[Reproductive and developmental toxicity study of suplatast tosilate (IPD-1151T) (1)--Fertility study in rats by oral administration]. The effect of suplatast tosilate (IPD-1151T) on reproductive ability and fetal development of the rat was studied. IPD-1151T was administered orally at dose levels of 0 (control), 200, 600, and 1800 mg/kg/day for the premating, mating and early pregnant period. For parent animals, IPD-1151T caused no abnormalities in clinical signs, reproductive ability, or autopsy findings; the females at 1800 mg/kg/day showed a reduction in body weight gain from one week after the start of administration and a decrease in food consumption from the day of the start of administration to day 6 of pregnancy. For fetuses, IPD-1151T produced no abnormalities in external, visceral or skeletal examinations; embryofetal mortality was increased at 1800 mg/kg/day. The results suggest that the non-effective dose level of IPD-1151T is 1800 mg/kg/day for males and 600 mg/kg/day for females in general toxicity, 1800 mg/kg/day for both sexes in reproductive ability, and 600 mg/kg/day for fetuses under the conditions of this study.


[Reproductive and developmental toxicity study of suplatast tosilate (IPD-1151T) (2)--Teratological study in rats by oral administration]. A teratological study of suplatast tosilate (IPD-1151T), a new anti-allergic agent which has a suppressive action on IgE antibody formation, was conducted with pregnant Wistar rats. Dosage levels of IPD-1151T 0, 300, 900 and 2700 mg/kg/day were administered to dams orally by gavage on days 7 through 17 of gestation. Two-thirds of dams per group was caesarean-sectioned on day 20 of gestation and their fetuses were removed for examination of external, visceral and skeletal anomalies. The remaining one-third was allowed to deliver naturally. F1 neonates were examined developmental, functional and behavioral parameters and reproductive abilities. The results were as follows: 1. Toxicities on F0 dams in the 2700 mg/kg/day group were salivation, piloerection, and decreases in body weight and food consumption. Seven animals (19.4%) showed severe toxicity and were dead. Toxicity in the 900 mg/kg/day group was a slight decrease in food consumption. The dosage level of 300 mg/kg/day was non-toxic. 2. Toxicities on F1 fetuses in the 2700 mg/kg/day group were a decrease in body weight and an increase in visceral anomalies (main one was ventricular septal defect that might be related to developmental retardation). No toxicities were seen in the 300 and 900 mg/kg/day. 3. In F1 neonates, suppressions of body weight were observed clearly in the male and female 2700 mg/kg/day and slightly in the male 900 mg/kg/day groups. But no changes in parameters of development, function, behavior or reproductive ability were seen in any dosed groups. It was suggested that no effective dose levels of IPD-1151T were 300 mg/kg/day for F0 dams and F1 neonates, and 900 mg/kg/day for F1 fetuses.


[Reproductive and developmental toxicity study of suplatast tosilate (IPD-1151T) (3)--Teratological study in rabbits by oral administration]. A teratological study of suplatast tosilate (IPD-1151T), a newly developed anti-allergic agent, was carried out in pregnant NZW rabbits to assess the effects on dams and fetuses. IPD-1151T was administered to dams orally at dose levels of 0, 100, 300, 450 and 700 mg/kg/day from day 6 through day 18 of gestation, and their fetuses were removed for teratological evaluation. The results were as follows: 1. In dams, marked increase in the incidence of abortion, and decrease in body weight gain, food consumption and feces mass were shown in the 700 mg/kg/day group. Slight decrease in body weight gain and food consumption were seen in the 450 mg/kg/day group, but no toxicities were observed in the 300 mg/kg/day or less groups. 2. In fetuses, marked increase in embryo-fetal deaths and decrease in alive fetal body weights and placental weights, but no teratogenicity were shown in the 700 mg/kg/day group. There were no fetal toxicity or teratogenicity in the 450 mg/kg/day or less groups. 3. No effective dose levels were 300 mg/kg/day for maternal general toxicity and 450 mg/kg/day for maternal reproductive toxicity and for fetuses.


[Reproductive and developmental toxicity study of suplatast tosilate (IPD-1151T) (4)--Perinatal and postnatal study in rats by oral administration]. In order to assess the effect of suplatast tosilate (IPD-1151T) on pregnancy of the rat, and the post natal development to maturity of the F1 generation, daily doses of 0 (control), 200, 600 and 1800 mg/kg/day were administered orally to female Wistar rats from day 17 of pregnancy to day 21 after delivery. All females were allowed to give birth and rear their young to weaning. F0 dams showed no treatment-related changes in general conditions including the state of delivery or nursing, gestation period, birth rate, or autopsy findings. The dams at 1800 mg/kg/day showed a tendency to reduction in body weight gain and a decrease in food consumption. F1 offspring showed no treatment-related changes in clinical signs on the birth day or during the nursing period, external examination on the birth day, general condition, physical or reflex development, open-field test, water multiple T-maze test, autopsy findings, organ weights, skeletal examination, reproductive ability, or histopathological examination on the reproductive organs of the animals of both sexes that failed to produce pregnancy. The offspring at 1800 mg/kg/day showed a reduction or its tendency in body weight gain. There were no treatment-related changes in any of reproductive parameters of F1 dams including external examination of F2 fetuses. The results suggest that the non-effective dose level of IPD-1151T is 600 and 1800 mg/kg/day for F0 dams in general toxicity and reproductive ability, respectively, and 600 mg/kg/day for F1 offspring in post natal development under the conditions of this study.


[Mutagenicity tests of suplatast tosilate (IPD-1151T)]. 1. The reverse mutation test was carried out on suplatast tosilate (IPD-1151T) at dose range of 50-5000 micrograms/plate using Salmonella typhimurium strains TA100, TA1535, TA98 TA1538 and TA1537, and Escherichia coli strains WP2, WP2uvrA. In all tester strains no significant differences were observed in the number of revertant colonies as compared with solvent control in the absence or presence of mammalian metabolic activation system. 2. The chromosomal aberration test on IPD-1151T was carried out using cultured Chinese hamster lung cells (CHL). The cells were treated with IPD-1151T at the doses of 125, 250 and 500 micrograms/ml without S9 Mix and at the doses of 1250, 2500 and 5000 micrograms/ml with S9 Mix. The incidence of structural- and numeral-aberration was 0-4% in the absence or presence of mammalian metabolic activation system, no significant increases were observed in the incidence of chromosomal aberrations. 3. The micronucleus test using BDF1 male mice was conducted in order to evaluate the in vivo mutagenicity of IPD-1151T. IPD-1151T was orally administered at doses of 625, 1250, 2500 and 5000 mg/kg, with a sampling time of 24 hr. The frequency of polychromatic erythrocytes with micronuclei (MNPCE) was 0.23% in the lowest dose (625mg/kg) of IPD-1151T, but the frequency of MNPCE was 0.03-0.13% in the groups treated with 1250-5000 mg/kg of IPD-1151T and no significant increases were observed with dose dependence. The results indicated that IPD-1151T was negative, even in the assessment standard using our background data. 4. The present study indicates that IPD-1151T has no in vitro and in vivo mutagenic potential.


[Antigenicity tests of suplatast tosilate (IPD-1151T)]. Antigenicity of suplatast tosilate (IPD-1151T) was investigated in guinea pigs and mice. The results were as follows: 1. Homologous passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA), active cutaneous anaphylaxis (ACA) and Schultz-Dale reaction tests were carried out using guinea pigs which were immunized orally with IPD-1151T alone or subcutaneously with IPD-1151T and Freund's complete adjuvants (CFA). Positive reactions in these tests were not produced by eliciting injection of IPD-1151T or its metabolite, M-1. On the other hand, the sensitization of ovalbumin (OVA) with CFA produced positive reactions in all of PCA, ASA, ACA and Schultz-Dale tests. 2. Heterologous passive cutaneous anaphylaxis (PCA) test using rats was carried out using two strains of mice (C3H/He and BALB/c) which were immunized orally with IPD-1151T alone or intraperitoneally with IPD-1151T and aluminum hydroxide gel (Alum) as an adjuvant. No animals showed positive reaction to both eliciting antigens, IPD-1151T and M-1. On the other hand, a positive reaction in PCA test to eliciting antigen, OVA, was obtained in rats treated with sera of mice sensitized with OVA plus Alum. 3. These findings showed that IPD-1151T had no antigenicity in guinea pigs and mice.


[A thirteen-week oral repeated dose toxicity study of suplatast tosilate (IPD-1151T) in dogs]. A 13-week oral repeated dose toxicity study of suplatast tosilate (IPD-1151T), a new anti-allergic agent, as well as a 5-week recovery study were carried out at dose levels of 0 (control), 50, 150, 450 and 1350 mg/kg/day using male and female beagle dogs. The results were as follows: 1. In general conditions, soft feces and diarrhea with specific smell were dose-dependently observed in males and females given 450 mg/kg/day or more. Both sexes given 1350 mg/kg/day, revealed reeling with dropped head, abnormal gait, dysstasia, lying at lateral or prone position, sedation, and tremor, and one male and one female in this group died after showing respiratory depression, collapse and cyanosis. 2. There were no significant or remarkable changes in body weight, food consumption, water consumption, ophthalmology, electrocardiogram, urinalysis, hematology, biochemistry, fecal occult blood test, and absolute and relative organ weights. 3. Pathological examination in dead animals revealed hemorrhagic change in the heart and slight vacuolar changes in hepatocytes. In survived animals, there were no pathological changes attributable to the IPD-1151T. 4. In electron microscopic examination, there were no abnormalities in the liver and kidney attributable to the IPD-1151T. 5. After 5-week recovery period, above-mentioned changes disappeared. 6. From the above results, the non-effective dose level and the toxic dose level were estimated to be 150 mg/kg/day and 1350 mg/kg/day, respectively, and no sex differences were found.


