Evolution of the capsid protein genes of foot-and-mouth disease virus: antigenic variation without accumulation of amino acid substitutions over six decades. The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time.


Transient expression and mutational analysis of the rotavirus intracellular receptor: the C-terminal methionine residue is essential for ligand binding. Maturation of rotavirus involves an intracellular membrane budding event in which the single-shelled icosahedral particle interacts with a virus-encoded receptor glycoprotein, NS28, that is located in the rough endoplasmic reticulum membrane. The receptor is a tetramer and is oriented with the C-terminal 131 amino acids on the cytoplasmic side of the membrane (A.R. Bellamy and G.W. Both, Adv. Virus Res. 38:1-48, 1990). We have used the T7-vaccinia virus transient expression system to deliver mutant variants of the NS28 gene to CV1 cells in order to assess the effects of site-specific modifications on receptor function. Three types of mutant proteins have been constructed by altering the extreme C-terminal methionine, cysteine residues within the third hydrophobic domain, and internal residues located within the cytoplasmic portion of the receptor, respectively. Deletion or conservative substitution of the C-terminal methionine completely abolishes receptor activity. Substitution of cysteine residues has no effect on receptor activity or on the ability of the receptor to adopt its native oligomeric state. Internal deletions result only in a reduction in the level of binding. An N-terminally truncated form of the receptor, containing only the cytoplasmic domain, retains full receptor activity and can form membrane-associated tetramers.


Localization of cis-acting sequence requirements in the promoter of the latency-associated transcript of herpes simplex virus type 1 required for cell-type-specific activity. We have previously demonstrated (A. H. Batchelor and P. O'Hare, J. Virol. 64:3269-3279, 1990) the selective activity in human neuroblastoma cells (IMR-32) of a promoter located upstream of the latency-associated transcript of herpes simplex virus type 1. In this work, we provide evidence for the basis of the selective activity of this latency-associated promoter (LAP). Recombinant constructs containing sequences up to -143 (relative to the LAP cap site) linked to the chloramphenicol acetyltransferase gene retain strong activity in HeLa cells but exhibit extremely weak activity in IMR-32 cells. Sequences mapping within the 108 bp upstream of -143 to position -251 enhance LAP activity by over 15-fold, restoring optimal levels of expression in IMR-32 cells, but have little or no effect (1.5-fold) in HeLa cells. This cell-type-specific enhancement of promoter activity took place in two major steps, with sequences between -143 and -158 conferring a four- to fivefold effect and sequences between -177 and -251 conferring a further threefold effect. Furthermore, sequences mapping from -40 to -258 could transfer the ability to be expressed in neuroblastoma cells to the normally inactive immediate-early 110K promoter (IE110K), increasing levels of expression by 35-fold. By comparison, this region had a relatively minor effect (twofold) on the activity of the IE110K promoter in HeLa cells, even though this promoter is open to activation by other mechanisms. However, neither of the overlapping subregions from -40 to -143 or -138 to -258 could confer efficient IMR-32 cell expression on the IE110K promoter, and we present alternative models for multiple element requirements or the requirement for a critical site around -140 which is not retained in either subfragment. We provide consistent evidence for a site around -140 and demonstrate the presence selectively in IMR-32 cells of a DNA-binding factor which binds a probe spanning this region. We propose that this element and the cognate factor (IC-1) may be involved in the selective activity of the LAP in neuroblastoma cells.


Structural and functional analysis of the visna virus Rev-response element. The distantly related lentiviruses human immunodeficiency virus type 1 (HIV-1) and visna virus each encode a posttranscriptional regulatory protein, termed Rev, that is critical for expression of the viral structural proteins. We genetically mapped the cis-acting target sequence for visna virus Rev, the visna virus Rev-response element or RRE-V, to a complex 176-nucleotide RNA stem-loop structure that coincides with sequences encoding the N terminus of the transmembrane component of envelope. The computer-predicted structure of the RRE-V was validated by in vitro analysis of structure-specific RNase cleavage patterns. The visna virus Rev protein was shown to interact specifically with the genetically defined RRE-V in vitro but was unable to bind the HIV-1 RRE. Similarly, HIV-1 Rev was also unable to bind the RRE-V specifically. We therefore conclude that the HIV-1 and visna virus Rev proteins, while functionally analogous, nevertheless display distinct RNA sequence specificities. These findings provide a biochemical explanation for the observation that these two viral regulatory proteins are functional only in the homologous viral system.


Herpes simplex virus type 1-mediated induction of human immunodeficiency virus type 1 provirus correlates with binding of nuclear proteins to the NF-kappa B enhancer and leader sequence. Herpes simplex virus type 1 (HSV-1) infection induces expression of the human immunodeficiency virus type 1 (HIV-1) provirus in the chronically infected T-cell line ACH-2. The HSV-1-mediated induction correlates with the appearance of two NF-kappa B-specific proteins of 55 and 85 kDa in the nucleus and with the binding of 50-kDa nuclear protein to the LBP-1 binding site of the untranslated leader sequence of the HIV-1 long terminal repeat. The HSV-1-induced LBP-1 binding protein, designated HLP-1, is present exclusively in HSV-1-infected, but not in phorbol-12-myristate-13-acetate- or tumor necrosis factor alpha-treated ACH-2 cells. Both the NF-kappa B and LBP-1 target sequences, when inserted either alone or together 5' of a heterologous minimal promoter (thymidine kinase), confer inducibility by HSV-1 infection in a transient transfection assay. Thus, it appears that the HSV-1-mediated activation of HIV-1 provirus is brought about by the binding of both NF-kappa B and HLP-1 specific proteins to two distinct regions of HIV-1 long terminal repeat.


The herpes simplex virus 1 RNA binding protein US11 is a virion component and associates with ribosomal 60S subunits. The herpes simplex virus 1 US11 gene encodes a site- and conformation-specific RNA binding regulatory protein. We fused the coding sequence of this protein with that of beta-galactosidase, expressed the chimeric gene in Escherichia coli, and purified a fusion protein which binds RNA in the same way as the infected cell protein. The fusion protein was used to generate anti-US11 monoclonal antibody. Studies with this antibody showed that US11 protein is a viral structural protein estimated to be present in 600 to 1,000 copies per virion. The great majority of cytoplasmic US11 protein was found in association with the 60S subunit of infected cell ribosomes. US11 protein associates with ribosomes both late in infection at the time of its synthesis and at the time of infection after its introduction into the cytoplasm by the virion. US11 protein expressed in an uninfected cell line stably transfected with the US11 gene associates with ribosomal 60S subunits and localizes to nucleoli, suggesting that US11 protein requires no other viral functions for these associations.


The 11,600-MW protein encoded by region E3 of adenovirus is expressed early but is greatly amplified at late stages of infection. We have reported that an 11,600-MW (11.6K) protein is coded by region E3 of adenovirus. We have now prepared two new antipeptide antisera that have allowed us to characterize this protein further. The 11.6K protein migrates as multiple diffuse bands having apparent Mws of about 14,000, 21,000, and 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting as well as virus mutants with deletions in the 11.6K gene were used to show that the various gel bands represent forms of 11.6K. The 11.6K protein was synthesized in very low amounts during early stages of infection, from the scarce E3 mRNAs d and e which initiate from the E3 promoter. However, 11.6K was synthesized very abundantly at late stages of infection, approximately 400 times the rate at early stages, from new mRNAs termed d' and e'. Reverse transcriptase-polymerase chain reaction and RNA blot experiments indicated that mRNAs d' and e' had the same body (the coding portion) and the same middle exon (the y leader) as early E3 mRNAs d and e, but mRNAs d' and e' were spliced at their 5' termini to the major late tripartite leader which is found in all mRNAs in the major late transcription unit. mRNAs d' and e' and the 11.6K protein were the only E3 mRNAs and protein that were scarce early and were greatly amplified at late stages of infection. This suggests that specific cis- or trans-acting sequences may function to enhance the splicing of mRNAs d' and e' at late stages of infection and perhaps to suppress the splicing of mRNAs d and e at early stages of infection. We propose that the 11.6K gene be considered not only a member of region E3 but also a member of the major late transcription unit.


Receptor properties of two varicella-zoster virus glycoproteins, gpI and gpIV, homologous to herpes simplex virus gE and gI. The varicella-zoster virus (VZV) genome contains 70 reading frames (ORF), 5 of which encode the glycoproteins gpI, gpII, gpIII, gpIV, and gpV. ORF 67 and 68 lie adjacent to each other in the unique short region of the VZV genome and code for gpIV and gpI, respectively. These two genes, which are contained within the HindIII C fragment of the VZV genome, were subcloned in the correct orientation downstream from the promoter regions of the eukaryotic expression vectors pCMV5 and pBJ. After transfection, 5 to 20% of the Cos cells bound antibody specific for the given glycoprotein. In this study, it was shown that only the cells transfected with the gpI construct bound to the Fc fragment of human immunoglobulin G. Neither the transfected gpIV gene product nor the vector only bound to the Fc fragment. Thus, VZV gpI is confirmed to be the VZV-encoded Fc-binding glycoprotein. Like the wild-type form of gpI expressed in VZV-infected cells, gpI precipitated from transfected cells contained both N-linked and O-linked glycans and was heavily sialated. In addition, the transfected gpI gene product was phosphorylated both in cell culture and in protein kinase assays by mammalian casein kinases I and II. Extensive computer-assisted analyses of the VZV gpI sequence, as well as those of alphaherpesviral homolog glycoproteins, disclosed properties similar to those of other cell surface receptors; these included (i) exocytoplasmic regions rich in cysteine residues, (ii) membrane-proximal regions with potential O-linked glycosylation sites, and (iii) cytoplasmic domains with consensus phosphorylation sites.


