Biomedical subjects

R W Moyer

Publications and source records attributed to R W Moyer.

At least 37 records · Page 2Linked to original sources

PubMed1998

Functional comparisons among members of the poxvirus T1/35kDa family of soluble CC-chemokine inhibitor glycoproteins.

Many poxviruses express a 35-40-kDa secreted protein, termed "T1" (for leporipoxviruses) or "35kDa" (for orthopoxviruses), that binds CC-chemokines with high affinity but is unrelated to any known cellular proteins. Many previously identified poxvirus cytokine-binding proteins display strict species ligand-binding specificity. Because the T1 and 35kDa proteins share only 40% amino acid identity, we compared the abilities of purified myxoma virus-T1 (M-T1) and vaccinia virus (strain Lister)- and rabbitpox virus-35kDa proteins to inhibit human CC-chemokines in vitro. All three proteins were equally effective in preventing several human CC-chemokines from binding to target chemokine receptors and blocking subsequent intracellular calcium release. The inhibitory affinities were comparable (Ki = 0.07-1.02 nM). These proteins also displayed similar abilities to inhibit (IC50 = 6.3-10.5 nM) human macrophage inflammatory protein-1alpha-mediated chemotaxis of human monocytes. None of the viral proteins blocked interleukin-8-mediated calcium flux or chemotaxis of human neutrophils, confirming that the biological specificity of the T1/35kDa family is targeted inhibition of CC-chemokines. Despite the significant sequence divergence between the leporipoxvirus T1 and orthopoxvirus 35kDa proteins, our data suggest that their CC-chemokine binding and inhibitory properties appear to be species nonspecific and that the critical motifs most likely reside within the limited regions of conservation.

Calcium↗

PubMed1998

Serotonin 5-HT2c agonists mimic the effect of light pulses on circadian rhythms.

The serotonin agonist quipazine has been shown to cause phase shifts in melatonin and activity rhythms and to induce c-fos in the suprachiasmatic nucleus of rats. In this study, in vivo pharmacological characterisation of the phase shifting properties of serotonin agonists has been performed, with a view to determining the receptor sub-types involved. Agonists for the 5-HT2a/2c receptors, (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride (DOI, 0.1 mg/k), 1-(3-chlorophenyl)-piperazine HCl (mCPP, 2 mg/kg) and N-(3-trifluoromethylphenyl)-piperazine HCl (TFMPP, 2 mg/kg) injected at CT18 resulted in acute transient inhibition of melatonin production and delays in the onset of production on the following nights of 1.2+/-0.2, 1.7+/-0.3 and 1. 4+/-0.8 h respectively. Drugs specific for 5-HT1a/7 and 5-HT3 receptors failed to affect melatonin production. At a dose of 0.07 micromole/kg, the serotonin antagonist, ritanserin inhibited the DOI induced phase delay whereas ketanserin was ineffective at this dose, providing strong evidence that DOI was acting through 5-HT2c receptors. DOI (0.5 mg/kg) at CT18 provoked a phase delay in the core body temperature rhythm of similar magnitude to that following a light pulse. Administration of DOI but not agonists active at other receptor sites resulted in the appearance of c-Fos in the ventrolateral division of the suprachiasmatic nucleus (SCN) at CT18 but not at CT6. Ritanserin was more potent than ketanserin at inhibiting the DOI induced increase in c-Fos labelled cells in the SCN. When rats were pre-treated with metergoline (15 mg/kg), ritanserin (3 mg/kg) or LY 53,857 (3 mg/kg) prior to a 2 lx/ 1 min light pulse, none of the drugs significantly inhibited the responses to light. The results of these experiments indicate that serotonergic agonists active at the 5-HT2c receptor mimic the effects of light on 2 independent rhythms and activate SCN neurones in the rat.

Amphetamines↗

PubMed1998

The non-permissive infection of insect (gypsy moth) LD-652 cells by Vaccinia virus.

