Influence of the endometrium, protease inhibitors and freezing on antiviral activity of proteins secreted by pig conceptuses

In Exp. 1, only medium from cultures containing conceptus tissue had antiviral activity (P less than 0.05). Addition of Day-15 pregnant endometrium or Day-14 cyclic uterine flush proteins to cultures containing 200 mg conceptus tissue decreased antiviral activity (conceptus x endometrial protein interaction, P less than 0.06). Effects of endometrium (-54%) and uterine flush proteins (-40%) on antiviral activity of conceptus cultures did not differ from each other (P greater than 0.10). In Exp. 2, antiviral activity was only detected in cultures containing conceptus tissue (P less than 0.06). The amount of antiviral activity in cultures of Day-15 conceptus tissue was not influenced differently (P greater than 0.10) by culture in medium conditioned by endometrium from Day 10 or Day 12 of pregnancy. However, antiviral activity was undetectable in medium conditioned by endometrium from one of the Day-12 gilts. In Exp. 3, antiviral activity was present in medium from only 1 of 3 cultures from Day-12 gilts when assayed unfrozen. Antiviral activity was lower (P less than 0.01) in cultures of conceptuses from Day 12 than Day 14 of pregnancy; however, antiviral activity increased quadratically (P less than 0.05) when cultures contained 0, 0.01, 0.1 and 1.0 units/ml aprotinin, respectively. Freezing and thawing culture medium did not reduce (P greater than 0.10) antiviral activity compared to medium assayed unfrozen (1438 vs 1354 units/ml, respectively). These results suggest a regulatory influence of the endometrium on secretion of antiviral proteins by pig conceptuses in vitro.     

Mx proteins: GTPases with antiviral activity

Mx proteins are synthesized in interferon-treated vertebrate cells. They have attracted much attention because some of them can block the multiplication of influenza A virus and certain other negative-stranded RNA viruses. Recently, Mx proteins have been shown to be GTPases with significant homology to dynamins and yeast VPS1, enzymes involved in intracellular protein trafficking. Several biochemical properties of dynamin and VPS1 are similar to those of Mx, promoting new speculation about how Mx proteins might interfere with virus multiplication.     

Characterization of proteins produced in vitro by periattachment bovine conceptuses

Bovine conceptuses from Days 16 (n = 4), 19 (n = 6), 22 (n = 3), and 24 (n = 4), and chorion from Day 69 (estrus/mating = Day 0) were cultured for 24 h in modified minimum essential medium (MEM) in the presence of radioactive L-leucine [( 3H] leucine) to characterize de novo synthesis and release of proteins. Proteins released into MEM were identified by two-dimensional polyacrylamide gel electrophoresis, fluorography, and gel and ion exchange chromatography. Major polypeptides identified in MEM were different from those identified in conceptus and chorionic tissues. Both uptake of [3H] leucine and quality of polypeptides produced de novo and released into MEM were related to stage of conceptus development. Percent retention of [3H] leucine in MEM was lowest (P less than 0.01) in Day 16 cultures (1.2 +/- 4.1%), increased in Days 19 (16.8 +/- 3.7%) and 22 cultures (20.9 +/- 5.8%), and decreased (P less than 0.07) in Day 24 cultures (6.9 +/- 4.1%). Complexity of polypeptides increased after Day 16. Days 16, 19, 22 and 24 conceptus culture MEM was enriched in low-Mr, acidic polypeptides (Mr/isoelectric point ranges: 22K-26K/6.5-5.6, 20K-26K/5.5-5.4, and 16K-20K/5.0-4.5), which were not prominent products of Day 29 and 69 tissues. A high-Mr (Mr +/- SEM; 735K +/- 22K) glycoprotein was produced by all conceptus and chorionic tissues. The transient nature of production of low-Mr polypeptides suggests that they may be required during the periattachment period.     