[A fifty-two week oral chronic toxicity study of suplatast tosilate (IPD-1151T) in rats]. A 52-week oral repeated dose toxicity study of suplatast tosilate (IPD-1151T), a new anti-allergic agent, as well as a 5-week recovery study were carried out at dose levels of 0 (control), 50, 300 and 1800 mg/kg/day using male and female rats. The results were as follows: 1. In general conditions, transient salivation after each administration and excretions with peculiar smells were noted in both sexes given 1800 mg/kg/day. Since one male and six female rats given 1800 mg/kg/day showed bradypnea, clonic/tonic convulsions, lying on the belly and/or side, subnormal temperature, abnormal gait, paralysis of extremities, they were sacrificed in moribund. 2. The body weight was lowered from the early stage of administration period in both sexes given 1800 mg/kg/day. 3. There were no remarkable changes in food consumption, urinalysis, fecal examination, hematology and ophthalmology. 4. Biochemical examination revealed a decrease in triglyceride in males given 300 mg/kg/day or more. 5. In pathological examination, the animals sacrificed in moribund showed necrosis and degeneration of neurons and/or sponge-like change of neuropile in nucleus caudatus of the cerebrum, necrosis and partial disappearance of granular cells and Purkinje's cells, and swelling of Bergmann's cells in the cerebellum. In survived animals, the relative organ weight in the liver increased in males given 300 mg/kg/day or more and females given 1800 mg/kg/day, and histopathological examination revealed slight vacuolization, hypertrophy of centrilobular hepatocytes in males given 1800 mg/kg/day. Furthermore, in some females, similar changes of the cerebrum and the cerebellum, as mentioned above, were slightly observed. In electron microscopic examination, slight proliferation of smooth endoplasmic reticulum in hepatocytic cytoplasm was observed in males given 1800 mg/kg/day. The necrobiotic changes, such as condensation of nuclear chromatin, increased electron density of cytoplasm and nuclei, mitochondrial accumulation and vacuolization, in the cells possibly derived from small granular cells in the cerebellum were observed in females given 1800 mg/kg/day. The mitochondrial swelling, decreased and dilated rough endoplasmic reticulum, and increased electron density of cytoplasm and nuclei with formation of cytoplasmic vacuole and membranous degenerated structure in neurons of cerebral temporal lobe cortex were observed in females given 1800 mg/kg/day. 6. In a recovery study, electron microscopic examination revealed a slight degeneration of myelinated nerve fibers in the cerebellum in males given 1800 mg/kg/day. On the contrary, there were no remarkable changes in general condition, body weight and various clinical parameters. It was noted that these changes induced by IPD-1151T seemed to be reversible changes.(ABSTRACT TRUNCATED AT 400 WORDS)


Basis for receptor specificity of nonecotropic murine leukemia virus surface glycoprotein gp70SU. Murine leukemia viruses (MuLVs) initiate infection of NIH 3T3 cells by binding of the viral envelope (Env) protein to a cell surface receptor. Interference assays have shown that MuLVs can be divided into four groups, each using a distinct receptor: ecotropic, polytropic, amphotropic, and 10A1. In this study, we have attempted to map the determinants within viral Env proteins by constructing chimeric env genes. Chimeras were made in all six pairwise combinations between Moloney MCF (a polytropic MuLV), amphotropic MuLV, and 10A1, using a conserved EcoRI site in the middle of the Env coding region. The receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity seems to map to the N-terminal portion of surface glycoprotein gp70SU. The difference between amphotropic and 10A1 receptor specificity can be attributed to one or more of only six amino acid differences in this region. Nearly all other cases showed evidence of interaction between Env domains in the generation of receptor specificity. Thus, a chimera composed exclusively of MCF and amphotropic sequences was found to exhibit 10A1 receptor specificity. None of the chimeras were able to infect cells by using the MCF receptor; however, two chimeras containing the C-terminal portion of MCF gp70SU could bind to this receptor, while they were able to infect cells via the amphotropic receptor. This result raises the possibility that receptor binding maps to the C-terminal portion of MCF gp70SU but requires MCF N-terminal sequences for a functional interaction with the MCF receptor.


Transcription of the E6 and E7 genes of human papillomavirus type 6 in anogenital condylomata is restricted to undifferentiated cell layers of the epithelium. The E6 and E7 genes of human genital papillomaviruses (HPVs) appear to transform cells by different mechanisms. They seem to act synergistically but are not equally important when tested under diverse experimental conditions. We were therefore tempted to investigate the E6- and E7-specific transcription pattern in HPV6-infected condylomas separately, by in situ hybridization. Recent studies have identified three promoters within the E6-E7 region of HPV6 and HPV11 by applying S1, exonuclease VII, and cDNA analyses. On the basis of these data, we cloned subgenomic fragments of HPV6 into plasmid pBS to obtain riboprobes that differentiated between transcripts starting upstream of the E6 and E7 open reading frames, respectively. These different species of mRNAs were analyzed in serial thin sections of eight HPV6-positive anogenital condylomas. The E6 probe (nucleotides 7862 to 241) led to weak signals within the basal layer. In three cases, rather strong signals were confined to a few basal cells. The E7 probe (nucleotides 242 to 534) gave rise to a more pronounced labeling of all cells within the two to three lowest epidermal layers. In situ hybridization with a riboprobe for human c-fos revealed an expression pattern similar to that observed with the E7 probe. In contrast to the preferential expression of the transforming E6 and E7 genes in the lower epithelium, the major transcriptional activity of the virus was detected in the middle and upper third by probes colinear with the 3' moiety of the early region.


The Epstein-Barr virus nuclear protein 1 promoter active in type I latency is autoregulated. The only member of the Epstein-Barr virus family of nuclear proteins (EBNAs) expressed during type I and type II latent infections is EBNA-1. This is in contrast to type III latency, during which all six nuclear proteins are expressed from a common transcription unit. The exclusive expression of EBNA-1 during type I and II latency is mediated through a recently identified promoter, Fp. The objective of this study was to characterize Fp in the Burkitt lymphoma cell background, where it is known to be differentially utilized. Using a short-term transfection assay and reporter gene plasmids containing Fp linked to the human growth hormone, we examined Fp activity in type I and type III latently infected and virus-negative Burkitt lymphoma cells. The data suggested that Fp is predominantly regulated through two distinct elements located between +24 and +270 relative to the transcription start site. One element positively mediates Fp activity, probably at the level of transcription, and acts in a virus-independent manner. The second element contains the EBNA-1 DNA binding domain III and negatively regulates Fp-directed gene expression in trans with EBNA-1 in type III as well as type I latency. Thus, we have identified a third function of EBNA-1, i.e., that of a repressor of gene expression, in addition to its known role in viral DNA replication and its ability to trans-activate gene expression. The overall activity of Fp in type I latently infected Burkitt cells was approximately sixfold lower than in virus-negative Burkitt cells, in which there is no autoregulation, suggesting that there is a fine balance between these two opposing regulatory elements during type I latency.


Epstein-Barr virus BZLF1 transactivator is a negative regulator of Jun. The Epstein-Barr virus BZLF1 protein that can induce the lytic cycle in latently infected cells is a transcription factor partially homologous to Fos and binds not only the canonical TPA (tetradecanoyl phorbol acetate)-responsive element (TRE) site but also sequences deviating from the TRE consensus sequence. Thus, expression of cellular genes regulated by AP-1, including the autoregulated AP-1 family, should be affected by BZLF1. However, induction of only Fos by BZLF1 was observed in a gel mobility shift assay using an oligonucleotide probe containing the TRE sequence and the antibody against Fos protein. The c-jun promoter, which contains a binding site for Jun and BZLF1, was stimulated by Jun but not by BZLF1. Furthermore, BZLF1 inhibited stimulation of the c-jun promoter by Jun. Jun together with Fos effectively activated the collagenase promoter that contains a single TRE site. However, not only was BZLF1 unable to stimulate the collagenase promoter, but it also inhibited activation by Jun and Fos. On the other hand, BZLF1 stimulated constructs containing multimeric binding sites. These results and those of previous studies of Epstein-Barr virus promoters regulated by BZLF1 indicate that BZLF1 requires adjacent multiple DNA-binding sites for cooperative interaction to function as a transactivator and to repress the activation by Jun of promoters containing a single TRE site. This suggests that BZLF1 evolved to confer distinct regulatory patterns upon viral target genes and cellular AP-1-responsive genes.


Transcriptional synergy by the Epstein-Barr virus transactivator ZEBRA. ZEBRA is an Epstein-Barr virus (EBV) transcriptional activator that mediates a genetic switch between the latent and lytic states of the virus by binding to the promoters of genes involved in lytic DNA replication and activating their transcription. A computer survey revealed that 9 of 23 potential or known ZEBRA-responsive EBV genes contained two or more upstream binding sites; this suggested that ZEBRA can stimulate transcription synergistically. By using a series of synthetic promoters bearing one, two, three, five, and seven upstream recognition sites, we showed that ZEBRA activates transcription synergistically when templates bearing multiple sites were compared with a template bearing a single site. This phenomenon was observed in both uninfected and EBV-infected B-lymphoid cells and in vitro in a HeLa cell nuclear extract. DNase I footprinting was used to show that the synergy was not due to cooperative DNA binding mediated by direct contact between ZEBRA dimers. The in vitro experiments revealed two manifestations of synergy. One was seen when the levels of transcription observed with the same amounts of ZEBRA added to templates bearing different numbers of sites were compared. The other was observed when the two lowest concentrations of ZEBRA that stimulated measurable transcription from any given template were compared. On the basis of both the number of sites and the calculated Kd of ZEBRA for a single site, we estimated that the critical concentration of ZEBRA needed to elicit transcriptional synergy corresponds to a site occupancy of two or three bound ZEBRA dimers. Our results have biologic implications for both the EBV lytic cycle and other processes in which the concentration of an activator changes either temporally or spatially.


Transcriptional interaction between retroviral long terminal repeats (LTRs): mechanism of 5' LTR suppression and 3' LTR promoter activation of c-myc in avian B-cell lymphomas. Chicken syncytial viruses induce bursal lymphomas by integrating into the c-myc locus and activating myc expression by 3' long terminal repeat (LTR) promoter insertion. In contrast to wild-type proviruses, in which transcription initiates predominantly in the 5'LTR, these myc-associated proviruses exhibit a predominance of transcription from the 3' LTR and little transcription from the 5' LTR. Most of these proviruses contain deletions within the 5' end of their genome that spare the 5' LTR. We report the identification of a 0.3-kb viral leader sequence that modulates 5' and 3' LTR transcriptional activities. In the presence of this sequence, transcription from the 5' LTR predominates, but in its absence, the 3' LTR promoter becomes activated, resulting in a high level of myc expression. This viral sequence does not behave like a classical enhancer; it activates transcription only when located downstream from the promoter and in the sense orientation. In this regard, it resembles the recently described human immunodeficiency virus RNA enhancer. This study suggests that retroviruses contain internal sequences which directionally activate the 5' LTR promoter to facilitate transcription of the viral genome and that deletion of these sequences is one step in the activation of the 3' LTR of myc-associated proviruses in avian bursal lymphomas.