The members of Poxviridae family are among the most complex of animal viruses and subfamily members infect both vertebrate (Chordopoxvirinae) and invertebrate (Entomopoxvirinae) hosts, respectively. Vaccinia virus (VV) is the most commonly studied vertebrate virus and the entomopoxvirus of Amsacta moorei (AmEPV) is the prototypic insect virus. AmEPV, while not able to productively infect vertebrate cells, does enter vertebrate cells and expresses early genes after which the infection aborts although the cells survive (Y. Li, R. L. Hall, and R. W. Moyer. J.Virol. 71(12), 95579562, 1997). We show here that a recombinant VV, containing the lacZ gene regulated by the cowpox virus A-type inclusion (ATI) late promoter, likewise does not productively infect insect cells. Our results suggest that the recombinant VV enters insect cells, host protein synthesis is inhibited, early gene expression is normal, and viral DNA replication occurs as does late protein synthesis. However, little if any proteolytic processing of late viral proteins, typical of morphogenesis, is observed. Electron micrographs of infected cells suggest that while cytoplasmic virosomes (factories) are formed, there is little indication of further morphogenesis or any formation of mature virions. Therefore, while both orthopoxviruses and entomopoxviruses fail to replicate in heterologous hosts, the nature of abortive infections is quite different.

Animals↗

PubMed1998

Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B.

Cytototoxic T lymphocyte-induced apoptosis can occur either through the directed exocytosis of granzyme B and perforin or via ligation of Fas. Both pathways involve the activation of a family of cysteine proteinases, the caspases, that cleave substrates at aspartic acid and are themselves activated by cleavage at internal aspartate residues. Fas recruits caspase 8, which initiates the death program through the subsequent activation of caspase 3. Granzyme B can process both caspase 8 and 3 in vitro, suggesting that both Fas and granzyme B access the apoptotic program in the same way. Here we demonstrate that although the two mechanisms are similar, the events that lead to activation of caspase 3 can be distinguished in vivo on the basis of their sensitivities to both pharmacological and virus-encoded caspase inhibitors. In cytotoxic T lymphocytes-mediated death the initial cleavage event on caspase 3 is insensitive to benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (zVAD-fmk) inhibition in both mouse and human systems. During Fas-mediated death, however, activation of caspase 3 is completely inhibited to zVAD-fmk. In addition, the viral serpin SPI-2, a homologue of cytokine response modifier A (crmA), is an effective inhibitor of the Fas but not the granzyme pathway. Our results demonstrate that whereas Fas-mediated activation of caspase 3 requires an upstream caspase activity that is zVAD-fmk-sensitive, the initial cleavage of caspase 3 during granule-mediated cell death is insensitive to zVAD-fmk, suggesting that caspase 3 is cleaved directly by granzyme B in vivo.

Amino Acid Chloromethyl Ketones↗

PubMed1998

High-level expression of Amsacta moorei entomopoxvirus spheroidin depends on sequences within the gene.

Spheroidin (SPH) is the most highly expressed gene of the entomopoxvirus isolated from Amsacta moorei (AmEPV). The level of expression of poxvirus genes is believed to be governed in large part by the promoter. Poxvirus promoters generally consist of approximately 40 bp which frequently terminate at the 3' end with a translation initiating TAAATG sequence. We have examined the requirements for high levels of SPH gene expression by constructing AmEPV recombinants containing either the SPH promoter or the late vertebrate poxvirus promoter derived from the cowpox virus A-type inclusion (ATI) gene. In addition, we have examined SPH promoter derivatives which extend beyond the 3' TAAATG to include 2 or 20 bp of the 5' coding sequence of the SPH gene. Examination of insect cells infected with these AmEPV ATI-lacZ or SPH-lacZ recombinants suggests that ATI-lacZ expression begins 12 h before and is essentially complete prior to any SPH-lacZ expression, allowing functional distinction between the ATI and SPH promoters and implying that different factors regulate the two promoters within the insect environment. SPH promoter-regulated expression is significantly enhanced within infected insect cells by including the additional 20 bp of the N-terminal SPH coding sequences as part of the promoter. However, when any of the SPH promoter constructs, including those containing the downstream sequences, were inserted into vaccinia virus, only very low levels of beta-galactosidase expression were observed. These results imply that downstream coding sequences within the SPH gene enhance SPH gene expression only within the insect environment.

