[Differentiation between hepatoma and hemangioma by inversion recovery snapshot FLASH MR imaging with variable TIs]. To differentiate hepatoma from hemangioma, MR studies were performed for 18 patients with 21 untreated liver tumors (hepatoma 10, hemangioma 11). We obtained inversion recovery snapshot FLASH images of liver tumors with variable TIs (50, 100, 150, 200, 250, 300, 400, and 500 msec). 8 hemangiomas showed higher intensity than liver parenchyma on the images at 150 msec and less of TI, and lower intensity at 200 msec or more TI. In 7, signal intensity of hepatoma became low at 150 msec or less of TI or remained high with prolonged TI. Inversion recovery snapshot FLASH imaging with variable TIs will be useful for differentiating between hepatoma and hemangioma.


The cleavage of DNA at phosphorothioate internucleotidic linkages by DNA gyrase. We have constructed a plasmid which contains 22 copies of a 147 bp DNA fragment which contains the major DNA gyrase cleavage site from plasmid pBR322 (located at base-pair 990). We have found that this fragment is efficiently bound and cleaved by gyrase. The selectivity for the sequence corresponding to position 990 in pBR322 is maintained even when this site is located only 15 bp from one end of the 147 bp fragment. A strategy for the specific incorporation of a single thiophosphoryl linkage into the 147 bp fragment has been developed, and gyrase has been shown to catalyse efficient cleavage of fragments bearing phosphorothioate linkages at the gyrase cleavage site in one or both strands.


Characterization of an enhancer upstream from the muscle-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The muscle-type isozyme of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is encoded by a mRNA transcribed from the M promoter of a 55-kb gene, which also produces the liver-type isozyme from an alternative promoter. By transient transfection and in vitro protein-DNA binding assays we have delineated, within 4.7 kb of 5' flanking sequence, the M promoter proper and an enhancer located between -1615 and -1809. This enhancer stimulated up to 12-fold the activity of the promoter in the context of an intact 5' flanking sequence and close to 900-fold the activity of the minimal (+41 to -40) M promoter cloned directly downstream from it. A functional dissection of the enhancer by site-directed mutagenesis and use of oligonucleotides suggested that its activity involves the cooperative effect of six binding sites for trans-acting factors clustered within 150 bp. These sites contain either an EF-1A/E4TF1 motif (also known to bind the ets oncogene product) or a Sp1 motif, or both. The activity of the enhancer could be demonstrated in L6 myoblasts and myocytes and in FTO2B hepatoma cells. When left within the intact 5' flanking sequence, however, enhancer activity was inhibited upon differentiation of myoblasts into myocytes.


A new affinity reagent for the site-specific, covalent attachment of DNA to active-site nucleophiles: application to the EcoRI and RsrI restriction and modification enzymes. A modified oligodeoxyribonucleotide duplex containing an unnatural internucleotide trisubstituted 3' to 5' pyrophosphate bond in one strand [5'(oligo1)3'-P(OCH3)P-5'(oligo2) 3'] reacts with nucleophiles in aqueous media by acting as a phosphorylating affinity reagent. When interacted with a protein, a portion of the oligonucleotide [--P-5'(oligo2)3'] becomes attached to an amino acid nucleophilic group through a phosphate of the O-methyl-modified pyrophosphate linkage. We demonstrate the affinity labeling of nucleophilic groups at the active sites of the EcoRI and RsrI restriction and modification enzymes with an oligodeoxyribonucleotide duplex containing a modified scissile bond in the EcoRI recognition site. With the EcoRI and RsrI endonucleases in molar excess approximately 1% of the oligonucleotide becomes attached to the protein, and with the companion methyltransferases the yield approaches 40% for the EcoRI enzyme and 30% for the RsrI methyltransferase. Crosslinking proceeds only upon formation of a sequence-specific enzyme-DNA complex, and generates a covalent bond between the 3'-phosphate of the modified pyrophosphate in the substrate and a nucleophilic group at the active site of the enzyme. The reaction results in the elimination of an oligodeoxyribonucleotide remnant that contains the 3'-O-methylphosphate [5'(oligo1)3'-P(OCH3)] derived from the modified phosphate of the pyrophosphate linkage. Hydrolysis properties of the covalent protein-DNA adducts indicate that phosphoamide (P-N) bonds are formed with the EcoRI endonuclease and methyltransferase.