Direct determination of the point mutation rate of a murine retrovirus. The point mutation rate of a murine leukemia virus (MuLV) genome (AKV) was determined under conditions in which the number of replicative cycles was carefully controlled and the point mutation rate was determined by direct examination of the RNA genomes of progeny viruses. A clonal cell line infected at a low multiplicity of infection (2 x 10(-3)) was derived to provide a source of virus with high genetic homogeneity. Virus stocks from this cell line were used to infect cells at a low multiplicity of infection, and the cells were seeded soon after infection to obtain secondary clonal cell lines. RNase T1-oligonucleotide fingerprinting analyses of virion RNAs from 93 secondary lines revealed only 3 base changes in nearly 130,000 bases analyzed. To obtain an independent assessment of the mutation rate, we directly sequenced virion RNAs by using a series of DNA oligonucleotide primers distributed across the genome. RNA sequencing detected no mutations in over 21,000 bases analyzed. The combined fingerprinting and sequencing analyses yielded a mutation rate for infectious progeny viruses of one base change per 50,000 (2 x 10(-5)) bases per replication cycle. Our results suggest that over 80% of infectious progeny MuLVs may be replicated with complete fidelity and that only a low percentage undergo more than one point mutation during a replication cycle. Previous estimates of retroviral mutation rates suggest that the majority of infectious progeny viruses have undergone one or more point mutations. Recent studies of the mutation rates of marker genes in spleen necrosis virus-based vectors estimate a base substitution rate lower than estimates for infectious avian retroviruses and nearly identical to our determinations with AKV. The differences between mutation rates observed in studies of retroviruses may reflect the imposition of different selective conditions.


Transcription from varicella-zoster virus gene 67 (glycoprotein IV). Three transcripts map to the varicella-zoster virus (VZV) open reading frame (ORF) 67, which encodes glycoprotein IV (gpIV). All of these transcripts are polyadenylated and are transcribed from left to right towards the genomic terminal short repeats. Previous Northern (RNA) blot analyses suggested that the most abundant of these transcripts (1.65 kb) might code for gpIV. We performed S1 nuclease protection and primer extension assays and determined that the 5' terminus of the 1.65-kb transcript maps 91 bp upstream from the gpIV initiation codon. An AT-rich region (ATAAA), -28 bp from the cap site, is a potential TATA box, and at -71 bp there is a consensus CCAAT box motif. The 3' end of the 1.65-kb transcript is 20 bp downstream of two overlapping polyadenylation signals, AATAAA and ATTAAA, and just downstream of the 3' terminus is a GU-rich sequence. These results are reminiscent of data from our analysis of the VZV gpV gene, confirming that VZV appears able to use unusual TATA box motifs. Many canonical TATA sequences are present upstream from these VZV transcriptional start sites but, apparently, are not used. We tested sequences upstream from the gpIV cap site for promoter activity in transient expression experiments by cloning a DNA fragment (+63 to -343 bp) into pCAT3M, which contains a chloramphenicol acetyltransferase reporter gene. This clone showed little constitutive promoter activity but was activated more than 200-fold by infection with VZV and 5-fold with herpes simplex virus. The two known VZV transactivating genes (those for ORF 4 and ORF 62) were tested for their abilities to activate expression from the gpIV promoter by using their cognate promoters. The ORF 4 gene was minimally active, whereas the ORF 62 gene gave twofold induction; both genes, acting together, gave fivefold induction. However, replacement of the IE62 promoter with the immediate-early cytomegalovirus promoter in the ORF 62 construct gave over 40-fold induction of chloramphenicol acetyltransferase activity under the gpIV promoter in the same assay.


Epstein-Barr virus latent gene expression in uncultured peripheral blood lymphocytes. In this study of Epstein-Barr virus (EBV) latency, the polymerase chain reaction was used in modified form for amplification and detection of viral mRNA sequences in peripheral blood lymphocytes from healthy seropositive adults. Six known promoters for latent gene expression and eight known gene products were identified in in vitro-immortalized lymphocytes and in the cell lines established spontaneously from seropositive adults. We examined whether mRNA expression in uncultured B cells from four seropositive adults was the same as that which occurred in spontaneously established EBV-positive B-cell lines from the same individuals. A minimum of 17 polymerase chain reaction targets was required to circumscribe the known latent mRNA structures. Expression of the C promoter for the EBNA genes was detected in B-cell RNA from three of the four subjects. Transcripts initiated from the alternative W promoter for EBNA expression were not detected. The spliced transcripts detected in the B cells contained only the C2-to-W1 alternative splice, which was nonproductive for EBNA4 gene expression. None of the other EBNA open reading frames were detected spliced onto the 3' ends of the C promoter-initiated RNAs. Spliced RNA from the TP gene was detected in all four subjects. Expression of the TP gene was restricted to TP1 promoter-initiated RNAs, as no TP2 promoter-initiated transcripts were detected. Expression of RNA from the LMP gene was not detected. The F promoter which is active in the restricted expression latency that occurs in Burkitt's lymphoma cells was not detected being expressed in peripheral blood B cells. This pattern of latent gene expression is unique to uncultured B cells, indicating that there are profound differences between viral latent states in vitro and in situ and suggesting a central role for the TP gene in the latency of EBV.


Identification of a putative receptor for subgroup A feline leukemia virus on feline T cells. Retrovirus infection is initiated by the binding of virus envelope glycoprotein to a receptor molecule present on cell membranes. To characterize a receptor for feline leukemia virus (FeLV), we extensively purified the viral envelope glycoprotein, gp70, from culture supernatants of FeLV-61E (subgroup A)-infected cells by immunoaffinity chromatography. Binding of purified 125I-labeled gp70 to the feline T-cell line 3201 was specific and saturable, and Scatchard analysis revealed a single class of receptor binding sites with an average number of 1.6 x 10(5) receptors per cell and an apparent affinity constant (Ka) of 1.15 x 10(9) M-1. Cross-linking experiments identified a putative gp70-receptor complex of 135 to 140 kDa. Similarly, coprecipitation of 125I-labeled cell surface proteins with purified gp70 and a neutralizing but noninterfering anti-gp70 monoclonal antibody revealed a single cell surface protein of approximately 70 kDa. These results indicate that FeLV-A binds to feline T cells via a 70-kDa cell surface protein, its presumptive receptor.


A new retrovirus packaging cell for gene transfer constructed from amplified long terminal repeat-free chimeric proviral genes. The retroviral gene transfer system is a powerful tool for somatic gene therapy. A retroviral stock with a high viral titer and lacking replication-competent virus (RCV) is desirable for this type of gene transfer. To fulfill these requirements, we made a new packaging cell line, designated ampli-GPE. To reduce the homology between proviral DNA in the packaging cell and retroviral vector, the gag-pol and env genes of Moloney murine leukemia virus were separated onto two different plasmids, pGP-KV and pENV-KV, respectively, in which the 5' long terminal repeat and the 3' long terminal repeat had been replaced by the mouse metallothionein I promoter or the human beta-globin gene containing the polyadenylation site as control units for the gag-pol and env genes. In addition, these plasmids contained 69% of the bovine papillomavirus gene for gene amplification to obtain production of virus at a high titer. NIH 3T3 clones containing approximately 20 to 50 copies of the gag-pol and env genes were selected and designated ampli-GPE. When ampli-GPE was transfected with the N2 vector or pZipNeoSV(DHFR) derived from pZipNeoSV(X)1, we established clones producing titers of 5 x 10(6) and 1 x 10(6) CFU/ml, respectively. There was no sign of RCV generation in any virus-producing cells from ampli-GPE. However, virus-producing cells derived from psi 2 cells transfected with N2 did generate RCV. Thus, we showed that ampli-GPE, possessing the minimum complement of proviral genes, has potential for the development of a gene transfer system.


Two AP1 sites binding JunB are essential for human papillomavirus type 18 transcription in keratinocytes. The activity and epithelial tropism of the human papillomavirus type 18 P105 early promoter, which directs the synthesis of the E6 and E7 transforming genes, are controlled by cis elements included in the viral long control region. To identify potential cellular regulators of this promoter, we mutagenized one or both of the 5'-TGACTAA-3' cis elements capable of interacting with the AP1 transcription factor, which is composed either of homodimers or heterodimers of the Jun products or of heterodimers of Jun and Fos. Mutation of both elements completely abolished P105 promoter activity in human keratinocytes. We show that either AP1 site can interact efficiently in vitro with any of the three different Jun products as heterodimers with c-Fos. However, in nuclear extracts prepared from human keratinocytes, JunB was the predominant Jun component bound to the DNA probe containing this cis element. These results implicate JunB as an important factor in human papillomavirus type 18 transcription in keratinocytes and strongly suggest a potential role of this Jun gene product in the tissue-specific transcription of the genital papillomaviruses.


Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome. The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.


Cytomegalovirus determinant of replication in salivary glands. Murine cytomegalovirus carrying a deletion mutation disrupting the expression of a gene dispensable for growth in cultured cells was found to disseminate poorly in the mouse. The mutation resulted in a dramatic decrease in the expression of a 1.5-kb major and a 1.8-kb minor beta transcript from a region adjacent to the ie2 gene in the viral genome. Nucleotide sequence determination indicated that 323 bp, including a predicted polyadenylation signal, was deleted from this beta gene. In cultured cells, the plaque morphology and growth characteristics of the mutant were similar to those of parental or rescued wild-type viruses. Following intraperitoneal inoculation of BALB/c mice, growth of the mutant in the salivary gland was dramatically reduced 10,000-fold, while growth in the liver and spleen was not dramatically affected. The beta gene was thus denoted sgg1 (salivary gland growth gene 1). Neither intranasal infection nor direct inoculation into the salivary glands completely overcame the restriction of growth in this organ, suggesting that the sgg1 gene encoded a determinant of tissue tropism. To investigate the impact of the sgg1 mutation on virus dissemination via the blood, the virus titer in peripheral blood leukocytes was determined. No difference was found between the sgg1 mutant and rescued wild-type virus. Thus, murine cytomegalovirus sgg1 gene products appear to be involved in entry or replication of virus in salivary gland cells.


A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress. The mouse L-cell mutant gro29 is defective for egress of herpes simplex virus type 1 (HSV-1) virions and is significantly reduced in HSV-1 glycoprotein export (B. W. Banfield and F. Tufaro, J. Virol. 64:5716-5729, 1990). In this report, we demonstrate that pseudorabies virus (PRV), a distantly related alphaherpesvirus, shows a distinctive set of defects after infection of gro29 cells. Specifically, we identify defects in the rate and extent of viral glycoprotein export, infectious particle formation, plaque formation, and virus egress. The initial rate of viral glycoprotein synthesis was unaffected in gro29 cells, but the extent of export from the endoplasmic reticulum to the Golgi apparatus was impaired and export through the Golgi apparatus became essentially blocked late in infection. Moreover, by using a secreted variant of a viral membrane protein, we found that export from the Golgi apparatus out of the cell was also defective in gro29 cells. PRV does not form plaques on gro29 monolayers. A low level of infectious virus is formed and released early after infection, but further virus egress is blocked. Taken together, these observations suggest that the gro29 phenotype involves either multiple proteins or a single protein used at multiple steps in viral glycoprotein export and virus egress from cells. Moreover, this host cell protein is required by both HSV and PRV for efficient propagation in infected cells.