Animals↗

PubMed1998

Activation of caspases in pig kidney cells infected with wild-type and CrmA/SPI-2 mutants of cowpox and rabbitpox viruses.

The cowpox virus (CPV) CrmA and the equivalent rabbitpox virus (RPV) SPI-2 proteins have anti-inflammatory and antiapoptosis activity by virtue of their ability to inhibit caspases, including the interleukin-1beta-converting enzyme (ICE; caspase-1). Infection of LLC-PK1 pig kidney cells with a CPV CrmA mutant, but not with wild-type (wt) CPV, results in the induction of many of the morphological features of apoptosis (C. A. Ray and D. J. Pickup, Virology 217:384-391, 1996). In our study, LLC-PK1 cells infected with CPV delta crmA, but not those infected with wt CPV, showed induction of poly(ADP-ribose) polymerase (PARP)- and lamin A-cleaving activities and processing of the CPP32 (caspase-3) precursor to a mature 18-kDa form. Surprisingly, infection of LLC-PK1 cells with either wt RPV (despite the presence of the SPI-2 protein) or RPV delta SPI-2 resulted in cleavage activity against PARP and lamin A and the appearance of the mature subunit of CPP32/caspase-3. The biotinylated specific peptide inhibitor Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone [AcYV(bio)KD-aomk] labeled active caspase subunits of 18, 19, and 21 kDa in extracts from LLC-PK1 cells infected with CPV delta crmA, wt RPV, or RPV delta SPI-2 but not wt CPV. Mixed infection of LLC-PK1 cells with wt RPV and wt CPV gave no PARP-cleaving activity, and all PARP cleavage mediated by SPI-2 and CrmA mutants of RPV and CPV, respectively, could be eliminated by coinfection with wt CPV. These results suggest that the RPV SPI-2 and CPV CrmA proteins are not functionally equivalent and that CrmA, but not SPI-2 protein, can completely prevent apoptosis in LLC-PK1 cells under these conditions.

Animals↗

PubMed1997

Quipazine and light have similar effects on c-fos induction in the rat suprachiasmatic nucleus.

The effects of the serotonin agonist, quipazine, on the induction of c-fos in the suprachiasmatic nucleus of the rat was examined at different times of the 24 h cycle. Quipazine administered at night induced Fos production in a dose dependent manner (1, 3, 10, 30 mumol/kg) in the ventrolateral portion of the suprachiasmatic nucleus at ZT18. Administration of the highest dose at other times resulted in c-fos induction at ZT15 but not at other times of the day or subjective day examined (CT6 and ZT12). When compared to the effects of light pulses (2 lux/1 min), quipazine only caused c-fos induction at times when light caused induction. Our results support a role of serotonergic pathways in the transmission or modulation of photic information from the retina to the suprachiasmatic nucleus of the rat.

Animals↗

PubMed1997

Attenuation of B5R mutants of rabbitpox virus in vivo is related to impaired growth and not an enhanced host inflammatory response.

The rabbitpox virus (RPV) B5R protein, synthesized late in infection, is found as a 45-kDa membrane-associated protein of the envelope of infectious extracellular enveloped virus (EEV) and as a 38-kDa protein secreted from the cell by a process independent of morphogenesis. The protein is not found associated with intracellular mature virus (IMV). Deletion of the gene attenuates the virus (RPV delta B5R) in animals (mice and rabbits), has relatively little effect on formation of IMV, prevents EEV formation in some but not all cells, and leads to a reduced host range. Analysis of the sequence of the protein suggests relatedness to factor H of the complement cascade. Collectively, these observations suggest that attenuation of the virus in vivo could be linked to an inhibition of the inflammatory response, a deficiency in growth, or both. In this report we have analyzed the behavior of RPV delta B5R in infected mice and rabbits and conclude that attenuation of the mutant virus likely results from simple failure to grow within the infected animal and that the inflammatory response probably contributes little to the observed attenuation.