Structural organization and diversification of Y-linked sequences comprising Su(Ste) genes in Drosophila melanogaster. Expression of the X-linked repeated Stellate (Ste) genes, which code for a protein with 38% similarity to the beta-subunit of casein kinase II, is suppressed by the Su(Ste) locus on the Y chromosome. The structure and evolution of the Y-linked repeats in the region of the Su(Ste) locus were studied. The 2800 bp repeats consist of three main elements: the region of homology to the Ste genes, an adjacent AT-rich, Y-specific segment, and mobile element 1360 inserted in the Ste sequence. Amplification of repeats was followed by point mutations, deletions, and insertions of mobile elements. DNA sequencing shows that these repeats may be considered as Ste pseudogenes or as damaged variants of a putative gene(s) encoding a protein quite different from the Ste protein as a result of an alternative splicing pattern. A comparison of 5 variants of the Y-Su(Ste) repeats shows a number of recombination events between amplified and diverged sequences that could be due to either multiple unequal mitotic sister-chromatid exchanges or to gene conversion. It is a first demonstration on a molecular level of these processes occurring in heterochromatic non-rDNA tandemly organized sequences in an eukaryotic genome.


Rapid shotgun cloning utilizing the two base recognition endonuclease CviJI. A new approach has been developed for the rapid fragmentation and fractionation of DNA into a size suitable for shotgun cloning and sequencing. The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions which alter the specificity of this enzyme (CviJI**) yield a quasi-random distribution of DNA fragments from the small molecule pUC19 (2686 base pairs). To quantitatively evaluate the randomness of this fragmentation strategy, a CviJI** digest of pUC19 was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI** restricts PyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation. Advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 micrograms instead of 2-5 micrograms), fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed), and higher cloning efficiencies are obtained (CviJI** digested and column fractionated DNA transforms 3-16 times more efficiently than sonicated, end-repaired, and agarose fractionated DNA).


123I-iomazenil: a quantitative study of the central benzodiazepine receptor distribution. Fourteen patients with temporal lobe epilepsy, 9 patients after amygdalohippocampectomy and 3 healthy volunteers were examined with the new benzodiazepine receptor marker 123I-Iomazenil and SPECT. For comparison perfusion SPECT studies with 99mTc-HMPAO were done and a quantitative ROI analysis of the data performed. This quantitative analysis consisted of calculation of right-to-left ratios for 123I-Iomazenil SPECTs, whereby values of 1 were obtained with narrow standard deviations. ROI measurements of the medial occipital, frontal and parietal cortex, the cerebellum and white matter showed a pattern of benzodiazepine receptor concentration in concordance with that previously found in PET and autoradiographic studies, if 123I-Iomazenil ROIs were normalized to the corresponding 99mTc-HMPAO ROIs. The abnormal distribution in the temporal lobes will not be discussed in this paper.


[Occurrence of antibodies against hepatitis C virus (HCV) among alcoholics]. 144 consecutive alcoholics attending an outpatient clinic were tested for the prevalence of anti-HCV with commercially available ELISA test (Abbott HCV EIA). Positive sera were further tested with neutralization assay (Abbott HCV Neutralization Assay). Anti-HCV were found in 35 (24%). There was no difference in the prevalence or mean titre of anti-CMV and anti-HSV between patients with and without anti-HCV what points to the specificity of tested antibodies.


[Occurrence of antibodies against hepatitis C virus (HCV) among drug addicts]. The prevalence of anti-HCV and serological markers of HBV infection was tested in 100 unselected intravenous drug addicts admitted to a detoxification ward. Anti-HCV were present in 78% addicts, and at least one markers of HBV infection was present in 69% addicts. No correlation was found between anti-HCV status and age, gender, duration of addiction or anti-HIV presence. Furthermore, there was no difference in mean titres of anti-CMV and anti-HSV between subjects with and without anti-HCV what suggests that the anti-HCV tested were specific.