Herpes simplex virus infection selectively stimulates accumulation of beta interferon reporter gene mRNA by a posttranscriptional mechanism. To study the mechanism of a novel herpes simplex virus (HSV) activity that stimulates expression of reporter genes containing beta interferon (IFN-beta)-coding sequences, we have established permanent DNA-transfected cell lines that each contain two distinct hybrid genes encoding mRNA species with different half-lives. These reporter genes comprised either the human IFN-beta- or bacterial chloramphenicol acetyltransferase (CAT)-coding and 3' untranslated regions placed under the transcriptional control of the powerful major immediate-early promoter-enhancer region (IE94) from simian cytomegalovirus. Most of the dual-transfected cell lines yielded significant levels of steady-state IE94-CAT mRNA and abundant constitutive synthesis of CAT enzyme activity, whereas no accumulation of IE94-IFN mRNA could be detected. However, infection with HSV type 1 resulted in a 300-fold increase in IE94-IFN-specific mRNA transcripts, compared with no more than 3- to 5-fold stimulation of IE94-CAT-specific mRNA. In contrast, cycloheximide treatment increased stable mRNA levels and transcription initiation rates from both the IE94-IFN and IE94-CAT hybrid genes. Run-on transcription assays in isolated nuclei suggested that induction of IE94-IFN gene expression by HSV type 1 occurred predominantly at the posttranscriptional level. Enhancement of the unstable IFN mRNA species after HSV infection was also observed in cell lines containing a simian virus 40 enhancer-driven IFN gene (SV2-IFN). Similarly, in transient-transfection assays, both SV2-IFN and IE94-IFN gave only low basal mRNA synthesis, but superinfection with HSV again led to high-level accumulation of IFN mRNA. Finally, substitution of the SV2-IFN gene 3' region with poly(A) and splicing signals from the SV2-CAT gene cassette led to stabilization of the IFN mRNA even in the absence of HSV. Therefore, we conclude that HSV infection leads to selective accumulation of IFN-beta mRNA by a posttranscriptional mechanism that is reporter gene specific and promoter independent.


Species-specific phosphorylation of mouse and rat p53 in simian virus 40-transformed cells. We have analyzed in detail the phosphorylation of p53 from normal (3T3) and simian virus 40 (SV40)-transformed (SV3T3) BALB/c mouse cells and from normal (F111) and SV40-transformed [FR(wt648)] rat cells by two-dimensional tryptic peptide mapping and phosphoamino acid analyses. To accommodate the different half-lives of p53 in normal (half-life, 15 min) and transformed (half-life, 20 h) cells and possible differences in the rates of turnover of phosphate at specific sites, cells were labeled for 2 h (short-term labeling) or 18 h (long-term labeling). Depending on the labeling conditions, either close similarities or marked differences were observed in the phosphorylation patterns of p53 from normal and transformed cells. After the 2-h labeling, the phosphorylation patterns of p53 from normal and transformed mouse cells were quite similar. In contrast, p53 from normal and transformed rat cells exhibited dramatic quantitative and qualitative differences under these labeling conditions. The reverse was found after an 18-h label leading to steady-state phosphorylation of p53 in transformed cells: while p53 in transformed mouse cells revealed a marked quantitative increase in phosphorylation compared with p53 from normal cells, the corresponding patterns of p53 from normal and transformed rat cells were similar. Our data thus indicate species-specific differences in the phosphorylation of mouse and rat p53 in SV40-transformed cells, reflected by (i) different turnover rates at specific sites in mouse and rat p53 and (ii) phosphorylation of nonhomologous serine and threonine residues in rat p53, as revealed by indirect assignment of phosphorylation sites to the phosphopeptides of rat p53. Analyses of p53 from the SV40 tsA58 mutant-transformed F111 cell lines FR(tsA58)A (N type) and FR(tsA58)57 (A type) yielded no conclusive evidence for a direct correlation between phosphorylation of p53, the metabolic stabilization of p53, and expression of the transformed phenotype.


Complementation studies with Rous sarcoma virus gag and gag-pol polyprotein mutants. Avian retroviruses (with the notable exception of spleen necrosis virus) express their protease (PR) both in their gag and their gag-pol polyprotein precursors, in contrast to other retroviruses, notably, the mammalian retroviruses, in which PR is encoded in the gag-pol polyprotein or in a separate reading frame as a gag-pro product. The consequence is that the avian PR is expressed in stoichiometric rather than catalytic amounts. To investigate the significance of the particular genome organization of the avian retrovirus prototype Rous sarcoma virus, we developed an assay that measures complementation between the gag and the gag-pol polyproteins by expressing them from two different plasmids in transfected cells. By using this assay, we showed that the protease PR from the gag-pol polyprotein is capable of autocatalytic self-cleavage and -activation when coexpressed with a protease-deficient gag protein and that the PR domain has a role in viral particle assembly. Furthermore, this complementation assay can be used to investigate the role of the gag domain in the gag-pol polyprotein by determining whether it can rescue a defect in the gag polyprotein. We report here the results of such an experiment, which studied a mutation in the N terminus of the gag gene.


Genomic variation and segregation of equine infectious anemia virus during acute infection. Equine infectious anemia virus (EIAV) is a lentivirus that infects and persists in the monocyte/macrophage populations of blood and tissues. We employed polymerase chain reaction to investigate the distribution and the level of genome variability of EIAV DNA in different tissues of a horse infected with a highly virulent variant of the Wyoming strain of the virus. Long terminal repeat, gag, and pol primer pairs were used to direct the amplification of EIAV DNA from the peripheral blood mononuclear cells and from cells, presumably the macrophage subtypes, of the kidney, spleen, liver, lymph node, and cerebellum and periventricular regions of the brain. Polymerase chain reaction analysis of four domains within the envelope showed that viral subtypes with particular envelope domains segregated to different tissues as defined by the presence or absence of a given type of domain. Collectively, these results show that virus variants in the env gene home to different tissues, presumably because of selection for tissue-specific envelope determinants.


Glycoprotein H of pseudorabies virus is essential for entry and cell-to-cell spread of the virus. To study the function of the envelope glycoprotein gH of pseudorabies virus, a gH null mutant was constructed. A premature translation termination codon was introduced in the gH gene by linker insertion mutagenesis, and a mutant virus was rescued by using a cell line that expresses the wild-type protein. Mutant virus isolated from complementing cells was unable to form plaques on noncomplementing cells, indicating that gH is essential in the life cycle of the virus. Immunological staining and electron microscopy showed that the mutant virus produced noninfectious progeny and was unable to spread from infected to uninfected cells by cell-cell fusion. Thus, similar to gH of herpes simplex virus, gH of pseudorabies virus is required for entry and cell-to-cell spread.


Characterization of regulatory functions of the varicella-zoster virus gene 63-encoded protein. Varicella-zoster virus (VZV) gene 63 encodes a protein (IE63) with a predicted molecular mass of 30.5 kDa which has amino acid similarities to the immediate-early (IE) protein 22 (ICP22) of herpes simplex virus type 1. ICP22 is a polypeptide synthesized in herpes simplex virus type 1-infected cells, and as is the case for its VZV counterpart, its regulatory functions are unknown. On the basis of the VZV DNA sequence, it has been shown that IE63 exhibits hydrophilic and acidic properties, suggesting that this protein could play a regulatory role during the infectious cycle. We report in this article cotransfection experiments which demonstrate that the VZV gene 63 protein strongly represses, in a dose-dependent manner, the expression of VZV gene 62. On the other hand, transient expression of the VZV gene 63 protein can promote activation of the thymidine kinase gene but cannot affect the expression of the genes encoding glycoproteins I and II. The results of transient expression experiments strongly suggest that the VZV gene 63 protein could play a pivotal role in the repression of IE gene expression as well as in the activation of early gene expression.


Efficient production of human immunodeficiency virus proteins in transgenic mice. Transgenic mice containing the complete human immunodeficiency virus (HIV) coding sequences fused to the mouse mammary tumor virus long terminal repeat were generated. They were found to produce high levels of authentic gag and env HIV proteins in several tissues known to support mouse mammary tumor virus-driven transcription. HIV proteins were also detected in serum and in body fluids (milk and epididymal secretions) known to be natural sites of retrovirus, and specifically of HIV, production. These results indicate that primary mouse cells from different tissues have the capacity to produce HIV proteins. These mice represent a novel animal model for HIV infection.


Predominant use of a T-cell receptor V beta gene family in simian immunodeficiency virus Gag-specific cytotoxic T lymphocytes in a rhesus monkey. To explore the structural basis for AIDS virus recognition by CD8+ lymphocytes, we sought to determine whether there is a diverse or restricted usage of T-cell receptors (TCR) by simian immunodeficiency virus of macaques (SIVmac) Gag-specific cytotoxic T lymphocytes (CTL) in the rhesus monkey. Six Gag-specific CTL clones were independently generated from an SIVmac-infected rhesus monkey. All six CTL clones recognized a single SIVmac Gag peptide in association with a single major histocompatibility complex class I gene product, Mamu-A*01. TCR alpha-chain sequences from these six CTL clones employed four different V alpha families and five different J alpha gene segments. In contrast, five of the six CTL clones expressed V beta genes that were members of the same family, a human V beta 23 homolog. Furthermore, only one J beta gene was expressed by four of the six CTL clones. These results indicate that TCR of SIVmac Gag-specific CTL from a rhesus monkey can exhibit a restricted usage of V beta gene families and J beta genes.


New member of the multigene family of complement control proteins in herpesvirus saimiri. A number of glycoproteins are regulators of the complement cascade and prevent damage to cells by inappropriate activation of complement. In humans, all of them are encoded by a multigene family on chromosome I and share a characteristic structural feature, the short consensus repeats of about 61 amino acids with a constant framework of cysteine, proline, and tryptophan. We found the gene for glycoproteins of analogous structure in herpesvirus saimiri, a T-lymphotropic tumor virus of New World primates. Unspliced transcripts code for a membrane-bound 65- to 75-kDa virion surface component, while spliced mRNA instructs a secreted glycoprotein of 47 to 53 kDa. Expression of complement control proteins suggests a novel mechanism of counteracting host immune defense to prevent elimination of a virus that is capable of persisting in circulating lymphocytes.


