despite alterations in glycosylation. While earlier experiments revealed that plasma membrane targeting of aglyco GLUT 1 transporter was significantly reduced, our data show for the first time that altered glycosylation provides sufficient information to drive appropriate trafficking.