[Cerebral glioblastoma: a new complication of HIV-1 infection]. A new case of supratentorial malignant glioma is reported in an HIV-1 infected male homosexual. Tumours of the nervous system account for only 5 to 10 percent of neurological complications of AIDS, and most of them are lymphomas or metastases from Kaposi's sarcomas. In fact, HIV-1 is a neurotropic lentivirus, not transforming by definition. Our patient had a frontal tumoral syndrome resistant to the conventional anti-toxoplasmic treatment. Pathological examination of a tumoral fragment obtained by stereotactic biopsy showed that according to the WHO criteria the tumour was a glioblastoma. The mechanism through which HIV infection results in malignant transformation of astrocytes is conjectural. There is no consensus on whether the virus is located in glial cells, but the transgenic animal technique suggests that the tat gene might play a certain role. Other hypotheses concerning the indirect neurotoxicity of HIV have been put forward, notably that of viral coinfection with viruses of the papova group.


[Choice and monitoring of the treatment of systemic mycoses. Value and limitations of in vitro tests]. Systemic fungal infections are an important cause of morbidity and mortality among immunocompromised patients. New antifungal agents, such as triazoles, are now available, and the place of in vitro tests has to be discussed. It has been shown that interlaboratory reproducibility of in vitro susceptibility tests against fungi was low, due to the lack of standardization. Recently, the NCCLS defined conditions permitting a good interlaboratory reproducibility. However, the predictive value of in vitro susceptibility tests on the therapeutic outcome remain to be demonstrated, and is now under investigation. At the present time, susceptibility testing can be useful: in patients treated by amphotericin B for a severe fungal infection and who do not improve under therapy; to detect resistance to 5-fluorocytosine; to compare the sensitivity to triazoles before and after treatment, in case of therapeutic failure. Serum levels monitoring is useful to prevent the toxicity due to 5-fluorocytosine and to control the digestive absorption of triazoles, especially the lipophilic compound itraconazole.


Mumps and Coxsackie B3 virus infection of human fetal pancreatic islet-like cell clusters. Islet-like cell clusters (ICCs) prepared from human fetal pancreases were infected with mumps or coxsackie B3 virus. Double-labeled antibody technique showed that the viruses infected both insulin-secreting and other pancreatic cells and that secretion of immunoreactive insulin into the culture medium of the mumps virus-infected cells had already ceased on day 7. The mumps virus-infected ICC clusters produced virus for 14 days, and the mumps virus antigen was detected in the ICCs through the whole 22-day observation period. The coxsackie B3 virus-infected ICCs contained cells highly positive for viral antigen during the first 2 days after infection, and the infectious virus was detected in the culture medium for 22 days. This in vitro model indicates that mumps and coxsackie B3 viruses infect human fetal pancreatic endocrine cells and are able to alter beta-cell function. Coxsackie B3 virus infection in ICCs is lytical and seems to lead to rapid cell destruction, but long-lasting, restricted mumps virus infection in human fetal pancreatic ICCs offers an interesting model to study the effects of viral infection in the endocrine pancreas and beta cell.


Cellular mechanisms of action of mineralocorticoid hormones. Mineralocorticoid hormones are a subset of steroid hormones that act primarily in epithelial tissues to regulate ion transport of Na+, K+ and H+. Cellular specificity is conferred by receptors which act in the nucleus to stimulate gene expression. Transcription and subsequent translation result in the production of new proteins which mediate the physiologic effects. The mechanisms involved in receptor specificity and localization, in regulation of gene activation, and in expression of transport effects are reviewed. The cellular actions of mineralocorticoids fit well with the general model of steroid hormone action but considerable questions remain at each step in the process.


The effect of triacylglycerol chain length on food intake in domestic fowl. Triacylglycerols of varying chain lengths were tested for their effects on food intake in fasted Single-Comb White Leghorn cockerels 9-12 weeks of age. Tributyrin, tricaprylin, triolein, or saline were intubated intragastrically or infused intrahepatically. All triacylglycerol administrations were in 10 ml volumes with 10 kcal being infused intrahepatically and 10 or 20 kcal being infused intragastrically. When administered intragastrically, the short-chained triacylglycerol tributyrin decreased food intake at most times tested; the medium chain-length tricaprylin and the long chain-length triolein affected food intake sporadically. When infused into the liver, tributyrin and tricaprylin decreased food intake at most times tested, whereas triolein decreased food intake at 60 and 180 minutes only. Satiating effectiveness was related to triacylglycerol chain length when intubated, which may reflect rate of absorption from the gut; however, hepatic satiety receptors were also differentially sensitive to triacylglycerol chain length indicating that more than rate of assimilation from the gut must be considered.