Heat stress-induced alterations in the synthesis and secretion of proteins and prostaglandins by cultured bovine conceptuses and uterine endometrium

Effect of in vitro heat stress on protein and prostaglandin synthesis and secretion by bovine conceptuses and endometrium was examined. Conceptuses (n = 11) and endometrium (n = 10) obtained on Day 17 of pregnancy were cultured at thermoneutral (39 degrees C, 24 h) or heat stress (39 degrees C, 6 h; 43 degrees C, 18 h) temperatures in medium supplemented with L-[4,5-3H]leucine (100 microCi) and arachidonic acid (10 micrograms/ml). Radiolabeled protein secreted into culture medium increased with time in both groups. Heat stress reduced (p less than 0.001) incorporation of [3H]leucine into intracellular and secreted proteins by conceptuses but did not alter incorporation of [3H]leucine by endometrium. In particular, heat stress reduced by 72% the secretion of bovine trophoblast protein-1, the conceptus polypeptide believed to cause extension of luteal lifespan. Two-dimensional, sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that heat stress altered the array of proteins in endometrial and conceptus tissues, as evidenced by the induction of "heat-shock proteins." Endometrial secretion of prostaglandin F (p less than 0.001) and conceptus secretion of prostaglandin E2 (p less than 0.05) increased in response to heat stress. Sensitivity of bovine conceptuses and endometrium to heat stress in vitro suggests that infertility associated with maternal heat stress may be caused, partially by alterations in signals required for maintenance of the corpus luteum during early pregnancy.     

Characterization of the antiviral activity constitutively produced by murine conceptuses: absence of placental mRNAs for interferon alpha and beta

Antiviral activity has been found in conceptus and placental tissues in numerous species, including mice, pigs, sheep, cattle and humans. In sheep and cattle, the antiviral activity is due to an interferon alpha (IFN-alpha), but in other species the nature of the protein(s) responsible for placental activity is unknown. The objectives of this study were to determine if the constitutive antiviral activity associated with the mouse conceptus is produced as early as the peri-implantation period, and to determine if the activity is due to an IFN-alpha or -beta. Conceptus and placental tissue explants released antiviral activity from Day 4 through at least Day 16 of gestation as measured in an agar overlay bioassay employing CHO cells challenged with vesicular stomatitis virus. This activity was neutralized by antiserum against MuIFN-alpha/beta. The same antiserum failed, however, to immunoprecipitate radiolabeled proteins from medium collected from Day 4 blastocysts cultured in the presence of L-[35S]-methionine. S1 nuclease analysis of placental RNA and screening of ectoplacental cone and extraembryonic ectoderm cDNA libraries with MuIFN-alpha and -beta probes failed to detect IFN related mRNAs, even under relatively non-stringent conditions of hybridization. Thus, while antiviral activity is produced by peri-implantation conceptuses in several diverse mammalian species, it does not appear to be due to a conserved type of IFN in all these species.     

干扰素诱导跨膜蛋白抗病毒感染研究进展

干扰素诱导跨膜蛋白(interferon-inducing transmembrane proteins,IFITM)是一类宿主限制性因子(host restrictive factor,HRF),在抗病毒感染过程中发挥重要作用。IFITM作为一类氨基酸高度保守的抗病毒蛋白,在机体内广泛分布。随着对其研究的深入,发现该IFITM以多种通路/机制产生抗病毒感染的作用,使其作为研发抗病毒治疗药物的作用靶点成为可能。现就IFITM拓扑结构,抗病毒机制和抗病毒谱的研究作一概述。     

Scenedesmus obliquus: Antioxidant and antiviral activity of proteins hydrolyzed by three enzymes

Purpose

To obtain protein hydrolysates from fresh water green algae Scenedesmus obliquus by three different enzymes and evaluate its antioxidant and antiviral activity.