The human fibroblast receptor for gp86 of human cytomegalovirus is a phosphorylated glycoprotein. A human embryonic lung (HEL) cell receptor for gp86 of human cytomegalovirus that functions in virus-cell fusion was further characterized. Anti-idiotype antibodies that mimic gp86 were used to immunoprecipitate the 92.5-kDa fibroblast membrane receptor for gp86, which was preincubated with various endoglycosidases. The receptor, which has a pI ranging from 5.3 to 5.6, appears to be a glycoprotein with primarily N-linked sugar residues, some of which have high concentrations of mannose and some of which are complex oligosaccharides. Western blots (immunoblots) of electrophoretically transferred receptor incubated with various biotinylated lectins confirmed the presence of sugar moieties, including N-acetylglucosamine, glucose or mannose, and galactose, but not fucose or N-acetylgalactosamine. This gp86 receptor from uninfected HEL cells also incorporated radiolabeled phosphate from orthophosphoric acid, indicating that it is a constitutively phosphorylated receptor.


Posttranslational modification and subcellular localization of the p12 capsid protein of herpes simplex virus type 1. We have previously shown that the 12-kDa capsid protein (p12) of herpes simplex virus type 1 (HSV-1) is a gamma 2 (true late) gene product encoded by the UL35 open reading frame (D. S. McNabb and R. J. Courtney, J. Virol. 66:2653-2663, 1992). To extend the characterization of p12, we have investigated the posttranslational modifications and intracellular localization of the 12-kDa polypeptide. These studies have demonstrated that p12 is modified by phosphorylation at serine and threonine residues. In addition, analysis of p12 by acid-urea gel electrophoresis has indicated that the protein can be resolved into three components, designated p12a, p12b, and p12c. Using isotopic-labeling and alkaline phosphatase digestion experiments, we have determined that p12a and p12b are phosphorylated forms of the protein, and p12c is likely to represent the unphosphorylated polypeptide. The kinetics of phosphorylation was examined by pulse-chase radiolabeling, and these studies indicated that p12c can be completely converted into p12a and p12b following a 4-h chase. All three species of p12 were found to be associated with purified HSV-1 virions; however, p12b and p12c represented the most abundant forms of the protein within viral particles. We have also examined the intracellular localization of p12 by cell fractionation and indirect immunofluorescence techniques. These results indicated that p12 is predominantly localized in the nucleus of HSV-1-infected cells and appears to be restricted to specific regions within the nucleus.


Mutational analysis of the ICP4 binding sites in the 5' transcribed noncoding domains of the herpes simplex virus 1 UL 49.5 gamma 2 gene. A previous report (P. Mavromara-Nazos and B. Roizman, Proc. Natl. Acad. Sci. USA 86:4071-4075, 1989) demonstrated that substitution of sequences of the thymidine kinase (tk) gene, a beta gene, extending from -16 to +51 with sequences extending from -12 to +104 of the gamma 2 UL 49.5 gene in viral recombinant R3820 conferred upon the chimeric gene gamma 2 attributes in the context of the viral genome in a productive infection. The UL49.5 gene sequences extending from -179 to +104 contain four DNA binding sites for the major regulatory protein ICP4. Of these sites, two map between nucleotides +20 and +80 within the sequence which confers gamma 2 regulation upon the chimeric gene. To determine the role of these ICP4 binding sites in conferring the gamma 2 gene attributes, sequences comprising the two ICP4 binding sites were mutagenized and used to reconstruct the R3820 recombinant virus. In addition, a new recombinant virus (R8023) was constructed in which tk sequences extending from -240 to +51 were replaced with wild-type or mutated sequences contained between nucleotides -179 to +104 of the UL 49.5 gene. Vero cells infected with the recombinant viruses in the presence or absence of phosphonoacetate, a specific inhibitor of viral DNA synthesis, were then tested for accumulation of tk RNA by using an RNase protection assay. The results indicate that in the recombinant R3820, a mutation which destroyed one of the two UL49.5 ICP4 DNA binding sites significantly reduced the accumulation of tk RNA at both early and late times after infection. The effect of this mutation was less pronounced in cells infected with the R8023 virus, whose chimeric tk gene contains the two upstream UL49.5 ICP4 binding sites. None of the mutations affected the sensitivity of the chimeric genes to phosphonoacetate. The mutated site appears to be involved in the accumulation of RNA.


The UL8 subunit of the herpes simplex virus helicase-primase complex is required for efficient primer utilization. The herpes simplex virus (HSV) type 1 helicase-primase is a three-protein complex, consisting of a 1:1:1 association of UL5, UL8, and UL52 gene products (J.J. Crute, T. Tsurumi, L. Zhu, S. K. Weller, P. D. Olivo, M. D. Challberg, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 86:2186-2189, 1989). We have purified this complex, as well as a subcomplex consisting of UL5 and UL52 proteins, from insect cells infected with baculovirus recombinants expressing the appropriate gene products. In confirmation of previous reports, we find that whereas UL5 alone has greatly reduced DNA-dependent ATPase activity, the UL5/UL52 subcomplex retains the activities characteristic of the heterotrimer: DNA-dependent ATPase activity, DNA helicase activity, and the ability to prime DNA synthesis on a poly(dT) template. We also found that the primers made by the subcomplex are equal in length to those synthesized by the UL5/UL8/UL52 complex. In an effort to uncover a role for UL8 in HSV DNA replication, we have developed a model system for lagging-strand synthesis in which the primase activity of the helicase-primase complex is coupled to the activity of the HSV DNA polymerase on ICP8-coated single-stranded M13 DNA. Using this assay, we found that the UL8 subunit of the helicase-primase is critical for the efficient utilization of primers; in the absence of UL8, we detected essentially no elongation of primers despite the fact that the rate of primer synthesis on the same template is undiminished. Reconstitution of lagging-strand synthesis in the presence of UL5/UL52 was achieved by the addition of partially purified UL8. Essentially identical results were obtained when Escherichia coli DNA polymerase I was substituted for the HSV polymerase/UL42 complex. On the basis of these findings, we propose that UL8 acts to increase the efficiency of primer utilization by stabilizing the association between nascent oligoribonucleotide primers and template DNA.


Complexes of Sendai virus NP-P and P-L proteins are required for defective interfering particle genome replication in vitro. We present evidence that the formation of NP-P and P-L protein complexes is essential for replication of the genome of Sendai defective interfering (DI-H) virus in vitro, using extracts of cells expressing these viral proteins from plasmids. Optimal replication of DI-H nucleocapsid RNA required extracts of cells transfected with critical amounts and ratios of each of the plasmids and was three- to fivefold better than replication with a control extract prepared from a natural virus infection. Extracts in which NP and P proteins were coexpressed supported replication of the genome of purified DI-H virus which contained endogenous polymerase proteins, but extracts in which NP and P were expressed separately and then mixed were inactive. Similarly, the P and L proteins must be coexpressed for biological activity. The replication data thus suggest that two protein complexes, NP-P and P-L, are required for nucleocapsid RNA replication and that these complexes must form during or soon after synthesis of the proteins. Biochemical evidence in support of the formation of each complex includes coimmunoprecipitation of both proteins of each complex with an antibody specific for one component and cosedimentation of the subunits of each complex. We propose that the P-L complex serves as the RNA polymerase and NP-P is required for encapsidation of newly synthesized RNA.


Transforming properties and substrate specificities of the protein tyrosine kinase oncogenes ros and src and their recombinants. To determine the sequences of the oncogenes src (encoded by Rous sarcoma virus [RSV]) and ros (encoded by UR2) that are responsible for causing different transformation phenotypes and to correlate those sequences with differences in substrate recognition, we constructed recombinants of the two transforming protein tyrosine kinases (PTKs) and studied their biological and biochemical properties. A recombinant with a 5' end from src and a 3' end from ros, called SRC x ROS, transformed chicken embryo fibroblasts (CEF) to a spindle shape morphology, mimicking that of UR2. Neither of the two reverse constructs, ROS x SRC I and ROS x SRC II, could transform CEF. However, a transforming variant of ROS x SRC II appeared during passages of the transfected cells and was called ROS x SRC (R). ROS x SRC (R) contains a 16-amino-acid deletion that includes the 3' half of the transmembrane domain of ros. Unlike RSV, ROS x SRC (R) also transformed CEF to an elongated shape similar to that of UR2. We conclude that distinct phenotypic changes of RSV- and UR2-infected cells do not depend solely on the kinase domains of their oncogenes. We next examined cellular proteins phosphorylated by the tyrosine kinases of UR2, RSV, and their recombinants as well as a number of other avian sarcoma viruses including Fujinami sarcoma virus Y73, and some ros-derived variants. Our results indicate that the UR2-encoded receptorlike PTK P68gag-ros and its derivatives have a very restricted substrate specificity in comparison with the nonreceptor PTKs encoded by the rest of the avian sarcoma viruses. Data from ros and src recombinants indicate that sequences both inside and outside the catalytic domains of ros and src exert a significant effect on the substrate specificity of the two recombinant proteins. Phosphorylation of most of the proteins in the 100- to 200-kDa range correlated with the presence of the 5' src domain, including the SH2 region, but not with the kinase domain in the recombinants. This corroborates the conclusion given above that the kinase domain of src or ros per se is not sufficient to dictate the transforming morphology of these two oncogenes. High-level tyrosyl phosphorylation of most of the prominent substrates of src is not sufficient to cause a round-shape transformation morphology.


Multiple complex families of endogenous retroviruses are highly conserved in the genus Gallus. We have analyzed the genome of the domestic chicken for the presence of genetic sequences related to the envelope protein-encoding genes of avian sarcoma/leukosis retroviruses to determine the organization, structure, potential functionality, and distribution of such sequences. We have previously identified in the genus Gallus an extensive group of endogenous avian retroviruses termed EAV-0. Southern blot and sequence analysis presented here of EAV-0 elements revealed that the majority of the EAV-0 elements in the domestic chicken genome have large deletions in their env genes. Screening of a line 0 chicken genomic DNA library for potential full-length env gene-containing endogenous elements yielded three provirus clones of a previously unrecognized group of endogenous retroviruses. These three clones, E13, E33, and E51, are more closely related to each other (80% or more sequence identity) than to other avian retroviruses (70% or less sequence identity). The E13 element has a large deletion in env, but the E51 element has full-length and highly divergent SU- and TM-coding domains. Complete sequence analysis of the E51 env gene region revealed a defective SU-coding domain and an intact TM-coding domain. Sequence analysis of the E51, E33, and E13 3' termini revealed highly distinctive long terminal repeats of approximately 360 bp which appear to be the products, in part, of long terminal repeat domain shuffling. Hybridization analysis with E51 and E33 env gene probes indicated that they are members of an extensive group of elements present in all Gallus species, and at least one element, E51, could be shown by polymerase chain reaction amplification and direct sequencing to have integrated prior to Gallus speciation.