Acute noise stress, ACTH administration, and blood pressure alteration. Two groups of male Wistar rats were submitted to a single noise exposure (2640 Hz, 30 W, 102 dB, 15 min) (St group) or to a single dose of ACTH (1.5 IU/100 g b.wt.) (Ac group), respectively. A control group of nontreated rats (Co group) was used. Blood pressure (BP) was measured using an indirect tail cuff method and corticosterone (B) levels were measured by specific RIA. Haematocrit (Hc) and blood pH values were also evaluated. Acute treatments of both noise exposure and ACTH administration produced corticosterone hypersecretion and blood pressure elevation, with lower haematocrit and higher blood pH values than those found in the Co group. No differences were found between St and Ac treatments.


Effects of acute swim stress on LiCl-induced conditioned taste aversions. The present study examined the effects of a 5-min period of swim stress experienced between a flavor (saccharin) and illness (LiCl) on conditioned taste aversion learning. Experiment 1 obtained a stress-induced attenuation of learning. Experiment 2 replicated the findings of Experiment 1, and also obtained a similar attenuation when stress was administered 30 min prior to the saccharin presentation. Experiment 3 examined the effects of swim stress either 15 min or 90 min after the LiCl had been administered. It was found that swim stress 15 min after LiCl significantly attenuated CTA, but swim stress 90 min after LiCl did not. These results are discussed with regard to current views of the relationship between external events and conditioned taste aversions.


Prolactin as a link between behavioral and immune differences between the Roman rat lines. Roman high (RHA)- and low (RLA)-avoidance rats are two lines of Wistar rats genetically selected on the basis of their active avoidance behavior in a shuttle-box. They also differ in several other behavioral responses, such as their locomotor activity in novel environments (open-field, circular corridor), with the RHA rats being more active than the RLA animals, as well as in endocrine reactivity and immune functions. These experiments were designed to investigate further the neuroendocrine characteristics of these animals as a possible link between the brain and immune functions. Despite the marked behavioral and immune differences observed, no between-lines variation could be found in basal hypothalamo-pituitary-adrenocortical axis activity or in its responses to different protocols of novel environment stress, or after corticotropin-releasing factor (CRF) challenge. On the other hand, stimulated prolactin levels were higher in the low avoidance line. These results exclude the pituitary-adrenocortical axis and suggest prolactin as a link between behavioral and immune differences between the Roman lines. Moreover, these results indicate that these rats may be an excellent model for the study of the relationships between the brain and the immune system.


Effects of controllable vs. uncontrollable chronic stress on stress-responsive plasma hormones. We have previously reported effects of chronic stress on circadian rhythms of temperature, eating, and locomotor activity. These studies were conducted using an around-the-clock signalled intermittent footshock paradigm in which some rats have control over shock termination while other rats are yoked to the rats with control. Although this paradigm is stressful, as suggested by decreases in food intake and disrupted circadian rhythms, rats tolerate the paradigm well, continuing to eat, drink, gain weight, and groom. In the present studies, rats were sacrificed following 3 or 14 days of stress, and plasma was collected for hormonal assays. After 3 days of stress, plasma corticosterone and prolactin levels were elevated in both stress groups compared to controls; yoked rats had higher levels of corticosterone than rats in the group with control over shock termination, while prolactin levels in both stressed groups were similar. ACTH levels were similar in stressed and control rats. After 14 days of stress, ACTH and corticosterone levels in both stress groups were similar to control levels. Prolactin levels were elevated in the yoked experimental group compared to levels in control or controllable stress groups. These data support previous studies suggesting that control over stressors attenuates the effects of stress on physiology and demonstrate that two hormones with diverse biological effects are elevated by chronic stress.