Mechanism of action of the papillomavirus E2 repressor: repression in the absence of DNA binding. Repression of papillomavirus E2-dependent gene expression was studied by using transient transfections into mouse embryo fibroblast cells. Cotransfection of a gene corresponding to the naturally occurring repressor E2-TR along with the full-length E2 gene resulted in up to 98% repression of E2-dependent reporter gene expression. A series of E2 DNA-binding domain mutants were transferred into the E2-TR form and characterized for their ability to repress E2-dependent transactivation. All mutants which were defective for DNA binding but were dimerization competent repressed E2 transactivation as well or nearly as well as the wild-type repressor. E2 mutants which lacked dimerization activity repressed transactivation poorly or not at all. These results indicate that the E2 repressor can inhibit transcription, in the absence of DNA binding, by forming heterodimers with full-length E2.


The Bel-1 protein of human foamy virus activates human immunodeficiency virus type 1 gene expression via a novel DNA target site. The Bel-1 protein of human foamy virus can activate transcription directed by the long terminal repeat (LTR) promoter of human immunodeficiency virus type 1 (HIV-1). The target sequence for Bel-1 is shown to lie within the HIV-1 LTR U3 region but does not coincide with any previously described factor-binding site. Gene expression directed by an HIV-1 LTR lacking functional sites for the inducible cellular transcription factor NF-kappa B was activated over 100-fold by coexpression of Bel-1. These observations suggest that Bel-1 has the potential to significantly enhance the level of HIV-1 gene expression in cells dually infected with HIV-1 and human foamy virus.


Protection of macaques against infection with simian type D retrovirus (SRV-1) by immunization with recombinant vaccinia virus expressing the envelope glycoproteins of either SRV-1 or Mason-Pfizer monkey virus (SRV-3). Rhesus macaques were immunized with live vaccinia virus recombinants expressing the envelope glycoproteins (gp70 and gp22) of simian type D retrovirus (SRV), serotype 1 or 3. All of the animals immunized with either the SRV-1 env or the SRV-3 env vaccinia virus recombinant developed neutralizing antibodies against the homologous SRV. In addition, both groups developed cross-reactive antibodies and were protected against an intravenous live-virus challenge with SRV-1. The four control animals immunized with a vaccinia virus recombinant expressing the G protein of respiratory syncytial virus were not protected against the same SRV-1 challenge. Although SRV-1 and SRV-3 immune sera showed cross-neutralization, they failed to neutralize a separate, more distantly related serotype, SRV-2, in an in vitro assay. These findings are consistent with the known degree of serologic and genetic relatedness of these three SRV strains.


[Relationship between the symptomatic cytomegalovirus infection and the fluctuation of anti-CMV antibodies in patient after renal transplantation]. Between Sept.1, 1989 and Feb.28, 1990, 19 patients received renal allografts, and all of them were examined by serological tests for CMV infection before and after the transplantation. Six patients who had some clinical symptoms such as fever and cough were monitored for anti-CMV antibodies by the ELISA method during the first 3 months after the transplantation. Five of the 6 patients complained of mild clinical symptoms but their condition was not serious. The other patients, a 42-year-old man developed serious CMV infection. His serological CMV antibody titer had decreased one month after the transplantation compared to that before the transplantation. By contrast, the other patients showed either elevation or no change of CMV antibody titer. Therefore, special attention should be paid to the case of decrease rather than increase in CMV antibody titer one month after the transplantation in patients with severe symptoms.


[Evaluation of 99mTc-PMT delayed imaging in diagnosis of hepatocellular carcinoma]. 99mTc-PMT delayed imaging was performed on 199 patients with hepatocellular carcinoma (HCC), and 72 patients with various hepatic diseases, from which HCC should be differentiated. Of the 199 patients with HCC, 128 (64.3%) showed positive results on 99mTc-PMT images. Of these 128, 102 (51.3%) showed increased uptake of radioactivity by the hepatic tumor as compared with the surrounding non-tumorous area of the liver, and 26 (13.1%) showed equal uptake. On the contrary, only 2 (9.1%) of the 22 patients with other malignant hepatic tumors (7 with cholangiocellular carcinoma and 15 with metastatic liver tumors) showed equal uptake of 99mTc-PMT. These findings indicated that 99mTc-PMT delayed imaging was useful for increasing the specificity in diagnosis of HCC. Of the patients with HCC showing increased uptake on 99mTc-PMT images taken 5-hour after the injection of the radioisotope, 26.3%, 69.6%, and 96.0% showed intense 99mTc-PMT uptake by hepatic tumor on 1-hour, 2-hour, and 3-hour images, respectively. These findings indicated that in diagnosing HCC, 5-hour image should be taken only in the patients with a hepatic tumor showing no increased uptake of radioactivity even on 3-hour image. Moreover, the rate of HCC to take up 99mTc-PMT intensely was higher in patients with hepatic tumor showing filling defect on colloid liver image than in those showing no filling defect (p less than 0.001). The points in the assessment of radioactivity uptake by hepatic tumor on 99mTc-PMT delayed image were as follows: Overlapping of radioactivity excreted into the gall bladder or intestine with the radioactivity of the liver tumors, radioactivity retention in the non-neoplastic portion of the liver, and the radioactivity of the dilated intrahepatic bile duct were noted in 6 (2.6%), 15 (6.5%) and 9 (3.9%), respectively, among the 230 patients with focal space occupying lesions. Further, a patient of giant nodular regenerative hyperplasia showed increased uptake of 99mTc-PMT consistent with the hepatic lesion.


[Hepatic blood flow in patients treated by continuous hepatic artery infusion chemotherapy]. Hepatic blood flow in tumor and nontumor regions was studied in four patients with hepatocellular carcinoma and in 16 patients with metastatic liver tumors. The regional hepatic blood flow was measured with the tissue clearance of 133Xe delivered through the implantable drug infusion system. The regional hepatic arterial/portal blood flow ratio was also measured by means of intravenous injection of 99mTc-stannous phytate. Mean hepatic arterial blood flow ratio of tumor regions was higher than that of nontumor regions (81.83 +/- 24.55% vs. 46.64 +/- 20.05%; p less than 0.01). This result suggests that hepatic arterial blood flow is increased in tumor regions of the liver. In tumor regions, the regional blood flow of the hepatic artery was inversely correlated with the mass reduction rate. In the lesions that showed a higher mass reduction rate by the continuous drug infusion treatment, the endothelial damage of the arterial wall and tumor necrosis seem to decrease blood flow. From this study, measurement of regional hepatic blood flow and hepatic arterial/portal blood flow ratio may be useful to evaluate the effectiveness of treatment for hepatic tumors.


Colonization of rabbits by Pasteurella multocida: serum IgG responses following intranasal challenge with serologically distinct isolates. Enzyme-linked immunosorbent assays (ELISAs) and immunoblots were used to measure serum IgG responses in rabbits which were intranasally challenged with Pasteurella multocida. The responses to two serologically distinct isolates (isolate 1, serotype 3:A and isolate 10, serotype 1:D) were compared and then correlated with the ability of the isolates to colonize the nasal passages. Five rabbits were challenged with each isolate (10(5) CFU); nasal washings and sera were collected weekly for 8 weeks. Serum IgG levels were measured by ELISA and immunoblots, using bacterial whole cells and lipopolysaccharides (LPSs) as antigens. The serum IgG response to isolate 1 was evident earlier and was significantly stronger than the response to isolate 10 (P less than 0.025). Immunoblots supported this observation and confirmed that both isolates elicited antibodies which reacted with bacterial protein and LPS antigens, with antibody to protein detectable before antibody to LPS. Results of weekly nasal cultures suggested that the antibody response data could be explained by a difference in the ability of the isolates to colonize the nasal passages: isolate 1 was recovered from four of five rabbits for 8 weeks, whereas isolate 10 was recovered for a maximum of 2 weeks, even when the challenge dose was increased tenfold. The strong response elicited by isolate 1 was therefore probably a result of persistent colonization, whereas the weak response to isolate 10 may have resulted from an inability to persistently colonize the nasal passages. The results of this study demonstrate that isolates of P. multocida elicit antibody responses of differing intensities and vary in their ability to colonize the nasal passages.(ABSTRACT TRUNCATED AT 250 WORDS)


Preliminary characterization of hereditary cerebellar ataxia in rats. A spontaneous model of Purkinje cell degeneration in rats is described. Breeding data indicate that the condition is hereditary and not sex linked. The breeding colony has remained free of common murine pathogens, including parvovirus. In older rats with pronounced ataxia, the major lesions consisted of greatly reduced numbers or complete absence of Purkinje cells (PCs), particularly in the anterior lobe of the cerebellum. There was a decreased thickness and increased cellular density of the molecular layer and degeneration of the inferior olivary nuclei. Morphometric analysis indicated that the anterior lobes of affected rats were 52% smaller than those of normal rats. In young rats, before severe signs of ataxia had developed, microscopic changes were minimal. The preliminary findings are discussed in relationship to human cerebellar ataxias and mouse models of Purkinje cell degeneration.


A new rat mutant with chronic conjugated hyperbilirubinemia and renal glomerular lesions. A new mutant strain of inbred Sprague Dawley rats with autosomal recessive hyperbilirubinuria, were studied by biochemical, histologic, and ultrastructural methods. The plasma bilirubin concentration in the homozygote was significantly higher than that of the heterozygote, and about 80% of the bilirubin was conjugated. Plasma BSP and ICG clearance were both severely delayed in the homozygote. Plasma BSP elimination kinetics suggested that the pathophysiologic defect was not hepatic uptake or storage but rather in secretion into bile. Histopathology of the liver demonstrated brown pigment in the hepatocytes that appeared to be lipofuscin. The electron microscopic features of the hepatic pigment resembled those of the Dubin-Johnson syndrome. Homozygote histopathology also revealed glomerular lesions with mesangial expansion and proliferation in the kidneys. Immunohistologic studies disclosed mesangial granular deposition of IgG, IgA, and to a lesser degree, IgM and C3. These renal changes resembled those of IgA nephropathy. The spontaneous hyperbilirubinuric rat (EHBR) may be a useful animal model for studying constitutive conjugated hyperbilirubinemia, bilirubin metabolism, cholestasis, and glomerulonephropathy subsequent to hepatic dysfunction.