A novel glycoprotein of feline infectious peritonitis coronavirus contains a KDEL-like endoplasmic reticulum retention signal. A new protein of feline infectious peritonitis coronavirus (FIPV) was discovered in lysates of [35S]cysteine-labeled infected cells. Expression of open reading frame (ORF) 6b of FIPV in recombinant vaccinia virus-infected cells was used to identify it as the 6b protein. Further characterization revealed that it is a novel type of viral glycoprotein whose function is not clear. It is a soluble protein contained in microsomes; its slow export from the cell is caused by the presence of an endoplasmic reticulum (ER) retention signal at the C terminus. This amino acid sequence, KTEL, closely resembles the consensus KDEL signal of soluble resident ER proteins. A mutant 6b protein with the C-terminal sequence KTEV became resistant to digestion by endo-beta-N-acetylglucosaminidase H with a half-time that was reduced threefold. In contrast, a mutant with the sequence KDEL was completely retained in the ER. The FIPV 6b protein is the first example of a viral protein with a functional KDEL-like ER retention signal.


Identification and genetic definition of a bovine papillomavirus type 1 E7 protein and absence of a low-copy-number phenotype exhibited by E5, E6, or E7 viral mutants. The bovine papillomavirus type 1 (BPV-1) genome replicates as a multiple-copy plasmid in murine C127 cells transformed to neoplasia by virus infection or by transfection with BPV-1 DNA. It was reported previously that BPV-1 genomes harboring frameshift mutations in the E6 or E7 open reading frame (ORF) replicated in C127 cells transformed by these mutants at a low copy number. Furthermore, the characterization of a BPV-1 mRNA in which the E6 and E7 ORFs were spliced together in frame has led to the assumption that an E6/7 fusion protein is expressed in virus-transformed C127 cells. To define the number and nature of the E6 and E7 gene products expressed in BPV-1-transformed cells, we performed immunoprecipitation experiments with antisera raised to bacterially expressed BPV-1 E6 and E7 fusion proteins. By employing cell culture conditions which induce BPV-1 E2 transactivator expression and viral early region transcription in virus-transformed C127 cell lines, we detected a single immunoprecipitated E6 protein species with an apparent molecular mass of 17 kDa and a single E7 protein species with an apparent molecular mass of 15 kDa. To characterize further these E6 and E7 proteins, C127 cells were transformed by transfection with BPV-1 genomes containing mutations predicted to prevent expression of specific E6 or E7 gene products, and the transformed cells were subjected to immunoprecipitation analysis with the E6 or E7 antiserum. The results of these experiments confirmed that the E6 and E7 ORFs encode distinct proteins and failed to establish the existence of an E6/7 fusion protein. We did not find a significant difference in the viral genome copy number between clonal C127 cell lines transformed by wild-type BPV-1 or by mutant viral genomes unable to express the E6 or the E7 protein. Furthermore, in contrast to two previous reports suggesting that expression of the BPV-1 E5 gene was required for the establishment or maintenance of a high viral plasmid copy number, we observed a two- to fourfold increase over wild-type BPV-1 plasmid copy number in C127 cells transfected with a BPV-1 E5-minus mutant and subsequently selected by neoplastic focus formation.


Epstein-Barr virus (EBV)-negative B-lymphoma cell lines for clonal isolation and replication of EBV recombinants. Previous experiments have demonstrated that positive selection markers recombined into the Epstein-Barr virus (EBV) genome enable the isolation of transforming or nontransforming mutant EBV recombinants in EBV-negative B-lymphoma (BL) cell lines (A. Marchini, J. I. Cohen, and E. Kieff, J. Virol. 66:3214-3219, 1992; F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). However, virus has been recovered from a BL cell clone (BL41) infected with an EBV recombinant in only one instance (Wang et al., J. Virol. 65:1701-1709, 1991). We now compare the utility of four EBV-negative BL lines, BJAB, BL30, BL41, and Loukes, for isolating EBV recombinants and supporting their subsequent replication. Transforming or nontransforming EBV recombinants carrying a simian virus 40 promoter-hygromycin phosphotransferase (HYG) cassette were cloned by selecting newly infected BL cells for HYG expression. Most of the infected BL clones contained EBV episomes, and EBV gene expression was largely restricted to EBNA-1. Although the BJAB cell line was a particularly good host for isolating EBV recombinants (Marchini et al., J. Virol. 66:3214-3219, 1992), it was largely nonpermissive for virus replication, even in response to heterologous expression of the BZLF1 immediate-early transactivator. In contrast, approximately 50% of infected BL41, BL30, or Loukes cell clones responded to lytic cycle induction. Frequently, a substantial fraction of infected cells expressed the late lytic infection viral protein, gp350/220, and released infectious virus. Since BL cells do not depend on EBV for growth, transforming and nontransforming EBV recombinants were isolated and passaged.


Capture of a cellular transcriptional unit by a retrovirus: mode of provirus activation in embryonal carcinoma cells. The expression of murine leukemia provirus in embryonal carcinoma (EC) cells is blocked by a mechanism still incompletely understood. The blockage is not overcome by deleting a large portion of the enhancer region (in U3) in recombinant retroviruses (M-MuLVneo delta Enh). This confirms the presence of negative elements outside the viral 82-bp repeats. However, a few sites in the genomes of EC cells permit M-MuLVneo delta Enh proviral expression. One such site, identified in PCC4, PCC3, and LT, was studied. The complete analysis of the mechanism of activation by Northern (RNA) blotting, cloning, and sequencing of partial cDNA copies of the viral transcript and of the site of integration establishes that viral transcripts are initiated from an upstream host-cell promoter and are spliced from a host donor to a cryptic viral acceptor at position 542 in the Moloney murine leukemia virus (M-MuLV) genome. In consequence, the mature transcripts are host cell-virus fusion transcripts from which M-MuLV sequences, including the cis-active negative elements of the 5' long terminal repeat-containing region, are absent. The provirus integrates apparently randomly into any of the three most proximal introns of the transcriptional unit. The host cell promoter contains a TATA box and 14 potential SpI binding sites included in a 1.0-kb GC-rich island. These elements promote gene expression of recombinant vectors in EC and differentiated cells. The mechanism described points to a mechanism by which retroviruses can be transcribed from upstream nonviral elements and can acquire host genes by 5' annexation of exons.


Herpes simplex virus type 1 entry through a cascade of virus-cell interactions requires different roles of gD and gH in penetration. We examined the entry process of herpes simplex virus type 1 (HSV-1) by using infectious virus and previously characterized noninfectious viruses that can bind to cells but cannot penetrate as a result of inactivation of essential viral glycoprotein D (gD) or H (gH). After contact of infectious virus with the cell plasma membrane, discernible changes of the envelope and tegument could be seen by electron microscopy. Noninfectious virions were arrested at distinct steps in interactions with cells. Viruses inactivated by anti-gD neutralizing antibodies attached to cells but were arrested prior to initiation of a visible fusion bridge between the virus and cell. As judged from its increased sensitivity to elution, virus lacking gD was less stably bound to cells than was virus containing gD. Moreover, soluble gD could substantially reduce virus attachment when added to cells prior to or with the addition of virus. Virus inactivated by anti-gH neutralizing antibodies attached and could form a fusion bridge but did not show expansion of the fusion bridge or extensive rearrangement of the envelope and tegument. We propose a model for infectious entry of HSV-1 by a series of interactions between the virion envelope and the cell plasma membrane that trigger virion disassembly, membrane fusion, and capsid penetration. In this entry process, gD mediates a stable attachment that is likely required for penetration, and gH seems to participate in fusion initiation or expansion.


The v-rel oncogene: insights into the mechanism of transcriptional activation, repression, and transformation. The v-rel oncogene product from the avian reticuloendotheliosis virus strain T corresponds to a member of the Rel-related family of enhancer-binding proteins that includes both the mammalian 50- and 65-kDa subunits of the NF-kappa B transcription factor complex. However, in contrast to NF-kappa B, v-Rel has been shown to function as a dominant-negative repressor of kappa B-dependent transcription in many mature cell types. We now demonstrate that a highly conserved motif within the Rel homology domain of v-Rel containing a consensus protein kinase A phosphorylation site is required for DNA binding, transcriptional repression, and cellular transformation mediated by this oncoprotein. However, replacement of the serine phosphate acceptor within the protein kinase A site with an alanine did not alter any of these functions of v-Rel, suggesting that phosphorylation at this site is not central to the regulation of this oncogene product. Rather, the inactive mutations appear to identify a functional domain within v-Rel required for these various biological activities. It is notable that these same mutations do not impair the ability of v-Rel to heterodimerize with the 50-kDa subunit of NF-kappa B, suggesting that v-Rel-mediated transcriptional repression likely involves direct nuclear blockade of the kappa B enhancer rather than indirect alterations in the composition of preformed cytoplasmic NF-kappa B complexes. Paradoxically, when introduced into undifferentiated F9 cells, v-Rel functions as a kappa B-specific transcriptional activator rather than as a dominant-negative repressor. These stimulatory effects of v-Rel require both the conserved protein kinase A phosphorylation site and additional unique C-terminal sequences not needed for v-Rel-mediated repression in mature cells. Retinoic acid-induced differentiation of these F9 cells restores the repressor function of v-Rel. These opposing biological actions of v-Rel occurring in cells at distinct stages of differentiation may have important implications for the mechanism of v-Rel-mediated transformation occurring in avian splenocytes.


trans-acting requirements for replication of Epstein-Barr virus ori-Lyt. Epstein-Barr virus (EBV) utilizes a completely different mode of DNA replication during the lytic cycle than that employed during latency. The latency origin of replication, ori-P, which functions in the replication of the latent episomal form of the EBV genome, requires only a single virally encoded protein, EBNA-1, for its activity. During the lytic cycle, a separate origin, ori-Lyt, is utilized. Relatively little is known about the trans-acting proteins involved in ori-Lyt replication. We established a cotransfection-replication assay to identify EBV genes whose products are required for replication of ori-Lyt. In this assay, a BamHI-H plasmid containing ori-Lyt was replicated in Vero cells cotransfected with the BamHI-H target, the three EBV lytic-cycle transactivators Zta, Rta, and Mta, and the EBV genome provided in the form of a set of six overlapping cosmid clones. By removing individual cosmids from the cotransfection mixture, we found that only three of the six cosmids were necessary for ori-Lyt replication. Subcloning of the essential cosmids led to the identification of six EBV genes that encode replication proteins. These genes and their functions (either known or predicted on the basis of sequence comparison with herpes simplex virus) are BALF5, the DNA polymerase; BALF2, the single-stranded DNA-binding protein homolog; BMRF1, the DNA polymerase processivity factor; BSLF1 and BBLF4, the primase and helicase homologs; and BBLF2/3, a potential homolog of the third component of the helicase-primase complex. In addition, ori-Lyt replication in this cotransfection assay was also dependent on one or more genes provided by the EBV SalI-F fragment and on the three lytic-cycle transactivators Zta, Rta, and Mta.