Protein production by cultures established from Day-14-16 sheep and pig conceptuses

Primary cell cultures were established from cells derived from dissociated Day-14 and -16 sheep and pig blastocysts. The appearance of cells in culture from both species was similar. Cultures contained a variety of cells with distinct morphologies, some were small and compact and formed clumps and multiple layers while others were large, flat and formed a monolayer. Within 4 h of culturing small floating fluid-filled spheres of cells were observed in the medium; some of these increased in size to greater than 1 cm diameter over 1-2 weeks. In addition, fluid-filled domes of cells arose from the underlying monolayer. Contractile cells became evident after about 8 days and some became organized into large patches of contracting tissue. Two-dimensional polyacrylamide gel electrophoresis and fluorography were performed on proteins released into the medium by confluent monolayers, floating spheres and floating cells that failed to attach during the first 24 h. All cultures produced as major products proteins with electrophoretic mobilities identical to certain fetal plasma proteins. In general, cultures did not produce proteins characteristic of short-term cultures of whole conceptuses harvested at Days 14-16. In cultures established from sheep blastocysts only the cells that failed to attach produced ovine trophoblast protein-1, a major polypeptide produced by the trophectoderm of the sheep conceptus between Days 13 and 21 of pregnancy.     

Mx proteins: antiviral proteins by chance or by necessity?

The interferon-inducible Mx1 protein is responsible for inborn resistance of mice to influenza. It is now recognized that this protein is a member of a family of interferon-inducible, putative GTP-binding proteins found in many organisms. Thus, these proteins, called the Mx proteins, are found in species that are naturally infected with influenza virus, and also in species that are not. Some Mx proteins display a broader antiviral profile than the one observed for Mx1 in mice. Others, however, may not be antiviral. Two recently discovered GTP-binding proteins, Vps1p in yeast and dynamin in rat, are also related to Mx1. These proteins are synthesized constitutively and serve basic cellular functions.     

Synthetic proteins with antiviral properties of alpha-interferons

Consensus sequence 30-36 LKDRHDF of human alpha-interferons is inserted into the C- and N-terminal sequences of the artificial protein albeferon using genetic engineering methods in order to obtain artificial proteins with antiviral activity. Albeferon, obtained by Dolgikh et al. (Protein Eng., 1996, vol. 9, pp. 195-201), already contains the IFN-alpha 2 130-137 fragment and possesses antiproliferative activity comparable with that of IFN-alpha 2. According to CD spectroscopy, both proteins have regular secondary structures similar to that of the precursor protein. They exhibit antiviral activities, and the activity of one of them is comparable with that of IFN-alpha 2. At the same time, their cytotoxic properties are displayed only at relatively high concentrations, which substantially exceed the minimal antiviral concentrations.     

Characterization of antiviral and antibacterial activity of Bombyx mori seroin proteins

Lepidopterans as other insects have a very potent innate immune system, which basically comprises cellular and humoral defence mechanisms against bacterial and fungal infections. In lepidopterans, not much is known about the defence mechanisms against viral pathogens, such as baculoviruses. Here we show that small silk proteins of the domesticated silkworm, Bombyx mori, called seroins, act as antiviral agents against a baculovirus pathogen, Bombyx mori nucleopolyhedrovirus (BmNPV). Involvement of these proteins in the inhibition of baculovirus infection was revealed by estimating the viral load upon their dsRNA‐mediated knockdown. Additionally, we found through antimicrobial assays that seroins are potent inhibitors of bacterial growth. Binding competition assays followed by antimicrobial assays showed that seroins bind to peptidoglycan, a cell wall component of bacteria. Analysis of bacterial load upon knockdown of seroins resulted in higher proliferation of bacteria. Phylogenetic analysis showed the recent origin of seroins in a few moth species and duplication only in Bombycids. The antiviral and antibacterial activity of seroins shown in this study using several biochemical and molecular biological assays provide strong evidence to characterize them as antimicrobial proteins. Hence, we hypothesize that seroins are potent candidates for use in development of transgene‐based disease resistant silkworm strains.     

Characterization of the epidermal growth factor receptor in preimplantation pig conceptuses.

Embryos recovered from sows on Days 9-13 of pregnancy (Day 0 = first day of estrus) exhibited saturable and time-dependent specific binding of 125I-epidermal growth factor (EGF). The specific binding (pg/mg protein) was greater (P less than 0.001) for Day 13 elongated conceptuses than for conceptuses of earlier stages. Scatchard analyses showed two classes of binding sites (Kd = 7.0 +/- 2.6 x 10(-11) M, Bmax = 6.2 +/- 1.4 fmol/mg protein and Kd = 3.4 +/- 0.2 x 10(-8) M, Bmax = 420 +/- 80 fmol/mg protein). The EGF receptor in Day 13 conceptus membranes is a 170-kDa protein and was phosphorylated in the presence of EGF and adenosine triphosphate. EGF stimulated protein tyrosine kinase activity about 1.6-fold over basal levels. The results show that the preimplantation pig conceptus possesses EGF-binding sites with the properties of functional EGF-receptors.     