Fasting hyperbilirubinemia in normal squirrel monkeys. The plasma of Bolivian squirrel monkeys, unlike that of Brazilian squirrel monkeys, is markedly yellow due to unconjugated hyperbilirubinemia after an overnight fast. The fasting hyperbilirubinemia in Bolivian squirrel monkeys is likely due to two mechanisms. First, a twofold increase in the bilirubin turnover/production rate occurs during a 24-hour fast. A second mechanism is the decreased hepatic conjugation potential for bilirubin due to the presence of a higher bilirubin UDP-glucuronosyltransferase UDPGAKm and a lower Vm; this results in higher steady-state plasma and hepatic bilirubin levels during a fast when hepatic UDP-glucuronic acid levels are low. The Bolivian squirrel monkey provides an excellent animal model for human Gilbert's syndrome type I in which to study rate-limiting mechanisms in the movement of bilirubin from plasma to bile.


Evaluation of the preference to and behavioral effects of an enriched environment on male rhesus monkeys. Two environments were provided to laboratory rhesus monkeys to determine if the animals spent more time (for the purposes of this study, defined as the cage side preference) in an enriched cage side than an unenriched cage side. The side (right or left) of a double-wide cage in which the animal spent the most time (as determined by Chi square analysis) was initially determined during baseline observations. The "nonpreferred" side was then enriched during the experimental phase of the study. The enrichment consisted of a perch, a Tug-A-Toy suspended inside the cage, a Kong toy suspended on the outside of the cage, and a grooming board mounted on the outside of the cage. No statistically significant changes in use of the enrichments were detected over time. Fifty percent of the animals switched cage side preference to the enriched side during the study. All subjects showed reduced behavioral pathology during exposure to the enriched environment with a return of behavioral pathology when the enrichments were removed.


Physiologic and immune responses associated with 48-hour fast of pigs. We sought to determine the effect of 48 hours of fasting on physiologic and immune responses of pigs. Sixteen crossbred barrows, approximately 8 weeks of age, were housed in a controlled environment (21 degrees C, 45% RH) with feed and water ad libitum. After 10 days, eight pigs were fasted for 48 hours with water available ad libitum; the remaining eight received feed and water ad libitum. Blood samples were obtained by venipuncture before fasting, at the end of 48-hour fasting, and 3 and 10 days later. No significant differences in responses to mitogens PHA or Con A were noted in whole blood or isolated lymphocyte cultures. Changes in numbers of leukocytes, neutrophils, and serum cortisol concentrations, but not of lymphocytes, were significant. The results suggested that short-term fasting transiently reduces the number of neutrophils and increases serum cortisol concentrations, with no effect on blastogenic responses of lymphocytes to selected mitogens.


Effects of yohimbine as a reversing agent for ketamine-xylazine anesthesia in budgerigars. Fourteen adult budgerigars (Melopsittacus undulatus) were anesthetized with a combination of ketamine hydrochloride (40 mg/kg) and xylazine hydrochloride (10 mg/kg) intramuscularly. Forty-five minutes after ketamine-xylazine injection, one of four yohimbine hydrochloride doses (0.0, 0.11, 0.275, or 0.44 mg/kg, IM) was administered in a 0.7% saline vehicle. Latencies were recorded in minutes from yohimbine injection until subjects' behavior indicated three different points of recovery: 1) lifting the head, 2) standing unaided without ataxia, and 3) perching. Means for all three recovery point latencies were significantly reduced by 0.275 mg/kg of yohimbine compared with saline vehicle alone. Mean latencies among treatment groups for each of the three recovery points were not significantly different, other than control versus treated groups. Based on these results, we recommend a yohimbine dose of 0.275 mg/kg as an effective reversing agent for ketamine-xylazine anesthesia in budgerigars.


Ketamine/xylazine/butorphanol: a new anesthetic combination for rabbits. Ketamine is often used in combination with tranquilizers to produce surgical anesthesia in rabbits. While generally effective, there is considerable variation in the depth and duration of anesthesia achieved with ketamine combinations. Butorphanol is a mixed agonist-antagonist opioid that is widely used in a variety of other species. In this study, the commonly used ketamine (35 mg/kg)/xylazine (5 mg/kg) combination is compared with ketamine (35 mg/kg)/xylazine (5 mg/kg)/butorphanol (0.1 mg/kg). Rabbits were anesthetized on consecutive weeks with one of the two regimens. Physiologic parameters including heart rate, respiratory rate, blood pressure and arterial blood gases (pH, PO2, PCO2) were measured throughout anesthesia. Loss of palpebral, pedal and righting reflexes were recorded and reflexes were subsequently evaluated. The addition of butorphanol prolonged reflex loss to 140% (X = 68 min +/- 20 SEM) of control for palpebral reflex; 506% (X = 52 min +/- 18 SEM) of control for pedal reflex; and 159% (X = 128 min +/- 21 SEM) of control for righting reflex. Addition of butorphanol to ketamine/xylazine resulted in mild alterations in the physiologic changes traditionally associated with this combination. Butorphanol can be safely added to the ketamine/xylazine combination in rabbits and results in moderate increases in the duration of reflex loss.


Chamber and gavage technique for metabolic studies of earthworms. Earthworms make very suitable laboratory animals for metabolic studies in vivo using radiolabeled test chemicals. We describe the construction and operation of a metabolic chamber to enable the collection of labeled CO2, volatile organics, material excreted into the bedding, and labeled material remaining in the worms. A gavage technique has been developed that permits the administration of water-soluble and lipid-soluble test chemicals in spite of the extremely low level of triglyceride lipase activity in the earthworm gut. This technique is less likely to puncture the worm tissue than previous methods. Radiolabeled DDT and diethylhexyl adipate were used to provide examples of the use of these techniques and the metabolic chamber. Results were qualitatively similar to those that have been noted in vertebrates.


Use of ameroid constrictors in the development of renin-dependent hypertension in dogs. We describe methods for using ameroid constrictors to develop renin-dependent hypertension in dogs. A vascular-access-port was implanted in each dog and mean arterial blood pressure was monitored. Via a small flank incision, an ameroid constrictor was placed on the left renal artery and mean arterial blood pressure, plasma renin activity, and the response to angiotensin-converting enzyme inhibition measured during the subsequent 4 weeks. Placement of the constrictor on the left renal artery resulted in a significant increase in plasma renin activity which was accompanied by a significant hypertension (blood pressure greater than 140 mmHg). The greatest increase in plasma renin activity and blood pressure occurred at week two which coincided with the greatest response to angiotensin-converting enzyme inhibition, when mean arterial blood pressure decreased more than 30 mmHg after administration of either captopril or enalapril (1 mg/kg, i.v.). The results of this study indicate that ameroid constrictors provide a reliable method for establishing renin-dependent hypertension in dogs.


Morphologic analysis of enteric lesions in conventional and streptomycin-treated inbred C3H/HeN mice infected with Serpulina (Treponema) hyodysenteriae. Oral administration of streptomycin is known to enhance the susceptibility of mice to enteric pathogens by altering the indigenous flora. We examined the effect of oral streptomycin treatment on the susceptibility of inbred C3H/HeN mice to infection with Serpulina (Treponema) hyodysenteriae. A total of 56 mice were randomly divided into four groups (A-D) of 14 each. From days 0 to 7, mice in groups A and B received streptomycin in their drinking water and mice in groups C and D served as controls. On day 7, mice in groups A and C were inoculated intragastrically with S. hyodysenteriae serotype 4, strain A1, and groups B and D served as uninoculated controls and received sterile trypticase soy broth. Clinical signs were monitored daily and body weights were recorded weekly. Mice were euthanized and necropsied for bacteriologic and histopathologic examinations on day 7 (2/group) and on days 14, 21, 28, and 35 (3/group) of the experiment. Soft fecal pellets were noticed in infected groups (A and C), but no significant differences in body weights were observed between groups (P greater than 0.05). Macroscopic changes were noted only in infected groups (A and C) beginning on day 21 of the experiment and consisted of catarrhal typhlitis, cecal emptiness, and atrophy. Histologically, the cecum and colon of mice in groups A and C had goblet cell hyperplasia, which preceded crypt epithelial cell hyperplasia, inflammatory cell infiltrates, and focal necrosis of mucosal epithelium. S. hyodysenteriae was reisolated from 10 of 12 mice in each infected group (A and C) from day 14 (7th day postinoculation) through day 35 (28th day postinoculation).(ABSTRACT TRUNCATED AT 250 WORDS)


Chromium-51 labeling of sheep red blood cells. The failure of sheep red blood cells (RBCs) labeled with Chromium-51 (Cr-51) using the ascorbic acid technique to act as a suitable intravascular marker of blood volume in a septic sheep model prompted us to investigate the technique of radiolabeling sheep erythrocytes with this isotope. Consequently, we studied thirteen sheep in which the labeling efficiency of Cr-51 as sodium chromate and hemoglobin typing was determined for each animal. Mean Cr-51 labeling efficiency of sheep RBCs was 67.5% (n = 13). Although 5 of the 13 sheep were discovered to have two types of hemoglobin (Hb) as determined by electrophoresis, overall labeling efficiency of sheep RBCs was determined to be independent of the type of hemoglobin present. However, when two types of Hb were present (Hb-A and Hb-B), Cr-51 had a higher affinity for Hb-B (80%) than Hb-A (20%) even though both Hb types are present in similar proportions (Hb-A = 53%, Hb-B = 46%). The results of this study indicate that sheep RBCs express a lower labeling efficiency for Cr-51 than do human RBCs and that Cr-51 has a higher affinity for Hb-B than for Hb-A when both hemoglobin types are present. This difference is noteworthy when interpreting Cr-51 RBC data in experimental sheep models. Furthermore, caution should be exercised when extrapolating established human protocols to animal models.


A new method for obtaining electrocardiograms in unrestrained crocodilian reptiles. A new procedure is described for acquiring measurements of electrocardiographic parameters in unrestrained crocodilians. These measurements are difficult to obtain in freely moving animals; hence, electrocardiographic activity under natural conditions has not been previously quantified. In this investigation, twelve American alligators were equipped with subcutaneous electrodes. The lead wires were sutured to each animal's skin and the extracutaneous wires coiled and held in place against the animals' dorsal surfaces with waterproof elastic bandages. The electrodes were connected to an ECG analyzer only at the time of measurement. The presence of the leads and harness did not appear to interfere with the movements of the animals either in the animal room or during testing. This method allows for more precise measurements of cardiac activity under conditions which closely resemble those of crocodilians in their natural state.