A poliovirus replicon containing the chloramphenicol acetyltransferase gene can be used to study the replication and encapsidation of poliovirus RNA. A poliovirus replicon, FLC/REP, which incorporates the reporter gene chloramphenicol acetyltransferase (CAT) in place of the region encoding the capsid proteins VP4, VP2, and part of VP3 in the genome of poliovirus type 3, has been constructed. Transfection of cells indicates that the FLC/REP replicon replicates efficiently and that active CAT enzyme is produced as a CAT-VP3 fusion protein. The level of CAT activity in transfected cells broadly reflects the level of FLC/REP RNA. A series of mutations in the 5' noncoding region of poliovirus type 3 were introduced into FLC/REP, and their effects were monitored by a simple CAT assay. These experiments helped to define further the stem-loop structures in the 5' noncoding region which are essential for RNA replication. The CAT-containing poliovirus replicon could also be packaged into poliovirus capsids provided by helper virus and was stable as a subpopulation of virus particles over at least four passages. The location of the CAT gene in FLC/REP excluded the presence of an encapsidation signal in the region of the poliovirus genome comprising nucleotides 756 to 1805.


Primary structure of the herpesvirus saimiri genome. This report describes the complete nucleotide sequence of the genome of herpesvirus saimiri, the prototype of gammaherpesvirus subgroup 2 (rhadinoviruses). The unique low-G + C-content DNA region has 112,930 bp with an average base composition of 34.5% G + C and is flanked by about 35 noncoding high-G + C-content DNA repeats of 1,444 bp (70.8% G + C) in tandem orientation. We identified 76 major open reading frames and a set of seven U-RNA genes for a total of 83 potential genes. The genes are closely arranged, with only a few regions of sizable noncoding sequences. For 60 of the predicted proteins, homologous sequences are found in other herpesviruses. Genes conserved between herpesvirus saimiri and Epstein-Barr virus (gammaherpesvirus subgroup 1) show that their genomes are generally collinear, although conserved gene blocks are separated by unique genes that appear to determine the particular phenotype of these viruses. Several deduced protein sequences of herpesvirus saimiri without counterparts in most of the other sequenced herpesviruses exhibited significant homology with cellular proteins of known function. These include thymidylate synthase, dihydrofolate reductase, complement control proteins, the cell surface antigen CD59, cyclins, and G protein-coupled receptors. Searching for functional protein motifs revealed that the virus may encode a cytosine-specific methylase and a tyrosine-specific protein kinase. Several herpesvirus saimiri genes are potential candidates to cooperate with the gene for saimiri transformation-associated protein of subgroup A (STP-A) in T-lymphocyte growth stimulation.


Nuclear localization of mouse Mx1 protein is necessary for inhibition of influenza virus. The interferon-induced Mx1 protein of mice confers selective resistance to influenza virus. It inhibits viral mRNA synthesis in the nucleus of influenza virus-infected cells. The related human MxA protein is localized in the cytoplasm and can inhibit influenza virus and vesicular stomatitis virus but not other viruses. MxA blocks a poorly defined cytoplasmic multiplication step of influenza virus that follows primary transcription of the viral genome. We previously showed that nuclear variants of MxA that carry an artificial nuclear translocation signal were also active against influenza virus. However, these variants blocked primary transcription of influenza virus. In the present study, we addressed the question of whether cytoplasmic forms of Mx1 were capable of mimicking the antiviral action of MxA by determining the antiviral activities of mutant mouse Mx1 protein. Cytoplasmic Mx1(E614), which differs from wild-type Mx1 by a single amino acid substitution in its nuclear transport signal, failed to inhibit the multiplication of influenza virus and vesicular stomatitis virus. Relocation of Mx1(E614) to the nucleus with the help of the simian virus 40 large T nuclear translocation signal attached to its amino terminus restored the influenza virus-inhibiting activity. Other changes in the carboxy-terminal region of Mx1 also abolished transport to the nucleus and simultaneously abolished antiviral activity. One of these variants, Mx1/A, gained activity against influenza virus upon relocation to the nucleus. These results demonstrate that unlike human MxA, the mouse Mx1 protein can function only in the nucleus. This finding has important implications regarding the mechanistic details of Mx protein action.


Linker scanning mutagenesis of the internal ribosome entry site of poliovirus RNA. The initiation of cap-independent translation of poliovirus mRNA occurs as a result of ribosome entry at an internal site(s) within the 5' noncoding region. A series of linker scanning mutations was constructed to define the genetic determinants of RNA-protein interactions that lead to high-fidelity translation of this unusual viral mRNA. The mutations are located within two distinct stem-loop structures in the 5' noncoding region of poliovirus RNA that constitute a major portion of a putative internal ribosome entry site. On the basis of our data derived from genetic and biochemical assays, the stability of one of the stem-loop structures appears to be essential for translation initiation via internal binding of ribosomes. However, the second stem-loop structure may function in a manner that requires base pairing and proper spacing between specific nucleotide sequences. By employing RNA electrophoretic mobility shift assays, an RNA-protein interaction was detected for this latter stem-loop structure that does not occur in RNAs containing mutations which perturb the predicted hairpin structure. Analysis of in vivo-selected virus revertants, in combination with mobility shift assays, suggests that extensive genetic rearrangement can lead to restoration of 5' noncoding region functions, possibly by the repositioning of specific RNA sequence or structure motifs.


Interaction of the human papillomavirus type 16 E6 oncoprotein with wild-type and mutant human p53 proteins. The E6 oncoproteins encoded by the cancer-associated human papillomaviruses (HPVs) can associate with and promote the degradation of wild-type p53 in vitro. To gain further insight into this process, the ability of HPV-16 E6 to complex with and promote the degradation of mutant forms of p53 was studied. A correlation between binding and the targeted degradation of p53 was established. Mutant p53 proteins that bound HPV-16 E6 were targeted for degradation, whereas those that did not complex HPV-16 E6 were not degraded. Since the HPV-16 E6-promoted degradation involves the ubiquitin-dependent proteolysis pathway, specific mutations were made in the amino terminus of p53 to examine whether the E6 targeted degradation involved the N-end rule pathway. No requirement for destabilizing amino acids at the N terminus of p53 was found, nor was evidence found that HPV-16 E6 could provide this determinant in trans, indicating that the N-terminal rule pathway is not involved in the E6-promoted degradation of p53.


Packaging system for rapid production of murine leukemia virus vectors with variable tropism. A method for rapidly producing helper-free murine leukemia virus (MLV) without using packaging cell lines is described. Viruses bearing ecotropic or amphotropic MLV or Rous sarcoma virus envelope glycoprotein and containing various retroviral vector genomes have been prepared with titers 30 to 40-fold higher than those produced by transient transfection of standard packaging cells. This system can be used to alter the cellular tropism of MLV by incorporating other envelope glycoproteins and to prepare retroviral vector stocks without establishing stable producer cell lines. This method will be particularly useful for preparing viruses that encode toxic proteins and for the rapid analysis of panels of mutant envelope glycoproteins.


Epstein-Barr virus-encoded small RNAs (EBERs) do not modulate interferon effects in infected lymphocytes. The recent derivation of otherwise isogenic Epstein-Barr virus (EBV) recombinants carrying or lacking the EBV small RNA (EBER) genes enabled us to test whether EBERs are similar to adenovirus VA RNAs in modulating interferon (IFN) effects on virus infection. EBER-positive and -negative EBV recombinants did not differ in their sensitivity to alpha interferon (IFN-alpha)- or IFN-gamma-mediated inhibition of lymphocyte growth transformation. In addition, EBERs did not decrease the inhibitory effects of IFN on vesicular stomatitis virus replication in EBV-transformed lymphocytes. EBER deletion also did not render EBV-transformed B lymphocytes susceptible to an IFN effect on cell proliferation or EBV replication.


Identification and characterization of the bovine immunodeficiency-like virus tat gene. A cDNA clone of the bovine immunodeficiency-like virus (BIV) trans-activator gene (tat) was identified and characterized. The tat cDNA clone was generated by splicing, and on the basis of sequence analysis, the Tat protein was found to be encoded entirely by the first exon. It is 103 amino acids in size and shares sequence homology with the human immunodeficiency virus (HIV) Tat. The BIV tat clone can trans activate the BIV promoter effectively, as measured by the expression of the bacterial chloramphenicol acetyltransferase gene, when transfected into bovine cells. Besides activating the BIV promoter, the BIV Tat can also trans activate the HIV promoter effectively. It is possible that BIV Tat and HIV Tat employ similar mechanisms in trans activation of the viral long terminal repeat-directed gene expression.


Human immunodeficiency virus type 1 gag-pol frameshifting is dependent on downstream mRNA secondary structure: demonstration by expression in vivo. The human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion polyprotein is produced via ribosomal frameshifting. Previous studies in vitro and in Saccharomyces cerevisiae have argued against a significant role for RNA secondary structure 3' of the shift site, in contrast with other systems, in which such structure has been shown to be required. Here we show, by expressing the HIV-1 gag-pol domain in cultured vertebrate cells, that a stem-loop structure 3' of the HIV-1 shift site is indeed important for wild-type levels of frameshifting in vivo.