Analysis of the in planta antiviral activity of elderberry ribosome-inactivating proteins.

Although the type-2 ribosome-inactivating proteins (SNA-I, SNA-V, SNLRP) from elderberry (Sambucus nigra L.) are all devoid of rRNA N-glycosylase activity towards plant ribosomes, some of them clearly show polynucleotide-adenosine glycosylase activity towards tobacco mosaic virus RNA. This particular substrate specificity was exploited to further unravel the mechanism underlying the in planta antiviral activity of ribosome-inactivating proteins. Transgenic tobacco (Nicotiana tabacum L. cv Samsun NN) plants expressing the elderberry ribosome-inactivating proteins were generated and challenged with tobacco mosaic virus in order to analyze their antiviral properties. Although some transgenic plants clearly showed antiviral activity, no clear correlation was observed between in planta antiviral activity of transgenic tobacco lines expressing the different ribosome-inactivating proteins and the in vitro polynucleotide-adenosine glycosylase activity of the respective proteins towards tobacco mosaic virus genomic RNA. However, our results suggest that the in planta antiviral activity of some ribosome-inactivating proteins may rely on a direct mechanism on the virus. In addition, it is evident that the working mechanism proposed for pokeweed antiviral protein cannot be extrapolated to elderberry ribosome-inactivating proteins because the expression of SNA-V is not accompanied by induction of pathogenesis-related proteins.     

Heterocyclic rimantadine analogues with antiviral activity

2-(1-Adamantyl)-2-methyl-pyrrolidines 3 and 4, 2-(1-adamantyl)-2-methyl-azetidines 5 and 6, and 2-(1-adamantyl)-2-methyl-aziridines 7 and 8 were synthesized and tested for their antiviral activity against influenza A. Parent molecules 3, 5, and 7 contain the alpha-methyl-1-adamantan-methanamine 2 pharmacophoric moiety (rimantadine). The ring size effect on anti-influenza A activity was investigated. Pyrrolidine 3 was the most potent anti-influenza virus A compound, 9-fold more potent than rimantadine 2, 27-fold more potent than amantadine 1, and 22-fold more potent than ribavirin. Azetidines 5 and 6 were both markedly active against influenza A H2N2 virus, 10- to 20-fold more potent than amantadine. Aziridine 7 was almost devoid of any activity against H2N2 virus but exhibited borderline activity against H3N2 influenza A strain. Thus, it appears that changing the five-, to four- to a three-membered ring results in a drop of activity against influenza A virus.     

Heterocyclic rimantadine analogues with antiviral activity

2-(1-Adamantyl)pyrrolidines 6, 7, 2-(1-adamantyl)piperidines 10, 12a–c, 15a,b and 2-(1-adamantyl)hexahydroazepines 19, 21, 22 were synthesized and tested for their antiviral activity against influenza A, B viruses and the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). The synthetic procedure followed for the preparation of the parent piperidine 10 represents a general method for the synthesis of 2-alkyl- or cycloalkyl-substituted piperidine alkaloids. Parent aminoadamantanes 6, 10 and 19 contain the 1-aminoethyl pharmacophore group of rimantadine drug 2, extended into a saturated nitrogen heterocycle: pyrrolidine, piperidine and hexahydroazepine, respectively. The ring size effect in anti-influenza A activity was investigated. Rimantadine analogues 6 and 10 were, respectively, 6- and 4-fold more active than the drug Rimantadine 2, whereas the hexahydroazepine derivative 19 was inactive. Thus, enlargement from a 5-(pyrrolidine)- or 6-(piperidine)- to a 7-(hexahydroazepine)- membered heterocyclic ring dramatically reduced the anti-influenza virus A activity. Substitution of piperidine 10 with a dialkyaminoethyl group led to the active compounds 15a and 15b: compound 15a was active against influenza A virus whereas both 15a and 15b were active against HIV-1.     