Prenatal diagnosis of systemic disorders of the respiratory chain in cultured amniocytes and chorionic villus fibroblasts by studying the formation of lactate and pyruvate from glucose. Formation of lactate and pyruvate from glucose was studied in cultured amniocytes and chorionic villus fibroblasts from controls, either untreated or treated with azide, an inhibitor of cytochrome c oxidase, or other inhibitors of the mitochondrial respiratory chain. Amniocytes with an established cytochrome c oxidase deficiency were also investigated. Control amniocytes treated with azide as well as cytochrome c oxidase deficient amniocytes displayed strongly increased lactate-to-pyruvate ratios after incubation with glucose, compared to control cells. Elevated lactate-to-pyruvate ratios were also found in chorionic villus fibroblasts in which complexes I, III or IV were inhibited by rotenone, antimycin or azide, respectively. We conclude that measurement of lactate and pyruvate production from glucose in cultured amniocytes and/or chorionic villus fibroblasts allows adequate prenatal diagnosis of systemic cytochrome c oxidase deficiency and presumably of other systemic deficiencies of mitochondrial respiratory chain enzymes.


Pretranslational regulation of extracellular matrix macromolecules and collagenase expression in fibroblasts by mechanical forces. In vivo, the extracellular matrix modulates the phenotype of the connective tissue cells both through its biochemical composition and the transfer of mechanical information. In this study, the mechanical effect was investigated in collagen gels populated by skin fibroblasts maintained under tension (bound lattices (BL)) compared with free retracting lattices (FL) and monolayer on plastic. The overall proteins and collagen synthesis of human skin fibroblasts, investigated by isotopic labeling, were decreased respectively by a factor of about 20 and 40 in FL compared with monolayers and increased by a factor of 4 and 6 in BL versus FL. As assayed by the degradation of [3H]collagen type I by trypsin-activated medium conditioned by fibroblasts under the three models of culture, collagenase activity was inversely regulated and increased in lattices when compared with monolayer culture. It was four times higher in FL than in BL. The steady-state level of mRNA coding for procollagen types I, III, and VI polypeptides, fibronectin, elastin, beta-actin, and procollagenase was determined by cDNA hybridization. The mRNA coding for beta-actin as well as for the various extracellular matrix macromolecules were increased in BL when compared with FL while the level of procollagenase mRNA was lower. These data demonstrate the existence of a modulation of the function of the fibroblasts performed by mechanical forces. This regulation operates, at least in part, at a pretranslational level.


[Lumbosacral polyradiculomyelitis caused by cytomegalovirus in a patient with acquired immunodeficiency syndrome]. The case of a HIV positive patient with lumbosacral polyradiculomyelitis by cytomegalovirus (PLS-CMV) is presented. The patient was a homosexual male receiving maintainance treatment with foscarnet for previous corioretinitis by CMV who consulted for paraparesia and sphincter disorders of a 3 week evolution. Neutrophilic pleocytosis and high levels of glucose and proteins were observed on LCR and CMV isolated discarding other entities. The LCR normalized upon treatment with gancyclovir although serious residual paraparesia persisted. PLS-CMV is an infrequent entity of typical clinic and liquoral characteristics. Recognition of the same is important since favorable response depends on early anti-CMV treatment.


[Resorbable antibiotic delivery systems in local treatment of chronic osteitis--polyglycolic acid/poly-L-lactide as drug carrier. Experimental studies in vitro]. Resorbable polyglycolic acid (PGA)- and poly-L-lactic acid (PLLA) cylinders were investigated in vitro to explore their properties as an antibiotic deposit (Ciprobay, Bayer Leverkusen) with prolonged release. PGA cylinders sized 3.2 x 5 mm, 4.5 x 5 mm and 4.5 x 7 mm respectively were shaped in monofil and polyfil technique. The Ciprofloxacin concentration varied from 0.5 mg to 5.0 mg of each cylinder. The cylinders were eluated in phosphate buffer, pH-value of 7.4, at 37 degrees C. The daily antibiotic release was measured by high performance liquid chromatography. Best combinations we could find demonstrated an initial delivery of Ciprofloxacin in vitro of 67 mg/l. The average daily release was about 16 mg/l during the first 36 days. After complete hydrolysis of the PGA carriers the recovery of Ciprofloxacin reached up to 6.5% and 11.6% respectively. For 40 bioactive cylinders (size phi 3.5 mm x 5 mm, containing 4 mg Ciprofloxacin) were eluated with phosphate buffer (pH 7.4 at 37 degrees C) respectively fresh human blood plasma and tested under various conditions. A gentamicin-polymethylmetacrylate (PMMA) chain (Septopal, E. Merck, Darmstadt) was exposed to equal test conditions for comparison. The quantities of released Ciprofloxacin and Gentamicin were analysed by a microbiological method (bioassay). Initially released rates of Ciprofloxacin were measured very high (up to 180 mg/l) but decreased rapidly within the first 5 days (4.2-22.5 mg/l). The release of Gentamicin produces an initial sharp decrease in concentration during the first 3 days (from 227.5 mg/l to 77.5 mg/l); this is then followed by an almost constant release over a long period of time (about 20 mg/l).(ABSTRACT TRUNCATED AT 250 WORDS)


Acute and late disease induced by murine coronavirus, strain JHM, in a series of recombinant inbred strains between BALB/cHeA and STS/A mice. To examine the genetic control of acute and late disease induced by a murine coronavirus, strain JHM (JHMV), BALB/cHeA, STS/A, F1 hybrids and 13 recombinant inbred (RI) strains between BALB/cHeA and STS/A mouse strains were inoculated intracerebrally with 100 pfu of JHMV. All the BALB/cHeA mice died within 2 weeks from acute encephalitis. In contrast, STS/A mice were shown to be partially resistant, with a mortality rate of 30%, longer survival times and lower rates of viral production. The mortality rates, survival times and viral titers of F1 hybrids and the RI strains varied, suggesting involvement of multiple genes. STS/A, F1 hybrid and RI mice surviving the acute infection occasionally developed severe paraparesis about 1 month post-infection. In these mice, vacuolar degeneration, astrocytosis, the absence of perivascular cuffing and minimal demyelination were found in the central nervous system. No infectious virus could be recovered from these mice. Although the paralysis of delayed onset was limited to STS/A, F1 hybrid and eight of the 13 RI strains, the incidence varied significantly among the RI strains. These results may suggest that JHMV-induced late disease is also under multifactorial control. The pathogenesis of JHMV infection is discussed.


Population growth makes waves in the distribution of pairwise genetic differences. Episodes of population growth and decline leave characteristic signatures in the distribution of nucleotide (or restriction) site differences between pairs of individuals. These signatures appear in histograms showing the relative frequencies of pairs of individuals who differ by i sites, where i = 0, 1, .... In this distribution an episode of growth generates a wave that travels to the right, traversing 1 unit of the horizontal axis in each 1/2u generations, where u is the mutation rate. The smaller the initial population, the steeper will be the leading face of the wave. The larger the increase in population size, the smaller will be the distribution's vertical intercept. The implications of continued exponential growth are indistinguishable from those of a sudden burst of population growth Bottlenecks in population size also generate waves similar to those produced by a sudden expansion, but with elevated uppertail probabilities. Reductions in population size initially generate L-shaped distributions with high probability of identity, but these converge rapidly to a new equilibrium. In equilibrium populations the theoretical curves are free of waves. However, computer simulations of such populations generate empirical distributions with many peaks and little resemblance to the theory. On the other hand, agreement is better in the transient (nonequilibrium) case, where simulated empirical distributions typically exhibit waves very similar to those predicted by theory. Thus, waves in empirical distributions may be rich in information about the history of population dynamics.


Detergent-shock response in enteric bacteria. Our work on bacterial detergent resistance started with the realization that bacteria growing in a sink full of soap must be resistant to the detergents in that soap. We chose sodium dodecyl sulphate (SDS) as a model detergent and decided to see how much SDS the bacterium growing in the sink could tolerate. The research program thus initiated has shown that bacteria such as Enterobacter cloacae can grow in up to 25% SDS and that SDS-shock proteins constitute c. 8% of the proteins synthesized by SDS-grown Escherichia coli. It has also provided explanations why enteric bacteria are oxidase negative, and how pyrroloquinoline quinone (PQQ) enters the periplasmic space. Finally, for E. coli, it has provided evidence for an alternate, phosphate-limited, aquatic life style which places greater emphasis on the Entner-Doudoroff pathway. Detergent resistance is important both medically and ecologically, e.g. entry of pathogens via bile-salt-containing intestinal tracts and biodegradation of detergent-like pollutants such as those resulting from oil spills. Our current research is focused on SDS-induced modifications of the cytoplasmic membrane and the presence of SDS in the periplasm.


Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step. When the genes coding for the outer membrane (OM) proteins OmpA and OmpF of Escherichia coli are fused to a signal sequence of a bacillar exoenzyme and expressed in Bacillus subtilis they remain cell-bound and the signal sequence is not cleaved. To identify the step of arrest in the export of these proteins we studied their accessibility to protease applied to intact protoplasts; they remained resistant indicating fully intracellular localization. Both proteins appeared associated with the cell membranes in sedimentation and flotation centrifugation experiments. However, OmpA and OmpF proteins synthesized in B. subtilis without a signal sequence were similarly associated with membranes in centrifugation experiments whereas electron microscopy showed the presence of intracytoplasmic inclusion bodies not obviously attached to the cytoplasmic membrane. We conclude that OmpA and OmpF proteins even when provided with a functional signal sequence do not enter the export pathway in B. subtilis, probably owing to lack of a specific export component in B. subtilis.


Geographic/ethnic differences in human herpesvirus-6 antibody patterns. Serum samples from healthy adults in four geographic/ethnic groups (Ghanaian Blacks, Malaysian Chinese, Malaysian Indians and United States Caucasians) were tested under code for antibodies to human herpesvirus-6 (HHV-6). The prevalence and titer of HHV-6 antibody in the healthy Ghanaians were significantly higher than in the Malaysian Chinese; United States Caucasians and Malaysian Indians had intermediate prevalence and titer of antibodies. Thus far, no specific differences in HHV-6-associated diseases have been noted between geographic/ethnic groups with these marked variations in antibody patterns.