Identification of a resin-dentin hybrid layer in vital human dentin created in vivo: durable bonding to vital dentin. The present study investigated the bond of 5% 4-methacryloxyethyl trimellitate anhydride in methyl methacrylate, initiated by partially oxidized tri-n-butyl borane in the presence of poly(methyl methacrylate) powder, to vital human dentin. In vivo dentinal substrates were pretreated for 10 or 30 seconds with an aqueous solution of 10% citric acid and 3% ferric chloride. Transmission electron microscopic examination of the bonded cross sections revealed the formation of a transitional, or "hybrid," layer of resin-reinforced dentin created by the impregnation, co-mingling and envelopment of collagen bundles, and encapsulation of hydroxylapatite crystals. The in vivo adhesion was assumed to be durable, because results of microscopic examinations were comparable to those of durable bonding of the same resin to extracted bovine dentin. Vital dentin exhibited greater resistance to demineralization by the acid solution than do extracted teeth. Carious extracted teeth were more easily dissolved in acid than were noncarious extracted teeth.


[Evaluation of brain tumors using positron emission tomography]. The clinical application of positron emission tomography (PET) for the evaluation of brain tumours has proved clinically valuable. Amino acid and FDG-glucose PET provide information on the degree of malignancy and the prognosis during the initial evaluation. After therapy, the residual tumour can be visualized and recurrence can be differentiated from necrosis. Amino acids have advantages over FDG for these clinical applications. Blood flow, oxygen extraction and metabolism and blood-brain barrier permeability are of minor relevance in clinical situations. Comparison of PET with MRI and MRS will provide new data. The quantitative information of the unique information yielded by PET will lead to a more important clinical role, as will the extrapolation of this experience to the SPECT technique.


GABA receptor alterations after chronic lithium administration. Comparison with carbamazepine and sodium valproate. 1. Effects of lithium, carbamazepine, sodium valproate and baclofen on GABA receptors were examined in several regions of the rat brain. 2. [3H]Muscimol (MUS) and [3H] (-)baclofen (BAC) were used to label GABAA and GABAB receptors, respectively, in synaptic membranes from rat brain. 3. Single treatment with lithium chloride, carbamazepine or sodium valproate did not change [3H]MUS or [3H]BAC binding in the frontal cortex, hippocampus and thalamus. 4. Following chronic treatment with lithium, carbamazepine or sodium valproate, [3H]BAC binding was significantly increased in the hippocampus but not in the frontal cortex, thalamus or striatum. 5. [3H]Muscimol binding did not change in any region examined after chronic treatment with lithium, carbamazepine or sodium valproate. 6. Single and chronic administration of baclofen did not change [3H]MUS or [3H]BAC binding. 7. One common mechanism of action of mood stabilizers may be mediated by GABAB receptors in the hippocampus.


Protein kinase C impairs the coupling of the GTP-binding protein to LTB4 receptor in neutrophil. In the present study, the mechanism of LTB4 receptor down regulation by protein kinase C (PKC) has been investigated using porcine neutrophil membranes. Pretreatment of intact porcine neutrophils with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 min prior to the preparation of plasma membrane, demonstrated a reduced binding sites (Bmax) for LTB4 without altering the receptor affinity (Kd). This effect of TPA on LTB4 receptor binding was found to be due to the activation of PKC as membrane treated with purified PKC (type III) produced the same effect. When membranes from neutrophils pretreated with TPA were exposed to non-hydrolyzable GTP analog, GTP-gamma S, or GMP-PNP, no further decrease in receptor Kd was observed, while the Bmax was reduced to the level observed in TPA treated samples. Treatment of isolated neutrophil membranes with purified PKC reduced the Bmax and blocked the effect of GTP analogs on the receptor affinity. These results suggest that, PKC interrupts the receptor binding to G-protein.


Cell-type specific responses in prostaglandin secretion by glandular and stromal cells from pig endometrium treated with catecholestrogens, methoxyestrogens and progesterone. The pig conceptus and endometrium possess the ability to convert estrogens into catecholestrogens and catecholestrogens into methoxyestrogens. Experiments were carried out to evaluate the effect of catecholestrogens, methoxyestrogens and progesterone on the secretion of prostaglandin (PG) E and F2 alpha by porcine endometrial glandular and stromal cells in vitro. Both 2-hydroxyestradiol (2-OH-E2, 0-20 microM) and 4-hydroxyestradiol (4-OH-E2, 0-20 microM) increased (P less than .05) PGE and PGF2 alpha secretion by stromal cells in a dose response manner. Two-hydroxyestradiol tended (P less than .1) to decrease PGF2 alpha production by glandular cells. Two-methoxyestradiol (20 microM) suppressed (P less than .05) PGF2 alpha secretion by glandular and stromal cells. Four-methoxyestradiol (20 microM) stimulated (P less than .05) PGE production and PGE:PGF2 alpha ratio. Progesterone (.1 microM) suppressed (P less than .05) PG secretion in both cell types. We conclude that catecholestrogens, methoxyestrogens, and progesterone may participate in the establishment of pregnancy by modulating PG production in the endometrium.