Postinfection treatment with antiviral serum results in survival of neural cells productively infected with virulent poliovirus. The death of human neuroblastoma cells undergoing productive infection with virulent poliovirus was prevented by addition of antiserum against the virus a few hours after the onset of infection; this treatment, however, did not prevent reproduction of the virus. Despite the presence of the viral antigen, the cells retained the ability to divide. Upon further cultivation in the absence of antiserum, the cells developed specific postinfection immunity or resistance to superinfection with poliovirus.


An Epstein-Barr virus transformation-associated membrane protein interacts with src family tyrosine kinases. In latently infected growth-transformed human lymphocytes, Epstein-Barr virus (EBV) encodes two integral plasma membrane proteins: LMP1, which constitutively induces B-lymphocyte activation and intercellular adhesion, and LMP2A, which associates with LMP1 and is a tyrosine kinase substrate. We now demonstrate that LMP2A associates with src family protein tyrosine kinases, particularly lyn kinase, in nonionic detergent extracts of transfected B lymphoma cells or in extracts of EBV-transformed B lymphocytes. The LMP2A and tyrosine kinase association is stable in nonionic detergents and includes a 70-kDa cell protein which is also an in vitro or in vivo kinase substrate. This LMP2A association with B-lymphocyte src family tyrosine kinases is likely to be an important pathway in EBV's effects on cell growth.


The UL11 gene of herpes simplex virus 1 encodes a function that facilitates nucleocapsid envelopment and egress from cells. The UL11 gene of herpes simplex virus 1 was reported to encode a myristylated protein (C. A. MacLean, B. Clark, and D. J. McGeoch, J. Gen. Virol. 70:3147-3157, 1989). To determine the function of the gene product, a recombinant virus (R7219) lacking 61% of the codons (176 bp of the 288-bp coding domain) was genetically engineered. The deletion mutant replicated in all cell lines tested, albeit to titers 30- to 250-fold lower than those obtained from cells infected with wild-type virus. Electron microscopic analyses indicated that both full and empty capsids accumulated in the nuclei, juxtaposed with the inner lamellae of the nuclear membranes, and that increased numbers of naked particles were present in the cytoplasm of cells infected with the mutant virus. There was a greater than 1,000-fold decrease in the amount of infectious extracellular virus released from Vero cells infected with the deletion mutant compared with that from cells infected with wild-type virus. Furthermore, the onset of release of infectious virus from cells infected with the UL11- mutant was significantly delayed: levels of extracellular UL11- virus increased 15-fold between 20 and 26 h after infection, while levels of wild-type extracellular virus increased 500-fold between 8 and 14 h after infection. A virus in which the UL11 gene was restored produced wild-type levels of total and extracellular virus and was indistinguishable from wild-type virus upon analysis by electron microscopy. Taken together, the data indicate that the absence of the UL11 gene causes a reduced capacity to envelope and transport virions into the extracellular space.


Changing trends in allogeneic bone marrow transplantation for leukemia in the 1980s. To identify changes in practice and outcome of bone marrow transplants for leukemia in the 1980s. Comparison of key explanatory and outcome variables in five 2-year cohorts, from 1980 through 1981 to 1988 through 1989, using a large database of detailed clinical information. Recipients (7788) of bone marrow transplants for acute lymphoblastic, acute myelogenous, or chronic myelogenous leukemia reported to the International Bone Marrow Transplant Registry, Milwaukee, Wis, by 185 transplant teams worldwide. Linear increases occurred during the periods 1980 through 1981 to 1988 through 1989 as follows with 95% confidence intervals: (1) transplants for chronic myelogenous leukemia from 14% +/- 2% to 35% +/- 2%; (2) transplants from unrelated donors from 1% +/- 1% to 7% +/- 1%; (3) preparative regimens without radiation from 3% +/- 1% to 30% +/- 2%; and (4) use of methotrexate plus cyclosporine to prevent graft-vs-host disease from 2% +/- 1% to 55% +/- 2%. Among recipients of human lymphocyte antigen-identical sibling bone marrow, the 2-year probability of treatment-related mortality decreased by 6% to 22%. The probability of relapse decreased from 46% +/- 6% to 38% +/- 6% in intermediate leukemia but did not change appreciably in early or advanced leukemia. Probabilities of leukemia-free survival improved from 51% +/- 4% to 57% +/- 3% in early leukemia, from 28% +/- 4% to 36% +/- 5% in intermediate leukemia, and from 12% +/- 4% to 18% +/- 5% in advanced leukemia. A separate analysis of a homogenous population of patients indicated that improvements in outcome in the 1980s were due to improvements in transplant practice rather than improved patient selection. Modest increases in leukemia-free survival rates occurred after human lymphocyte antigen-identical sibling bone marrow transplants in the 1980s. Improvements were due primarily to reductions in treatment-related mortality with little or no change in relapse risk. More effective antileukemia strategies and continued reductions in treatment-related toxic effects are needed.


Hepatitis C as a cause of chronic liver disease in northern Pakistan. The antibodies to hepatitis C virus (HCV) were tested in 45 histologically confirmed cases of chronic liver disease. Twelve cases had chronic hepatitis, 24 cirrhosis and 9 hepatocellular carcinoma. Anti-HCV was detected in 6 patients. Two (16.67%) were suffering from chronic hepatitis, 3 (12.5%) had cirrhosis and one (11.11%) hepatocellular carcinoma. None of the anti-HCV positive cases had past history of blood transfusion. The patients of chronic liver disease in this study had a much higher prevalence of HBV infection which indicates that in northern Pakistan hepatitis C virus infection is not a common cause of chronic liver disease whereas HBV infection plays an aetiological role in a much larger number of these cases.


[Auer rods-positive neutrophils observed at diagnosis increased after remission induction in patient with acute promyelocytic leukemia]. 50-year-old male was admitted to our hospital because of gingival bleeding and fever in August 1987. The leukocyte count was 13,300/microliters with 80.5% leukemic promyelocytes and bone marrow was hypercellular with 86.4% leukemic promyelocytes. A small number of mature neutrophils containing Auer rods were seen in bone marrow. On a diagnosis of acute promyelocytic leukemia and treated with induction chemotherapy consisting of behenoyl-arabinofuranosyl cytosine (BHAC), daunorubicin, 6-mercaptopurine (6-MP) and prednisolone (PSL) was reformed. After cytoreduction, leukemic cells reappeared in the peripheral blood, concomitant with mature neutrophils having Auer rods. Vitamin D3 was not effective as a differentiation inducing agent. Complete remission was obtained in November 1987 by the reinduction chemotherapy consisting of BHAC, aclarubicin, 6-MP and PSL. In this case, neutrophils with Auer rods might have been derived from the leukemic clone and differentiation of leukemic promyelocytes by intensive chemotherapy.


[Pulmonary complications in patients with adult T-cell leukemia]. In order to investigate the character of pulmonary complications in patients with adult T-cell leukemia (ATL), a pathological and bacteriological study was performed in 92 autopsy cases with hematologic malignancies including 17 cases of ATL and 103 autopsy cases with solid malignancies from 1981 to 1990. Among 17 cases with ATL, pulmonary complications were seen in 16 cases (94.1%); pulmonary infection in 14 (82.3%), leukemic cell pulmonary infiltration in 9 (52.9%), pulmonary hemorrhage in 5 (29.4%), pulmonary alveolar calcinosis in 2 (11.8%), and idiopathic interstitial pneumonia in 2 (11.8%). The causative microorganisms were virus in 10; 9 of which were cytomegalovirus, followed by bacteria infection in 4 cases, mainly pseudomonas aeruginosa, and fungal infection in 3, mainly cryptococcus. pneumocystic carinii and mycobacterium tuberculosis were not detected. It is suggested that patients with ATL are severely compromised with chiefly cellular immunodeficiency, and administration of sulfamethoxazole-trimethoprim and isoniazid is very effective in prevention of pneumocystis carinii pneumonia and pulmonary tuberculosis.


[A case of idiopathic interstitial pneumonia with cytomegalovirus infection]. A 67-year-old female was admitted to our hospital because of fever, dry cough, and exertional dyspnea. The findings of chest X-ray, transbronchial lung biopsy, and bronchoalveolar lavage were compatible with the diagnosis of idiopathic interstitial pneumonia. Prednisolone was administered and she felt better for a while. However, she developed severe dyspnea, and marked diffuse infiltrative shadows were observed on chest X-ray after 3 months of steroid therapy. In spite of pulse therapy with methylprednisolone, she died of severe respiratory failure. Complement fixation test and IgG antibody enzyme immunoassay for cytomegalovirus were positive, but there was no change the titers between admission and death. IgM antibody was negative. The lung findings at autopsy compatible with usual interstitial pneumonia and diffuse alveolar damage, moreover, cytomegalovirus infection was observed. We consider that recurrent cytomegalovirus pneumonia had been present due to secondary immunodeficiency caused by administration of steroid hormones.


[A case of influenza B viral bronchopneumonia followed by CT]. A 69-year-old male with bronchial asthma was admitted to a hospital with fever, dyspnea, and productive cough. Arterial blood gas analysis revealed sever hypoxemia (PaO2 54.8 torr, PaCO2 28.8 torr). Chest roentgenogram showed diffuse reticulonodular shadows predominantly in the upper filed and a small amount of bilateral pleural effusion. CT image of the lung showed nodular opacities at the peripheral branches of the pulmonary arteries and bronchi, some of which had become confluent. The bronchoarterial bundle had become thicker compared with a CT taken 18 months before this admission. Three days treatment with antibiotics and gamma globulin did not change the symptoms or radiologic findings. After commencing methylprednisolone therapy, the pneumonia showed rapid improvement. Based upon the significant elevation of serum influenza B (B/Singapore/79) virus antibody titer, the patient was diagnosed as having influenza B viral bronchopneumonia. Twenty-three days after initiation of steroid therapy, slight nodular opacities were observed on CT. This finding suggests that bronchiolitis has a relatively prolonged course in influenza viral bronchopneumonia.