Comparison of indirect immunofluorescence (IF) test with enzyme-linked immunosorbent assay (ELISA) in screening of hybridomas to very virulent Marek's disease virus. For identifying virus-specific antigens of Marek's disease virus (MDV), monoclonal antibodies (MAbs) against strain Md5 of serotype 1, which is known to be a very virulent MDV (vvMDV), were isolated. Fifty-eight hybridoma clones that secreted MAbs against vvMDV were obtained. Of these MAbs, 36 gave positive reactions in an immunofluorescence (IF) test, and 22 gave positive reactions on enzyme-linked immunosorbent assay (ELISA). None of these MAbs gave positive reactions in both the IF test and ELISA. Of the MAbs that gave positive reactions in the IF test, 33 clones reacted with MDV1-specific epitopes, the other three reacting with MDV1-HVT intertypic epitopes. None of the clones reacted with MDV1-MDV2 intertypic epitopes. Three virus-specific polypeptides were identified by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or immunoblotting. These polypeptides were recognized by 12 MAbs giving positive reactions by IF, but by none of those giving positive reactions by ELISA. In addition, size heterogeneity of the MDV1-specific phosphorylated polypeptides in the MDV1 strains was shown using the MAbs against Md5.


Search for the origin of multiple sclerosis by first identifying the vector. By intensive study of environmental conditions during the final stages in an active cluster area, an attempt is made to crack the enigmatic deadlock concerning the origin and etiological cause of Multiple Sclerosis (MS). Previous investigations of other cluster areas have emphasized the probability that the outbreaks of MS were most likely caused by an unknown exogenous environmental agent, but unfortunately, the studies were conducted several years after the epidemics had occurred, making it virtually impossible to observe changing environmental conditions. This study attempts to overcome such defects, and in the process identifies not only the probable exogenous viral agent(s), but the here-to-fore unknown avian vectors as well.


AIDS and SLE: a reverse process of the same disease. Acquired immunodeficiency syndrome (AIDS) and systemic lupus erythematosus (SLE) are two diseases with striking similarities as well as contrasting differences. There is evidence that AIDS and SLE have the same or similar viruses as their etiologic agent. Genetic, clinical and immunological studies suggest that the two diseases are the same, with their only difference being the stimulatory effects of the AIDS virus on cortisol production versus the inhibitory effects of the proposed SLE virus on cortisol production. Possible mechanisms of the disease process for both AIDS and SLE are discussed, as well as proposed treatments for AIDS, including the use of procainamide and ascorbic acid.


The 5'-flanking region of the rat luteinizing hormone/chorionic gonadotropin receptor gene confers Leydig cell expression and negative regulation of gene transcription by 3',5'-cyclic adenosine monophosphate. The LH/CG receptor is a G protein-coupled receptor present on gonadal cells whose levels are modulated by a number of hormones, growth factors, and second messenger analogs. With the recently cloned cDNA for the LH/CG receptor, it has been shown that changes in the levels of the cognate mRNA are involved, at least in part, in the observed changes in receptor density. In order to study the transcriptional regulation of the LH/CG receptor we have isolated a 2-kilobase region of the 5'-flanking region of the rat LH/CG receptor gene and subcloned nucleotide -1 (relative to the translational initiation codon) to -1370 into a luciferase reporter plasmid. We show here that this region of the LH/CG receptor gene is able to enhance luciferase activity in MA-10 cells, a line of Leydig tumor cells that normally express LH/CG receptors, as opposed to human kidney 293 cells, which do not. Furthermore, the addition of 8-bromo-cAMP to MA-10 cells, under conditions known to decrease LH/CG receptor numbers and receptor mRNA levels, decreases the relative luciferase activity to about 26% of control. This decrease in reporter gene activity is severely blunted in a subclone of MA-10 cells with a cAMP-resistant phenotype. Our studies show, for the first time, that sequence(s) present with 1370 base pairs of the translational start site of the rat LH/CG receptor gene are sufficient for conferring expression of this gene in Leydig cells and for the negative modulation of LH/CG receptor gene transcription by high concentrations of cAMP.


Effects of truncations of the cytoplasmic tail of the luteinizing hormone/chorionic gonadotropin receptor on receptor-mediated hormone internalization. The LH/CG receptor is a member of the family of G protein-coupled receptors and consists of a large N-terminal extracellular domain (which is responsible for binding hormone) attached to a region that spans the plasma membrane seven times, ending with an intracellularly located C-terminus. Binding of LH or human CG (hCG) to the LH/CG receptor causes a stimulation of adenylyl cyclase, presumably via activation of Gs. The binding of hormone also leads to its subsequent internalization by receptor-mediated endocytosis. In order to investigate the role of the cytoplasmic tail of this receptor in these events, we prepared a series of mutants in which progressively larger portions of the cytoplasmic tail were deleted. Deletion of 58 amino acids from the C-terminus, in which only 11 cytoplasmic residues remain, resulted in a receptor that was not expressed on the plasma membrane. Receptors rat LHR (rLHR)-t653 and rLHR-t631, in which 21 or 43 amino acids were removed, respectively, were properly expressed. These results suggest that a region(s) between residues 616 and 631 of the rLH/CG receptor are required for proper insertion and/or targeting of the receptor into the plasma membrane. Cells expressing rLHR-t653 or rLHR-t631 bound hCG with the same high affinity as cells expressing the full-length receptor, and basal levels of cAMP were the same among the cells. However, cells expressing the truncated receptors responded to hCG with approximately 2-fold greater levels of maximal cAMP accumulation than cells expressing the full-length receptor. Deletion of up to 43 amino acids from the C-terminus of the rLH/CG receptor had no deleterious effect on hCG internalization. In fact, mutants lacking 21 and 43 amino acids exhibited progressively faster rates of hCG internalization as compared to the full-length receptor. Once internalized, hCG was also degraded at a faster rate in cells expressing the truncated LH/CG receptors. Since hCG-stimulated cAMP stimulation and hCG internalization are retained by rLHR-t631, it can be concluded that the residues, not necessarily the same, required for these functions reside within the 26 amino acids of the cytoplasmic tail closest to the seventh transmembrane helix and/or residues within the intracellular loops. Our data show, however, that both hCG-stimulated cAMP production and hCG internalization are enhanced by the removal of the distal portion of the cytoplasmic tail.


Amino acid substitutions in the DNA-binding domain of the human androgen receptor are a frequent cause of receptor-binding positive androgen resistance. In some subjects with genetic and endocrine evidence of androgen resistance, no defect is demonstrable in the binding of androgen to its receptor in cultured genital skin fibroblasts. We have defined the molecular defect in the androgen receptor in four unrelated subjects in this category (termed receptor positive) with the phenotype of compete or incomplete testicular feminization. In these patients we detected amino acid substitutions in either exon 2 or exon 3, which encodes the DNA-binding domain of the androgen receptor. In one patient with incomplete testicular feminization, two separate mutations were present in exon 3. Introduction of these amino acid substitutions into the androgen receptor-coding segment leads to the expression of receptor proteins that bind ligand in a normal fashion but do not activate the transcription of the androgen-responsive mouse mammary tumor virus promoter. Mobility shift assays using androgen receptor fusion proteins produced in E. coli indicate that these mutations impair binding of the receptor to specific DNA sequences. In the subject with incomplete testicular feminization, a Ser-Gly substitution at amino acid residue 595 is able to partially restore DNA-binding activity to a mutant receptor protein that carries an Arg-Pro substitution at position 615. These findings indicate that mutations in amino acid residues crucial to the binding of the androgen receptor to target DNA sequences are a common cause of receptor-binding positive androgen resistance and that variable impairment of DNA binding can lead to distinctive phenotypes.


Half-site spacing and orientation determines whether thyroid hormone and retinoic acid receptors and related factors bind to DNA response elements as monomers, homodimers, or heterodimers. The receptors for thyroid hormone (T3R) and retinoic acid (RAR) are members of a nuclear receptor subfamily that are capable of recognizing similar DNA sequences. Native response elements for T3R and RAR consist of two or more putative half-site binding motifs organized as imperfect direct or inverted repeats separated by different sized nucleotide gaps. To clarify how T3R, RAR, and related factors recognize DNA response elements, we analyzed the interaction of purified receptors with a series of inverted and direct repeats of an idealized AGGTCA half-site separated by different sized nucleotide gaps. Our results indicate that RAR and T3R can bind to half-sites as monomers and, depending on the orientation and distance between half-sites, also bind as homodimers or T3R-RAR heterodimers. T3R also binds to certain DNA elements as a heterodimer with one or more nuclear factors from eucaryotic cells. Thus, the orientation and spacing of half-sites play a central role in determining which configuration of receptors and nuclear factors will interact with a specific DNA element. This along with the ability of these factors to participate in reversible protein-protein interactions serve to broaden and diversify the responses mediated by T3R, RAR, and related members of this nuclear receptor subfamily.


The effect of glucocorticoid on the subcellular localization, oligomerization, and processing of mouse mammary tumor virus envelope protein precursor Pr74. Mouse lymphoma cell line W7MG1 is stably infected with mouse mammary tumor virus and produces the viral envelope glycoprotein precursor Pr74, but the mature envelope proteins gp52 and gp33, which are derived from Pr74 by posttranslational processing, are produced only when the cells are cultured with a glucocorticoid agonist. The current study demonstrated that even when W7MG1 cells are grown with hormone, the conversion of Pr74 to gp52 and gp33 is an inefficient process in this cell line. At least 2 h of exposure to glucocorticoid were required to induce the appearance of gp52 and gp33; furthermore, Pr74 labeled in the absence of hormone was not converted to gp52 and gp33 upon subsequent addition of hormone. RNA synthesis inhibitors blocked the hormonal induction of gp52 and gp33, indicating that the hormone acts by promoting the expression of a new gene(s) required for the production of gp52 and gp33, rather than by inhibiting the expression of a gene(s) that prevents processing of Pr74. Subcellular fractionation studies demonstrated that Pr74 produced in either the presence or absence of hormone was associated primarily with the ER, whereas gp52 and gp33 were found in the Golgi and plasma membrane fractions. The Pr74 molecules from W7MG1 cells grown either with or without glucocorticoid coimmunoprecipitated with BiP/GRP78 and sedimented as aggregates of heterogeneous size. In contrast, Pr74 from virus-producing GR3A mouse mammary tumor cells, which process Pr74 more efficiently, sedimented as apparent monomers, dimers, and trimers.(ABSTRACT TRUNCATED AT 250 WORDS)


