Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.     

Biochemical characteristics,metabolism, and antitumor activity of several acetylated hexosamines

We have synthesized several potential inhibitors and/or modifiers of the carbohydrate portion of plasma membrane glycoconjugates. These include fluorinated and actylated analogs of D-glucosamine, D-galactosamine, and D-mannosamine. These compounds have been tested to determine their effects on both [¹⁴C] glucosamine and [³H] leucine incorporation into glycoconjugate and on cell growth and viability using P-288 murine lymphoma cells maintained in tissue culture. The most cytotoxic agent tested was 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-β-D-glucopyranose or simply β-pentaacetylglucosamine which prevented cell growth at 10^?4–10^?3 M. β-Pentaacetylglucosamine cytotoxicity was correlated with its high lipid solubility, having an octanol/water partition coefficient of 0.424 as compared with 0.278 for the β-anomer and 0.017 for N-acetylglucosamine. In vitro metabolism studies with [¹⁴C]-and/or [³H]-labeled pentaacetylglucosamine have indicated intracellular de-O-acetylation leading to the biosynthesis of UDP-N-acetylglucosamine, followed by the incorporation of this sugar into cellular glycoprotein. Concomitant with the formation of increased amounts of this nucleotide sugar, intracellular UTP and CTP pools fell to one third normal within 3 h after the administration of 1 mM pentaacetylglucosamine. At present it is unclear whether the cytotoxicity of β-pentaacetylglucosamine or other similar agents is due to alterations in nucleotide and nucleotide-sugar pools causing a decrease in energy charge and polynucleotide biosynthesis or is due to a direct effect on membrane glycoconjugate biosynthesis.     

Purification and characterization of ribulose-5-phosphate kinase from spinach

An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44,000 Mr subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH2-terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation.     

Generation of transgenic plants expressing plasma membrane-bound antibodies to the environmental pollutant microcystin-LR

In this paper we describe the engineering and regeneration of transgenic tobacco plants expressing a recombinant plasma membrane-retained antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by cyanobacteria. The antibody was created by a genetic fusion of the antigen binding regions of the microcystin-specific single chain antibody, 3A8, with the constant regions from the murine IgG1κ, Guy’s 13, including a membrane retention sequence at the C-terminal end of the antibody heavy chain. The antibody produced in the leaves was shown to be functional by binding to MC-LR in an ELISA with antibody yields in transgenic plant leaves reaching a maximum of 1.2 μg g⁻¹ leaf f.wt (0.005% total soluble protein). Antibody-MC-LR complexes formed in leaves after addition of MC-LR to hydroponic medium around the roots of transgenic plant cultures.     

Functional insights from structural genomics

Structural genomics efforts have produced structural information, either directly or by modeling, for thousands of proteins over the past few years. While many of these proteins have known functions, a large percentage of them have not been characterized at the functional level. The structural information has provided valuable functional insights on some of these proteins, through careful structural analyses, serendipity, and structure-guided functional screening. Some of the success stories based on structures solved at the Northeast Structural Genomics Consortium (NESG) are reported here. These include a novel methyl salicylate esterase with important role in plant innate immunity, a novel RNA methyltransferase (H. influenzae yggJ (HI0303)), a novel spermidine/spermine N-acetyltransferase (B. subtilis PaiA), a novel methyltransferase or AdoMet binding protein (A. fulgidus AF_0241), an ATP:cob(I)alamin adenosyltransferase (B. subtilis YvqK), a novel carboxysome pore (E. coli EutN), a proline racemase homolog with a disrupted active site (B. melitensis BME11586), an FMN-dependent enzyme (S. pneumoniae SP_1951), and a 12-stranded β-barrel with a novel fold (V. parahaemolyticus VPA1032).     

Activity of some enzymes in Physarum polycephalum

Summary The activity levels of seven enzymes were studied in growing plasmodia of Physarum polycephalum. The enzymes were isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, acid phosphatase, phosphodiesterase, -glucosidase, and histidase. Six of the enzymes showed a continuous increase in activity during the mitotic cycle; glutamate dehydrogenase exhibited a stepwise increase about 5 h after mitosis. Cycloheximide immediately inhibited the activity of all enzymes. Actinomycin D was ineffective in inhibiting enzyme activity until after one mitotic cycle had been completed; this indicates that mRNA was stable for all of these enzymes during the G2 period. Attempts to induce enzyme activity were unsuccessful.

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Zusammenfassung Der Aktivitätsverlauf von sieben Enzymen wurde in wachsenden Plasmodien von Physarum polycephalum untersucht. Es handelte sich um die Enzyme: Isocitrat-Dehydrogenase, Glucose-6-Phosphat-Dehydrogenase, Glutamat-Dehydrogenase, saure Phosphatase, Phosphodiesterase, -Glucosidase und Histidase. Sechs dieser Enzyme wiesen einen kontinuierlichen Aktivitätsanstieg während des Mitosecyclus auf; Glutamat-Dehydrogenase zeigte einen stufenförmigen Anstieg etwa 5 Std nach der Kernteilung. Cycloheximid hemmte sofort die Aktivität der Enzyme, während Actinomycin D erst nach Ablauf eines halben Teilungscyclus inhibierend wirkte. Dies deutet auf eine relativ stabile mRNA hin. Versuche, die Aktivität zu induzieren, schlugen fehl.