Animals↗

PubMed1997

Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8.

The activation of multiple interleukin-1beta converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic signaling pathways. Here we used an affinity-labeling agent, YV(bio)KD-aomk, to investigate the caspases activated during apoptotic cell death. YV(bio)KD-aomk identified six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these findings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.

Affinity Labels↗

PubMed1997

Characterization of a DNA topoisomerase encoded by Amsacta moore entomopoxvirus.

We have identified an Amsacta moorei entomopoxvirus (AmEPV) gene encoding a DNA topoisomerase. The 333-amino acid AmEPV topoisomerase displays instructive sequence similarities to the previously identified topoisomerases encoded by five genera of vertebrate poxviruses. One hundred nine amino acids are identical or conserved among the six proteins. The gene encoding AmEPV topoisomerase was expressed in bacteria and the recombinant enzyme was partially purified. AmEPV topoisomerase is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA. Like the vaccinia, Shope fibroma virus, and Orf virus enzymes, the AmEPV topoisomerase forms a covalent adduct with duplex DNA at the target sequence CCCTT decreases. The kinetic and equilibrium parameters of the DNA cleavage reaction of AmEPV topoisomerase (Kobs = 0.08 sec-1; Kcl = 0.22) are similar to those of the vaccinia virus enzyme.

Amino Acid Sequence↗

PubMed1997

The T1/35kDa family of poxvirus-secreted proteins bind chemokines and modulate leukocyte influx into virus-infected tissues.

Immunomodulatory proteins encoded by the larger DNA viruses interact with a wide spectrum of immune effector molecules that regulate the antiviral response in the infected host. Here we show that certain poxviruses, including myxoma virus. Shope fibroma virus, rabbitpox virus, vaccinia virus (strain Lister), cowpox virus, and raccoonpox virus, express a new family of secreted proteins which interact with members of both the CC and CXC superfamilies of chemokines. However, swinepox virus and vaccinia virus (strain WR) do not express this activity Using a recombinant poxviruses, the myxoma M-T1 and rabbitpox virus 35kDa secreted proteins were identified as prototypic members of this family of chemokine binding proteins. Members of this T1/35kDa family of poxvirus-secreted proteins share multiple stretches of identical sequence motifs, including eight conserved cysteine residues, but are otherwise unrelated to any cellular genes in the database. The affinity of the CC chemokine RANTES interaction with M-T1 was assessed by Scatchard analysis and yielded a Kd of approximately 73 nM. In rabbits infected with a mutant rabbitpox virus, in which the 35kDa gene is deleted, there was an increased number of extravasating leukocytes in the deep dermis during the early phases of infection. These observations suggest that members of the T1/35kDa class of secreted viral proteins bind multiple members of the chemokine superfamily in vitro and modulate the influx of inflammatory cells into virus-infected tissues in vivo.

Amino Acid Sequence↗

PubMed1997

Effect of variable temperatures, darkness and light on the secretion of melatonin by pineal explants in the gecko, Christinus marmoratus.

This study examined the combined effect of thermocycles with either variable or constant photic conditions on melatonin production by pineal organs in vitro in the gecko, Christinus marmoratus. A 30 degrees C:15 degrees C thermocycle elicited a rhythm of melatonin production under conditions of 12L:12D, constant light or constant darkness when the cryophase coincided with the dark phase of the photocycle or with the subjective night. A 6 h advance of the thermocycle with respect to the photocycle produced an advance in the onset and offset of melatonin production in subsequent nights. When the thermocycle was 180 degrees out of phase with the photoperiod, the rhythm of melatonin production was disrupted, suggesting a differential pattern of sensitivity to photothermal stimuli. It was concluded that both light and temperature are important modulators of pineal function although their combined effects on pineal melatonin production is complex and unclear.

Animals↗

PubMed1997

Transient, nonlethal expression of genes in vertebrate cells by recombinant entomopoxviruses.