[A case of hCG-producing large cell carcinoma of the lung--clinical utility of serum hCG levels]. We describe a case of hCG-producing large cell carcinoma of the lung in a 73-year-old man who had gynecomastia associated with high serum concentration of hCG. Serum hCG levels fluctuated in parallel with the response of the cancer to surgery, chemotherapy, and radiotherapy. The patient was admitted to our hospital with a huge mass shadow in the right lung filed on chest X-ray film on July 31, 1989. Physical examination revealed bilateral gynecomastia. Serum hCG and beta-hCG were 1108.0 mIU/ml (Normal less than 2.0) and 31.9 ng/ml (Normal less than 1.0), respectively. Clinical staging was T2N1M0, determined by radioisotope scanning of bone, and CT scans of the chest, brain and upper abdomen. Right upper and middle lobectomy with mediastinal lymph node dissection was performed on August 18, 1989. The tumor, 6 x 6 x 8 cm in size, was located in the middle lobe and was histologically confirmed to be large cell carcinoma of the lung. A few of small nodules found on the surface of the middle lobe at thoracotomy were histologically proved to be pleural dissemination. Metastatic involvement was present in the hilar and mediastinal lymph nodes. The pathological stage was concluded to be T4N2M0. Immunohistochemistry showed positive staining reaction for hCG within some of the tumor cells. Three weeks after the operation, serum hCG had decreased rapidly but did not reach the normal range. Two courses of DDP, VDS, and MMC were given at four week intervals. Following chemotherapy, serum hCG decreased to the normal range. He was discharged from our hospital on November 29, 1989.2+ useful parameter for the evaluation of treatment and the prediction of prognosis.


Adrenocorticotrophin and steroid-induced hypertension in humans. ACTH and adrenocortical steroids have been known to raise blood pressure since their introduction into clinical practice. Our experimental studies in normal subjects show that ACTH reproducibly increases blood pressure in association with a rise in cardiac output, plasma and extracellular fluid volumes and exchangeable sodium. The rise in pressure is adrenally dependent and appears due to ACTH-induced increases in cortisol secretion. When ACTH is given by constant intravenous infusion, rates as low as 50 micrograms/day raise pressure. The increase in blood pressure is not dependent on, but modified by, dietary sodium content. Synthetic steroids (prednisolone, methylprednisolone, triamcinolone, dexamethasone) raise pressure in the absence of any increase in plasma volume or urinary sodium retention. Cortisol increases pressor responsiveness to endogenous and exogenous catecholamines, without evidence of any increase in sympathetic nervous activity. The role of this increased pressor responsiveness in ACTH/steroid-induced hypertension remains to be determined. There is some evidence from human studies that steroids may raise pressure by a hypertensinogenic mechanism, distinct from classical mineralocorticoid or glucocorticoid effects.


Effect of CRF, ACTH and adrenal steroids on sodium intake and excretion of rabbits. The effect of CRF, ACTH and adrenal steroid hormones on the sodium intake and excretion of wild and laboratory rabbits was studied in our laboratory in detail. All these hormones are known to play important roles in the initiation and maintenance of stress-reaction. Intracerebroventricular (icv) infusion of CRF increased both sodium intake and excretion of rabbits on the day of the infusion, and the stimulated sodium turnover persisted for several days after the infusion stopped. Systemic administration of the same dose of CRF did not influence sodium intake or excretion. Icv infusion of CRF was accompanied by a rise in plasma cortisol and plasma corticosterone concentration. Plasma sodium concentration was unchanged despite the increased turnover. The elevated plasma concentration of adrenal steroid hormones indicates that icv infused CRF resulted in ACTH and consequent cortisol and corticosterone release. Earlier studies in our laboratory established that ACTH, and similarly cortisol and corticosterone, when injected systemically, also elicited increased sodium intake, which was accompanied by increased sodium excretion. The rise in sodium turnover occurred on the second or third day of ACTH or steroid administration. The difference in the time of onset of sodium appetite between icv CRF and ACTH or adrenal steroids indicates that CRF influences sodium intake by other mechanisms as well. These other contributing mechanisms are probably activated by the binding of CRF to the specific binding sites demonstrated earlier in the rabbit's brain. Its is possible, that the small natriuresis accompanying icv infusion of CRF has some role in the initiation of the sodium appetite.(ABSTRACT TRUNCATED AT 250 WORDS)


[Production of ACTH and cortisol in patients with diffuse purulent peritonitis]. In 91 patients with generalized purulent peritonitis, peritoneal dialysis (in 44) and laparotomy with programmed lavage of the abdominal cavity (in 47) were performed. ACTH and cortisol content in the blood serum, was measured by means of the radioimmunologic method. In favourable course of the disease, hormonal production is preserved, in severe course, hormonal disbalance occurs.


ESR spin label and ultrastructural monitoring of protein-lipid interactions in the lens fiber-cell plasma membranes in relation to human ageing and cataractogenesis. The Electron Spin Resonance (ESR) technique and the protein spin labels 2,2,4,4,9-pentamethyl-1,2,3,4-tetrahydro-gamma-carboline-3-oxyl and 4-(N-maleimido)-2,2,6,6-tetramethylpiperidine-1-oxyl were used in this study to probe the fluidity and binding ability to protein functional groups in human lens membranes. The image and the stage of cataract as well as the ultrastructural characteristics of the lens fiber cell membranes were evaluated in parallel studies. ESR measurements in membrane structures of the transparent lenses of different ages have shown that the sorbtion parameter of the carboline label on the surface of protein-lipid components was usually weakly expressed but increased with aging and the extent of immobilization of the bound label was not significant. At different stages of the lens opacification the carboline analogue spin label increasingly bound to the lens membranous structures. A spin label signal can be enhanced by addition to the samples of the paramagnetic probe K3Fe(CN)6. The maleimide spin label bound to the protein SH-groups in the cataractous lens membranes at a slow rate, however in transparent lenses a gradual increase of the rapidly binding phase of this label could be detected. The results show an appearance of at least two types of the reactive SH-groups in membranes of human transparent lenses. The electron microscopic studies suggested that the age-related and cataractogenic changes in the lens matter are accompanied by deterioration of the lenticular fiber plasma membranes and formation of the coalescing globules with a diameter of 220-500 nm. At the stage of cataract with advanced opacities, which is biochemically characterized by the increase in number of the carboline label binding sites with the surface proteins and annular lipids of membranes, a mass of amorphous aggregates filled with the electron-grey debris is formed contributing to significant scatter of light. It appears, therefore, that the suggested ESR spin label technique can be effectively used to monitor the aggregation process of protein membrane components which takes place during human cataractogenesis.


Age-related events in active T subpopulation. Changes in polyphosphoinositide metabolism during mitogenic activation. The active E rosette test allows the characterisation of a lymphocyte subset with high affinity receptors for sheep red blood cells. In this study, we compared active T lymphocytes of healthy subjects over 65 and under 30 years of age after mitogenic stimulation. Active E rosettes increased in both age groups after 48 h of phytohemoagglutinin (PHA) stimulation, mainly in young subjects. In addition, analysing the in vitro phosphorylation of inositol lipids, changes occurred in the incorporation of radioactivity in both phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bis phosphate (PIP2) in the early steps of the mitotic response in the elderly subjects as compared to young ones, hinting that modifications of this signal transduction system are related to mitogenic stimulation in this peculiar T subpopulation.


[A genetic and hormonal study of 5 patients with the nonclassical form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency]. To study the association between the 21-hydroxylase deficiency with the HLA histocompatibility complex in a mediterranean ethnic group. 5 patients with late-onset 21-hydroxylase deficiency, diagnosed on the basis of a high plasma level of 17-hydroxyprogesterone, along with 23 family members were typed. 17-hydroxyprogesterone response to iv ACTH stimulus was measured too in the family members. We found a genetic linkage disequilibrium between the late-onset 21-hydroxylase deficiency and the HLA antigen B51. Moreover, similar biologic profiles were observed in the patients and those of their siblings who were HLA identical. The heterozygous carriers showed a intermediate 17-hydroxyprogesterone response to ACTH between propositus and homozygotes and their family members who had no HLA haplotype identical to those of the propositus. These observations tend to confirm that a close linkage exits between the gene (or genes) for 21-hydroxylase deficiency in late-onset adrenal hyperplasia and the HLA genes. This association may change in the different ethnic groups.


[A change in the epidemiologic pattern of hepatitis A in Spain]. Seroepidemiologic studies carried out in different countries show that the prevalence of markers for infection by hepatitis A (HVA) is progressively decreasing in relation with improvement in the level of health care. The prevalence of anti-HVA in Catalunya and the factors related with infection by HVA were studied in order to obtain bases for designing prevention strategies adequate for the epidemiology of this infection. Anti-HVA was determined by a method of enzyme immunoassay in a randomized sample of the general population in Catalunya. Investigation of the influence of socio/demographic variables in the prevalence of HVA infection was carried out. The global prevalence of anti-HVA was of 36%. In the population from 6 to 14 years of age it was of 13% and that of those over the age of 15 it was 86%. The prevalence of anti-HVA increases with age and is related with the social level of the families, the level of education and the place of birth. The incidence was higher in children of the lowest social class, in adults with the least education and in those born outside Catalunya. These factors constituted independent risk factors of HVA infection. The present study confirms the association between HVA infection and socio-economic factors and demonstrates that the infection is not commonly found in infancy, an increase in the number of susceptible adults is foreseeable.


Triester glycerothiophosphates of cholecalciferol (vitamin D3). Tris(N,N-dimethyl)amide of phosphorous acid activated by addition of iodine at an optimal molar ratio of 1.05:0.05 was used as a phosphorylating reagent to synthesize cholecalciferyl-3-O-(1-2-isopropylidene-rac-glycero-3-O-)-thiophos phate and cholecalciferyl-3-O-(1,2-dipalmitoyl-rac-glycero-3-O-)thiophosphat e of methyl alcohol, 2-dimethylaminoethanol, 3-dimethylamino-1-propanol and 1,2-isopropylidene-rac-glycerol in a "one-pot procedure" in overall yields of 60-80%. Activation of the reaction with an equimolar mixture of imidazole and carbon disulfide at the triester formation step permits selective phosphorylation at room temperature. The compounds synthesized represent new triester phospholipid model compounds in which (in addition to glycerol and another requisite alcohol) a steroid and an element other than oxygen are bond to phosphorus.