     

Immunocytochemical and ultrastructural study of the frog (Rana pipiens) pars distalis with special reference to folliculo-stellate cell function during in vitro superfusion

Summary The colloidal gold immunocytochemical technique was used to determine the ultrastructural features of the glandular cells in the pituitaries of male frogs, Rana pipiens, both in vivo and after superfusion in vitro. Specific reactions to antisera against bullfrog gonadotropins, human prolactin, and synthetic 1–39 corticotropin allowed identification of the 3 corresponding types of glandular cells. No immunoreaction was obtained with antisera against human or ovine-growth hormone, human -thyrotropin hormone, and bovine S-100 protein. General morphological features of these immunocytochemically identified glandular cells were similar to those of equivalent cells previously described in other amphibian species. Non-glandular folliculo-stellate cells were distinctive. In freshly removed pituitaries, these folliculo-stellate cells contained lysosome-like structures, but did not show phagocytic vacuoles in the cytoplasm; they contained many mitochondria, and the Golgi complex and endoplasmic reticulum were relatively undeveloped. After 4 or 18 h of superfusion, some immunoreactive gonadotropic, prolactin, and corticotropic cells showed degeneration and destruction. In the same gland, folliculo-stellate cells retained a viable appearance, but showed phagocytic vacuoles containing secretory granule-like structures which were immunoreactive to gonadotropic, prolactin, and corticotropic antibodies. Some folliculo-stellate cells showed phagocytic vacuoles containing complete glandular cells. These results suggest that superfusion causes a destruction of some of the glandular cells, and that folliculo-stellate cells act as phagocytes when cellular debris or moribund cells are present in the intercellular space in the pituitary parenchyma.Supported by grant DCB 8710462 from the National Science Foundation, grant 2148-83 from the CAICYT (Spain) and the Junta de Andalucia (Spain)     

Expression and activity of the Fas antigen in bovine ovarian follicle cells

The Fas antigen is a cell surface receptor that triggers apoptosis when bound to Fas ligand (FasL). Studies were undertaken to determine whether the cow provides a suitable model to study the role of the Fas pathway in inducing apoptosis of ovarian cells during follicular atresia. Expression of Fas antigen mRNA and responsiveness to FasL-induced killing in vitro were measured. Effects of the cytokines tumor necrosis factor (TNF)-alpha and interferon-gamma (IFN) were studied because of previous demonstrations of their role in Fas-mediated apoptosis in other cell types. Fas antigen mRNA was detectable in cultured granulosa and theca cells, and expression was increased by treatment with IFN but not TNF. Granulosa and theca cells were resistant to FasL-induced killing unless pretreated with IFN. TNF had no effect on FasL-induced killing. Granulosa and theca cell cultures in which killing occurred in response to FasL stained positively for annexin V, an early marker for cells undergoing apoptosis. These results provide a basis for further studies using the bovine ovary to examine the role of the Fas antigen in follicular atresia.     

A Model System for the Study of the Release of Luteinizing Hormone-Releasing Hormone from Isolated Storage Granules

Abstract: We have developed an in vitro system for the study of the release of luteinizing hormone-releasing hormone (LH-RH) from its storage granules. In this system, homogenates of hypothalamic tissue are subjected to hypoosmotic shock, and the LH-RH-containing granules are isolated by means of differential centrifugation. The isolated granules are then incubated in a buffered medium, and the incubation is terminated by passing the incubation mixture through LH-RH affinity columns. The LH-RH associated with the granules passes freely through the columns, whereas the LH-RH released into the medium binds to the columns and is subsequently eluted with an acid solution. LH-RH is quantified by radioimmunoassay (RIA). We tested the effects of various concentrations of KCl on LH-RH release, which was found to be dependent on the concentration of KCl in the medium over the range 40–160 mM. We then studied the effects of pH on the release of LH-RH. Incubation of granules at pH 7.8 in the presence of 160 mM-KC1 resulted in the release from the granules of 14% of the stored LH-RH, whereas incubation at pH 6.2 resulted in the release of approximately 30% of the LH-RH. In addition, granules were incubated at pH 7.8 with MgATP and KCl. MgATP elicited a marked release of LH-RH that was approximately twice that seen in the absence of MgATP. In summary, in this in vitro system, granules containing LH-RH are stable under defined biochemical conditions, and LH-RH release from these granules is stimulated by ions and MgATP.     

大苞藤黄化学成分研究及其互变异构体超高效液相色谱质谱联用分析

从大苞藤黄枝叶的混合粉碎物中分离到11个化合物,运用光谱手段分别鉴定为neobractatin(1),brasixanthone B (2),5-O-methylxanthone V1 (3),10-O-methylmacluraxanthone (4),isobractatin (5),xanthone V1(6),xerophenone A (7),xerophenone B (8),bractatin (9),macluraxanthone (10)和3-O-methylneobractatin (11).本文首次应用超高效液相色谱-质谱联用技术分离了异构体7和8并测定了其精确分子量.其中化合物6～8为首次从该植物中发现.