Two different genes coding for processable and nonprocessable forms of a viral envelope protein can account for the apparent hormonal stimulation of protein processing in W7MG1 lymphoma cells. In the mouse mammary tumor virus (MMTV)-infected mouse T-lymphoma cell line W7MG1, glucocorticoid hormone regulates two aspects of MMTV gene expression: hormone stimulates MMTV gene transcription and increases the ratio of mature envelope proteins to envelope precursor protein produced. To separate these two effects and determine the mechanism by which hormone regulates the conversion of the envelope precursor Pr74 to the mature cleaved products gp52 and gp33, we constructed expression vectors in which the envelope gene is constitutively transcribed. Surprisingly, the envelope precursor protein Pr74 encoded by two independently isolated, allelic envelope genes behaved differently. Pr74-P (encoded by the ENV/P gene) was processed efficiently to the mature products gp52 and gp33, independently of the level of expression, hormonal induction of cellular genes, or the presence of other MMTV proteins. In contrast, under the same conditions, Pr74-N (encoded by the ENV/N gene) was not processed further despite being relatively stable. In sucrose gradient analyses, Pr74-P sedimented as monomers, whereas Pr74-N was found in high mol wt aggregates of heterogeneous size. Coimmunoprecipitation analysis determined that Pr74-N associated with BiP, whereas Pr74-P did not. This is indicative of improper folding of Pr74-N in the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)


Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary. PC1 and PC2 are enzymes involved in the activation of prohormones via the cleavage of pairs of basic amino acids. The expression levels of each of these enzymes were evaluated in the rat anterior and neurointermediate pituitary lobes by in situ hybridization and Northern gel analysis and after various pharmacological manipulations. All intermediate lobe melanotrophs expressed high levels of PC2 mRNA and lower levels of PC1 mRNA. PC1 mRNA was highly expressed throughout the anterior lobe; however, appreciable PC2 mRNA levels were also found. Based on colocalization studies, anterior lobe corticotrophs were found to express PC1 mRNA, but very little PC2 mRNA. Neurointermediate lobe levels of PC1, PC2, and POMC mRNA increased 2- to 6-fold in rats treated with haloperidol, while they decreased to 10-25% of their control values after bromocriptine treatment. These results indicate that in the intermediate lobe, dopamine is involved in the regulation of PC1 and PC2. In the anterior lobe, haloperidol had a strong effect on PC2 mRNA, increasing its levels by 8- to 12-fold compared to the control value, while PC1 mRNA was unaffected. Both PC1 and PC2 mRNA levels were increased 5- to 9-fold in animals made hypothyroid by treatment with 6-n-propyl-2-thiouracil. Adrenalectomy had no significant effect on anterior lobe PC1 mRNA levels. However, both PC1 and PC2 mRNA levels were responsive to dexamethasone treatment in the AtT-20 cell lines. Our results indicate that dopamine, thyroid hormones, and corticosteroids are involved in PC1 and/or PC2 gene expression. These data are also consistent with the role of PC1 and PC2 as prohormone-processing enzymes.


Identification and characterization of the GC-rich and cyclic adenosine 3',5'-monophosphate (cAMP)-inducible promoter of the type II beta cAMP-dependent protein kinase regulatory subunit gene. A rat genomic clone containing 4.5 kilobases of 5'-flanking DNA and the first exon of the type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase was isolated, restriction mapped, and sequenced. The proximal 400-basepair promoter region was GC rich, lacked TATA/CAAT box motifs, and initiated transcription at multiple sites. Bandshifting and DNase-I footprinting experiments using this region of the RII beta promoter detected several related specific DNA-protein complexes formed using crude and fractionated nuclear extracts from rat ovary, brain, adrenal gland, and liver. All binding in these experiments mapped to a domain within the same region found to confer cAMP inducibility to a chloramphenicol acetyltransferase (CAT) reporter gene when transfected into primary cultures of rat granulosa cells. Although GC boxes (putative SP1-binding sites) and activator protein-2 (AP-2) elements were present in this functional region, and although expression vectors containing AP-2 sites conferred high levels of cAMP regulation of the CAT gene in cultured ovarian cells, neither the GC boxes nor the AP-2 sites were protected by footprint analyses or required for band shift activity of nuclear extract protein. These known regulatory elements, therefore, may be involved in functional activity of the RII beta promoter, but additional cis-acting DNA and trans-acting factors (yet to be characterized) also appear to interact with the functional promoter of the RII beta gene and regulate the hormone-specific expression of the A-kinase subunit in ovarian and neuronal cells.


A recombinant calcitonin receptor independently stimulates 3',5'-cyclic adenosine monophosphate and Ca2+/inositol phosphate signaling pathways. Calcitonin (CT), a polypeptide hormone, regulates calcium homeostasis by activating surface receptors coupled to stimulation of adenylyl cyclase in bone and kidney cells. CT has also been reported to increase cytoplasmic Ca2+ in osteoclasts and renal tubule cells. Signaling pathways activated by a recombinant porcine renal calcitonin receptor transiently expressed in HEK-293 cells were studied. In cells expressing the recombinant CT receptor, salmon CT stimulated cAMP accumulation (EC50, 0.16 nM) and synthesis of inositol phosphates (IP; EC50, 3.7 nM). Two other recombinant receptors, the m1-muscarinic acetylcholine receptor and the LH receptor, activated synthesis of either IP or cAMP, respectively, but not both. Stable expression of the CT receptor in a CT receptor-deficient cell line, M18, restored the cells' ability to increase cytoplasmic Ca2+ in response to salmon CT. These results show that a single recombinant CT receptor can independently activate effector pathways mediated by cAMP and IP/Ca2+.


Characterization of 1,25-dihydroxyvitamin D3 receptor interactions with target sequences in the rat osteocalcin gene. The sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor and mediate its effects on gene transcription contain three copies of a motif homologous to those found in other steroid response elements. To evaluate the relative importance of these motifs and their flanking sequences, functional properties and receptor binding of a series of mutant response elements were analyzed. Mutations in the third motif, including its deletion, have no effect on receptor binding and only modestly detrimental effects on the ability of the upstream motifs to confer 1,25-(OH)2D3 responsiveness in transfected cells. Mutations in the first two motifs have negative effects on both receptor binding and gene activation. Mutagenesis of bases upstream from each of the first two motifs alters receptor binding and gene activation; these bases are, therefore, an integral part of the rat osteocalcin 1,25-(OH)2D3 response element. Two direct hexameric repeats (GGGTGA ATG AGGACA) and not the palindromic motif thus characterize this 1,25-(OH)2D3 response element.


Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors. Human progesterone receptors (PR) in T47D breast cancer cells are synthesized as two different sized proteins, PR-A [94 kilodaltons (kDa)] and PR-B (120 kDa). Progestin addition to cells (in vivo) causes a 2-fold increase in total phosphorylation of PR and an increase in the apparent mol wt of both PR-A and PR-B on sodium dodecyl sulfate (SDS)-gels. Time-course experiments showed that increased PR phosphorylation that results from hormone addition is a multistep process and involves a rapid increase into total 32P labeling that takes place before the more slowly occurring phosphorylation(s) responsible for the change in electrophoretic mobility of PR on SDS-gels. As an approach to test whether phosphorylation is involved in regulating PR activity, we have examined the effects of cellular modulators of protein phosphorylation on PR-mediated target gene transcription in vivo using a T47D cloned cell line containing a stably transfected mouse mammary tumor virus-chloramphenicol acetyltransferase construct. Treatment with 8-bromo-cAMP (activator of cAMP-dependent protein kinases) or okadaic acid (protein phosphatase-1 and -2A inhibitor) did not stimulate target gene expression in the absence of progestin. When added together with progestin, either compound augmented PR-mediated target gene transcription by 3- to 4-fold. The cyclic nucleotide-dependent protein kinase inhibitor H8 completely blocked target gene responsiveness to hormone. Neither 8-bromo-cAMP, okadaic acid, nor H8 altered the hormone- or DNA-binding activities of PR, as measured in vitro or affected cellular concentrations of PR. These agents, therefore, appeared to selectively modulate PR transcriptional activity. Moreover, none of these compounds altered expression from a control reporter gene, pSV2CAT, indicating that these agents affect PR-mediated processes directly and are not acting through a general effect on transcription. Effects on PR phosphorylation were assessed by measuring 32P labeling of PR in vivo. None of these treatments had a substantial effect on the extent of total 32P labeling of immune isolated PR or on the phosphorylation(s) responsible for PR up-shifts on SDS-gels. This suggests that these agents modulate PR transcriptional activity either through phosphorylation of another protein intimately involved in PR-mediated transcription or through modification of a key site(s) not measurable as a change in total PR phosphorylation or electrophoretic mobility on SDS gels.


Low ligand binding activities of two distinct types of Fc gamma receptor on guinea-pig peripheral blood polymorphonuclear leukocytes are differentially improved by proteolysis or platelet activating factor. The expression and ligand binding activity of Fc receptors for IgG (Fc gamma R) on guinea-pig peripheral blood polymorphonuclear leukocytes (blood PMN) have been compared with those on casein-elicited peritoneal PMN (exudate PMN). Both the PMN were found to express two distinct types of Fc gamma R, one specific for IgG1 and IgG2 (Fc gamma 1/gamma 2 R) and the other for IgG2 alone (Fc gamma 2 R), when evaluated by their reactivity to monoclonal antibodies (mAb) directed against each type of Fc gamma R. The surface density of Fc gamma 1/gamma 2 R was not significantly different between the two cell types, whereas exudate PMN expressed five times as many Fc gamma 2 R as blood PMN. Moreover, IgG immune complex (IC) binding activities of both the Fc gamma R on blood PMN were markedly low as compared with those on exudate PMN. In addition, blood PMN could not significantly generate superoxide anion (O2-) when exposed to IC. However, these lower activities were improved by protease treatment of the cells or by incubation with platelet activating factor (PAF). Fc gamma 2 R on blood PMN was found to be sensitive to pronase, whereas Fc gamma 1/gamma 2 R was resistant. Pronase-treated blood PMN showed a marked IC binding activity, though they lacked Fc gamma 2 R. This activity was completely blocked by anti-Fc gamma 1/gamma 2 R mAb, indicating that the proteolysis augments the ligand binding capacity of Fc gamma 1/gamma 2 R. In contrast, PAF was found to specifically modulate Fc gamma 2 R. The Fc gamma 2 R expression was significantly increased within 5 min incubation with PAF, whereas that of Fc gamma 1/gamma 2 R was not affected. The cells also exhibited enhanced IgG2-IC binding and subsequent O2- generating activities. These results indicate that both the Fc gamma R on blood PMN are functionally immature and are converted to exhibit intrinsic activities by proteases and PAF; such changes may occur in vivo during exudation and at inflammatory sites.