A stochastic analysis of three viral sequences. This paper analyzes the nucleotide sequences of three viruses: Kunjin, west Nile, and yellow fever. Each virus has one long open reading frame of greater than 10,200 nucleotides that codes for four structural and seven nonstructural genes. The Kunjin and west Nile viruses are the most closely related pair, when assessed on the basis of matches between their nucleotide sequences. As would be expected, the matching is least for bases at third-position codon sites and is greatest for second-position sites. Statistics are presented for the numbers of mismatches that are transitions or transversions. Nucleotide base usage is also reported. To each of the 33 virus-gene segments, nonhomogeneous Markov chain models have been fitted to describe the sequences of nucleotide bases. The models allow for different transition probabilities ("transition" is used in the mathematical sense here) and for different degrees of dependency, at the three sites in the codons. Reasonably satisfactory fits can be obtained for many of the genes by using models that are first order for both first- and second-position sites in the codon but that are second order for third-position sites. One consequence of such a model is that the correlation between one amino acid and the next is limited to the correlation of the last base of the former with the first base of the latter. Other consequences are that the model can (and does) prohibit the occurrence of stop codons within a gene and that subsequences of only first-position bases, or only third-position bases, are also first-order Markov chains. In theory, second-position subsequences may not be Markov chains at all. In practice, the data suggest that each of these subsequences is effectively a zero-order Markov chain, i.e., bases spaced three apart are statistically independent. Stationarity of nucleotide base distributions can be interpreted in either of two ways: (1) spatially along the sites or (2) temporally at each site. These interpretations must often be inconsistent, when the former allows for Markov dependence between adjacent sites whereas the latter assumes independence between sites. The inconsistency can be overcome, for these viruses, if subsequences at different codon positions are analyzed separately.


Inverted repeats in the long-terminal repeats of the wheat retrotransposon Wis 2-1A. The wheat insertion sequence Wis 2-1A possesses all the structural features characteristic of retrotransposons. Its long-terminal repeats (LTRs) are unusually long (1,755 bp) compared with those of other retrotransposons. Sequence analysis revealed that they differ from each other by only six point mutations. They contain a few tandem direct repeats, which could be explained by slippage mechanisms during replication. Almost half (44%) of the length of the LTRs is occupied by hairpin structures, which may relate to their large size. Possible origins of these inverted repeats are proposed, including the insertion and imprecise excision of transposable elements and errors when the DNA replication intermediate switches RNA template during retrotransposon replication.


Differential regulation of two genes encoding lysyl-tRNA synthetases in Escherichia coli: lysU-constitutive mutations compensate for a lysS null mutation. Lysyl-tRNA synthetases are synthesized in Escherichia coli from two distinct genes, lysS and lysU, which are regulated differentially. A strain which is null for lysS, the constitutive gene, was created by gene disruption (lysS1) and exhibited cold-sensitive lethality. Hence, lysS is dispensable at high temperatures. This cold sensitivity was suppressed by a multi-copy plasmid carrying lysU, the inducible gene. These data are interpreted as indicating that lysS is functionally replaceable by lysU for cell growth, and that the cold sensitivity of lysS1 is caused by insufficient expression of lysU at low temperatures. To investigate the mechanism of lysU expression, cold-resistant bypass mutations were isolated from lysS1, and named als (for abandonment of lysS). Two als mutations which were linked to lysU contain IS2 insertions upstream of the lysU promoter. They caused a 16-19-fold increase in the lysU-mRNA level. Furthermore, deletion mutations created immediately upstream of the lysU promoter restored growth of lysS1. These results suggest that transcription of lysU is negatively controlled by a cis-element located upstream of the promoter.


Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. A 2.6 kb plasmid, named pBBR1, was isolated from Bordetella bronchiseptica S87. After insertion of an antibiotic resistance marker, this plasmid could be transferred into Escherichia coli, Bordetella pertussis, B. bronchiseptica, Vibrio cholerae, Rhizobium meliloti, and Pseudomonas putida by transformation or conjugation. Conjugation was possible only when the IncP group transfer functions were provided in trans. As shown by incompatibility testing, pBBR1 does not belong to the broad-host-range IncP, IncQ or IncW groups. DNA sequence analysis revealed two open reading frames: one was called Rep, involved in replication of the plasmid, and the other, called Mob, was involved in mobilization. Both the amino-terminal region of Mob and its promoter region show sequence similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. In spite of these sequence similarities, pBBR1 does not replicate via the rolling-circle mechanism commonly used by small Gram-positive plasmids. We therefore speculate that pBBR1 may combine a mobilization mechanism of Gram-positive organisms with a replication mechanism of Gram-negative organisms. Determination of the plasmid copy number in E. coli and B. pertussis indicated that pBBR1 has a rather high copy number, which, in conjunction with its small size and broad host range, renders it particularly interesting for studies of broad-host-range replicons and for the development of new cloning vectors for a wide range of Gram-negative bacteria.


PhoA gene fusions in Legionella pneumophila generated in vivo using a new transposon, MudphoA. To enable effective use of phoA gene fusions in Legionella pneumophila, we constructed MudphoA, a derivative of the mini-Mu phage Mu dII4041, which is capable of generating gene fusions to the Escherichia coli alkaline phosphatase gene (EC 3.1.3.1). Although an existing fusion-generating transposon, TnphoA, has been a useful tool for studying secreted proteins in other bacteria, this transposon and other Tn5 derivatives transpose inefficiently in Legionella pneumophila, necessitating the construction of a more effective vector for use in this pathogen. Using MudphoA we generated fusions to an E. coli gene encoding a periplasmic protein and to an L. pneumophila gene encoding an outer membrane protein; both sets of fusions resulted in alkaline phosphatase activity. We have begun to use MudphoA to mutate secreted proteins of L. pneumophila specifically, since this subset of bacterial proteins is most likely to be involved in host-bacterial interactions. This modified transposon may be useful for studies of other bacteria that support transposition of Mu, but not Tn5, derivatives.


Prevalence of hepatitis C antibodies in patients with clotting disorders in Victoria. Relationship with other blood borne viruses and liver disease. To elucidate the seroepidemiology of hepatitis C in patients with clotting disorders in comparison with other blood borne infections; to examine the effects of hepatitis C on liver function; and to assess the effectiveness of current screening and inactivation procedures used in preventing the transmission of blood borne viruses by clotting factor preparations. A retrospective analysis of the prevalence of antibodies to hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV) by means of commercially available enzyme immunoassays (for antibodies to HCV and HIV) or radioimmunoassays (for HBV antibodies and surface antigen). An analysis was made of serum transaminase levels where such information was available and this was correlated with HCV status. Panels of sera were collected from adults and children with clotting disorders attending two Melbourne haemophilia treatment centres in 1973 (n = 33), 1980 (n = 33), 1984-1985 (n = 111) and 1987-1990 (n = 217) and tested for antibodies to HCV, HBV and HIV. The prevalence of antibodies to HCV in the four panels tested was 45%, 74%, 75% and 76%, and the prevalence of markers of infection with HBV was 66%, 74%, 62% and 65% respectively. No antibodies to HIV were found in sera in Panels I and II but the prevalence in Panels III and IV was 23% and 36% respectively. In subjects in whom liver function test results were available, there was a significant association between the presence of antibodies to HCV and raised transaminase levels. Since heat inactivation of clotting factors was commenced in Australia in 1984, no new cases of transmission of HIV by clotting factors has been detected, but transmission of HCV in 19 subjects and HBV in one subject could not be excluded. Hepatitis C infection in haemophiliacs has been a very frequent event, and the presence of antibodies to HCV is associated with an increased incidence of raised transaminase levels. Screening and heat inactivation of clotting factors has prevented further HIV transmission, but exposure to HBV and HCV has not been eliminated.


[Clinical evaluation of myocardial protection by oxygenated crystalloid cardioplegic solutions with DBcAMP]. Possible enhancement of myocardial protection by adding DBcAMP and oxygenation of a crystalloid cardioplegic solution (CCS) was evaluated in a three group study. The patients having coronary bypass operation or valvular operation were divided into three groups, each consisting of 15 patients, and differing only in the type of CCS employed. Group I was protected by nonoxygenated CCS (PO2 190 mmHg, PCO2, 32 mmHg, pH 7.78, K 30 mmEq/L), Group II by adding DBcAMP to nonoxygenated CCS and Group III by adding DBcAMP to oxygenated CCS (PO2 790 mmHg, PCO2 26 mmHg, pH 7.87). Group III had significantly improved CI and double product (p less than 0.05) compared with Group II. However, CPK, CPK-MB, and myoglobin in the serum were similar in each group. Lactate and pyruvate ratio (L/P) in the coronary sinus bloods were improved to lower value after the pump than before the pump only in Group III. Base excess in the coronary sinus held on alkalosis after aortic declamp only in Group III. The refunction time was significantly shortest with Group III than with other groups (p less than 0.01, 0.05) and Group II was significantly shorter than Group I (p less than 0.05). It is concluded that oxygenation and adding DBcAMP to CCS are effectual for the myocardial metabolism and protect the myocardial damage during cardiac arrest.


Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mutations were targeted to the Hprt locus of mouse embryo-derived stem cells by using 22 different sequence replacement and sequence insertion vectors. The targeting frequency was examined at two sites within the Hprt locus as a function of the extent of homology between the targeting vector and the target locus. The targeting frequency was also compared by using vectors prepared from isogenic and nonisogenic DNA sources. With one exception, all of the vectors showed the same exponential dependence of targeting efficiency on the extent of homology between the targeting vector and the target locus. This was true regardless of whether they were sequence replacement or sequence insertion vectors, whether they were directed toward either of the two different sites within the Hprt locus, or whether they were prepared from isogenic or nonisogenic DNA sources. Vectors prepared from isogenic DNA targeted four to five times more efficiently than did the corresponding vectors prepared from nonisogenic DNA. The single case of unexpectedly low targeting efficiency involved one of the vectors prepared from nonisogenic DNA and could be attributed to an unfavorable distribution of heterology between the Hprt sequences present in the targeting vector and the endogenous Hprt gene.


Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid. The gene coding for apolipoprotein AI (apoAI), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine. Liver-specific expression is controlled by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located between nucleotides -222 and -110 upstream of the apoAI gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXR alpha over the previously characterized RA receptors RAR alpha and RAR beta. In this study we investigated the combined effects of ARP-1 and RXR alpha on apoAI gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXR alpha-mediated transactivation of the apoAI gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXR alpha bind to site A with similar affinities. These assays also revealed that ARP-1 and RXR alpha bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXR alpha alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAI gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXR alpha has very little effect on the levels of expression regardless of the presence or absence of RA. However, while ARP-1 alone or ARP-1 and RXR alpha together dramatically repress expression in the absence of RA, the repression by ARP-1 and RXR alpha together, but not ARP-1 alone, is almost completely alleviated in the presence of RA. These results indicate that transcriptional repression by ARP-1 sensitizes apoAI gene responsiveness to RXR alpha and RA and suggest that the magnitude of this responsiveness is regulated by the intracellular ratio of ARP-1 to RXR alpha. These observations raise the possibility that transcriptional repression is a general mechanism for switching gene transcription between alternative transcription activation pathways.