The group B entomopoxvirus (EPV) from Amsacta moorei (AmEPV) productively infects only insect cells. A series of AmEPV-lacZ recombinants was constructed in which the lacZ gene was regulated by either late (the AmEPV spheroidin or the cowpox virus A-type inclusion [ATI]) or early (the AmEPV esp [early strong promoter; derived from a 42-kDa AmEPV protein] or the Melolontha melolontha EPV fusolin, fus) virus promoters. When the AmEPV recombinants were used to infect vertebrate cells, beta-galactosidase expression occurred (in >30% of the cells) when lacZ was regulated by either the fus or esp early promoters but not when lacZ was regulated by the late promoters (spheroidin or ATI). Therefore, AmEPV enters vertebrate cells and undergoes at least a partial uncoating and early, but not late, viral genes are expressed. Neither viral DNA synthesis nor cytopathic effects were observed under any infection conditions. When an AmEPV recombinant virus containing the Aequorea victoria green fluorescent protein gene (gfp) under the control of the esp promoter was used to infect vertebrate cells at a low multiplicity of infection, single fluorescent cells resulted, which continued to divide over a period of several days, ultimately forming fluorescent cell clusters, suggesting that vertebrate cells survive the infection and continue to grow. Therefore, AmEPV may prove to be a highly efficient, nontoxic method of gene delivery into vertebrate cells for transient gene expression.

Animals↗

PubMed1996

CrmA/SPI-2 inhibition of an endogenous ICE-related protease responsible for lamin A cleavage and apoptotic nuclear fragmentation.

CrmA, a poxvirus gene product with a serpin-like structure, blocks a variety of apoptotic death events in cultured cells. Based on the ability of CrmA to inhibit the interleukin-1beta converting enzyme in vitro, it has been speculated that interleukin-1beta converting enzyme-related proteases (caspases) essential for apoptosis are the cellular targets of CrmA. Here we found that rabbitpox virus CrmA/SPI-2 inhibits the cleavage of lamin A mediated by a caspase in our cell-free system of apoptosis. In the presence of CrmA/SPI-2, nuclear apoptosis in vitro was blocked at an intermediate stage after collapse of the chromatin against the nuclear periphery and before nuclear shrinkage and disintegration into apoptotic body-like fragments. Using N-(acetyltyrosinylvalinyl-Nepsilon-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone, which derivatizes the active forms of caspases, we could show that one of five caspases active in the extracts is inhibited both by CrmA/SPI-2 and by a peptide spanning the lamin A apoptotic cleavage site. These results reveal that CrmA/SPI-2 can inhibit a caspase responsible both for lamin A cleavage and for the nuclear disintegration characteristic of apoptosis.

Animals↗

PubMed1996

The Amsacta moorei entomopoxvirus spheroidin gene is improperly transcribed in vertebrate poxviruses.

The Amsacta moorei entomopoxvirus (AmEPv) spheroidin is the most highly expressed late viral gene product in infected insect cells. However, when a cassette containing the spheroidin gene and putative promoter was inserted into cowpox (CPV) or vaccinia viruses, only very low levels of spheroldin gene expression were observed. Primer extension analysis suggests much lower spheroidin gene transcript levels than seen either for the highly expressed CPV A-type inclusion gene or for the spheroidin gene within infected insect cells, indicating that in vertebrate cells, the spheroidin promoter functions poorly if at all. Examination of the spheroidin mRNA synthesized in recombinant CPV shows that the 5' start site of the spheroidin transcript was also unexpectedly imprecise and upstream (approximately 31 bp) of the well-defined start site normally observed in AmEPV-infected insect cells. Sequencing of the 5' terminus of the CPV recombinant spheroidin mRNA suggested that 5' poly(A), a characteristic feature of late poxvirus mRNAs and spheroidin mRNA derived from insect cells, was absent, despite the presence of the typical vertebrate poxvirus late promoter consensus sequence, TAAATG. Our results indicate that insect and vertebrate poxvirus promoters may not be universally interchangeable and imply that there are regulatory features of gene expression unique to the infected insect cell environment.