Capacitation is associated with tyrosine phosphorylation and tyrosine kinase-like activity of pig sperm proteins

Capacitation represents the final maturational steps that render mammalian sperm competent to fertilize, either in vivo or in vitro. Capacitation is defined as a series of events that enables sperm to bind the oocyte and undergo the acrosome reaction in response to the zona pellucida. Although the molecular mechanisms involved are not fully understood, sperm protein phosphorylation is associated with capacitation. The hypothesis of this study is that protein tyrosine phosphorylation and kinase activity mediate capacitation of porcine sperm. Fresh sperm were incubated in noncapacitating or capacitating media for various times. Proteins were extracted with SDS, subjected to SDS-PAGE, and immunoblotted with an antiphosphotyrosine antibody. An M(r) 32 000 tyrosine-phosphorylated protein (designated as p32) appeared only when the sperm were incubated in capacitating medium and concomitant with capacitation as assessed by the ionophore-induced acrosome reaction. The p32 was soluble in Triton X-100. Fractionation of sperm proteins with Triton X-114 demonstrated that after capacitation, this tyrosine phosphoprotein is located in both the cytosol and the membrane. Enzyme renaturation of sperm proteins was conducted in gels with or without either poly glu:tyr (a tyrosine kinase substrate) or kemptide (a protein kinase A substrate). An M(r) 32 000 enzyme with kinase behavior was observed in all gels but was preferentially phosphorylated on tyrosine, as assessed by phosphorimagery and by thin layer chromotography to identify the phosphoamino acids. Indirect immunolocalization showed that the phosphotyrosine residues redistribute to the acrosome during capacitation, which is an appropriate location for a protein involved in the acquisition of fertility.     

Two-dimensional polyacrylamide gel electrophoresis of proteins synthesized and released by conceptuses and endometria from pony mares

Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with L-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography. Conceptuses obtained before Day 14 after ovulation released a characteristic pattern of labelled proteins. These included two groups of apparent isoelectric variants of relative molecular weights (Mr) 30,000-40,000 (pI values 4.5-5.5 and 6-7), one group of Mr approximately 22,000 (pI 6.5-7), and large protein(s) that did not enter the 10% polyacrylamide gel. After Day 14 the array of labelled proteins had changed and resembled that produced by isolated yolk sac at the later stages of pregnancy studied. Included amongst these were several acidic polypeptides with Mr 20,000 (pI 5-6). The endometrial samples released an array of non-dialysable polypeptides into the culture medium. Fluorograms could be assigned to one of three general groups, with endometrium from mares within each group producing similar patterns of labelled proteins. The first group consisted of anoestrous, transitional and ovariectomized mares, and mares at oestrus or Day 1 or Day 18 after ovulation. The second group was comprised of mares at Days 12-16 of dioestrus or Days 12-18 of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of two species of interferon by Large White and Meishan pig conceptuses during the peri-attachment period.

Antiviral activities present in uterine flushings from pregnant Large White, Large White 'hyperprolific', and prolific Meishan gilts, between Days 8 and 20 of gestation were compared. Flushings (20 ml) from all gilts between Days 14 and 20 were positive in an in-vitro interferon (IFN) assay using vesicular stomatitis virus as a challenge infection. Highest antiviral activities (of up to 400,000-1,200,000 total Units/flushing) were obtained at Day 16 of gestation, i.e. clearly after the beginning of attachment. There was no major difference between breeds although, at Day 14, flushings from Meishan gilts yielded significantly higher titres than those from the other two, suggesting a correlation with the previously described earlier trophoblast elongation in Meishan gilts. Conceptus cultures contained antiviral activity, with values very close to those obtained in vivo, but the difference between breeds was not significant. Cultures from Day 20 on contained very little antiviral activity. The antiviral activity was associated with a mixture of at least two IFNs, one of which was IFN-alpha like, and the other was serologically identified as an IFN-gamma, that is an 'immune IFN', previously found to be secreted only by T lymphocytes. This finding may have implications for our understanding of the immunology of early pregnancy.