Animals↗

PubMed1996

Differential inhibition of the Fas- and granule-mediated cytolysis pathways by the orthopoxvirus cytokine response modifier A/SPI-2 and SPI-1 protein.

Cytotoxic T lymphocytes are important effectors of antiviral immunity, and they induce target cell death either by secretion of cytoplasmic granules containing perforin and granzymes or by signaling through the Fas cell surface antigen. Although it is not known whether the granule-mediated and Fas-mediated cytolytic mechanisms share common components, proteinase activity has been implicated as an important feature of both pathways. The orthopoxviruses cowpox virus and rabbitpox virus each encode three members of the serpin family of proteinase inhibitors, designated SPI-1, SPI-2, and SPI-3. Of these, SPI-2 (also referred to as cytokine response modifier A in cowpox virus) has been shown to inhibit the proteolytic activity of both members of the interleukin 1 beta converting enzyme family and granzyme B. We report here that cells infected with cowpox or rabbitpox viruses exhibit resistance to cytolysis by either cytolytic mechanism. Whereas mutation of the cytokine response modifier A/SPI-2 gene was necessary to relieve inhibition of Fasmediated cytolysis, in some cell types mutation of SPI-1, in addition to cytokine response modifier A/SPI-2, was necessary to completely abrogate inhibition. In contrast, viral inhibition of granule-mediated killing was unaffected by mutation of cytokine response modifier A/SPI-2 alone, and it was relieved only when both the cytokine response modifier A/SPI-2 and SPI-1 genes were inactivated. These results suggest that an interleukin 1 beta converting enzyme-like enzymatic activity is involved in both killing mechanisms and indicate that two viral proteins, SPI-1 and cytokine response modifier A/SPI-2, are necessary to inhibit both cytolysis pathways.

Animals↗

PubMed1995

The effect of inhibitors on the growth of the entomopoxvirus from Amsacta moorei in Lymantria dispar (gypsy moth) cells.

Within the family of Poxviridae, the entomopoxviruses are the most distant relatives of the more well-known and intensively studied orthopoxviruses (vaccinia and variola). The growth of the entomopoxvirus from Amsacta moorei (AmEPV) has been characterized in cell culture and compared to that of vaccinia virus (VV), the prototypic orthopoxvirus. The overall characteristics of infected cell cultures were generally similar between the two viruses. One striking difference noted was the apparent absence of proteolytic processing of late AmEPV viral proteins, a hallmark of vertebrate poxvirus infections associated with viral morphogenesis. AmEPV, like VV, was found to be sensitive to all the inhibitors of viral infection tested including phosphonoacetic acid, 1-beta-D-arabinofuranosylcytosine, and isatin-beta-thiosemicarbazone (IBT), a compound associated with the rather specific inhibition of vertebrate poxviruses. While both VV and AmEPV are inhibited by IBT, the inhibition of AmEPV, unlike that of VV, is not accompanied by either a breakdown of ribosomal RNA or a global inhibition of late viral protein synthesis. Instead, in the presence of IBT, AmEPV enveloped, immature virions form devoid of a well-differentiated core, which unlike mature virions fail to insert into occlusion bodies.

Animals↗

PubMed1995

Effect of constant temperatures, darkness and light on the secretion of melatonin by pineal explants and retinas in the gecko Christinus marmoratus.

The effects of temperature and lighting conditions on the secretion of melatonin by the pineal organ of the nocturnal gecko Christinus marmoratus was studied using in vitro perifusion. In a 12L:12D lighting regime, a high-amplitude melatonin rhythm was detectable at a constant temperature of 20 and 30 degrees C but not at 10 or 37 degrees C. There were sustained high levels of melatonin in constant darkness and sustained low levels in constant light. No retinal melatonin was detected using static and perifusion culture techniques. These results show that the pineal organ of C. marmoratus maintains light sensitivity in vitro but does not contain an oscillator coupled to the melatonin synthetic pathway.

Animals↗