Cloning of Campylobacter jejuni genes required for leucine biosynthesis, and construction of leu-negative mutant of C. jejuni by shuttle transposon mutagenesis. Campylobacter jejuni is a Gram-negative pathogen responsible for diarrhoeal diseases in humans. To date, very little is known about the genetic organization and molecular biology of this microorganism. The cosmid vector pHC79 was used to construct a genomic library from the total genomic DNA of C. jejuni strain C31 in Escherichia coli and recombinant cosmids capable of complementing the auxotrophic defect in leucine biosynthesis of E. coli HB101 were identified. Three of 400 clones tested were found to be capable of complementing the nutritional defect of E. coli HB101 as well as those of independent leuB mutants of E. coli strains. These results indicated that the cloned genes responsible for leucine complementation encoded an enzyme analogous to the beta-isopropylmalate dehydrogenase specified by the leuB gene in E. coli strains. The sizes of the recombinant cosmids which became stabilized in E. coli cells ranged from 12.9 to 15.4 kb compared to the expected, originally packaged, 45- to 50-kb molecules, attesting to major rearrangements occurring in this background. The recombinant plasmid pILL547 was shown to carry genes that were analogous to the leuB gene and also to the leuC and leuD genes of E. coli. The gene required for leuB complementation was subcloned on a 1.6-kb restriction fragment and was mapped more precisely by insertional mutagenesis using as transposon a newly constructed (MiniTn3-Km) element engineered to mutagenize Campylobacter genes. The leuB gene of C. jejuni was shown to be expressed from its own promoter in E. coli cells. In E. coli minicells, the cloned insert encoded a polypeptide with an apparent molecular weight of 40 kDa. A leucine auxotrophic mutant of C. jejuni strain C31 was constructed in vitro by allelic exchange, replacing the original copy of the leucine gene by an allele mutated by the insertion of the kanamycin transposable element.


Effect of Bdellovibrio bacteriovorus infection on the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli: evidence for activation of cytoplasmic proteolysis. Intact cells of Bdellovibrio bacteriovorus strain 109J were found to be incapable of taking up 14C-methyl alpha-glucoside, mannitol or fructose, and extracts derived from these cells exhibited negligible activities of the protein components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Escherichia coli strain ML35 cells exhibited high in vivo sugar uptake activities that were progressively lost over a period of 2 h at 30 degrees C following the entry of B. bacteriovorus into the periplasm of E. coli. In vitro complementation assays revealed that the E. coli PTS enzymes, enzyme I, HPr, and the glucose- and mannitol-specific enzymes II, were all lost almost in parallel with the disappearance of uptake activity. Thus, loss of activity in vivo was not due to membrane leakiness, energy depletion, or preferential inhibition or inactivation of any one protein component of the PTS. Instead, loss of PTS activity was attributed to digestion of the protein constituents of the system by proteases present in the cytoplasm of the host cell after bdellovibrio entry. Both ethylenediaminetetraacetate and phenylmethylsulphonyl fluoride partially protected against inactivation in vitro, and the two inhibitors together gave full protection, suggesting that both metallo- and seryl-proteases were responsible for the inactivation. Protease activity increased progressively with time following bdellovibrio entry and appeared to degrade the E. coli PTS enzymes in vivo. Preliminary evidence suggested that the proteases responsible for PTS enzyme degradation may be encoded by the B. bacteriovorus chromosome.


Human papillomavirus involvement in esophageal precancerous lesions and squamous cell carcinomas as evidenced by microscopy and different DNA techniques. A series of 71 surgically resected esophageal squamous cell carcinomas, including 51 cases of formalin-fixed samples and 20 cases of fresh biopsy specimens derived from the high-incidence area of esophageal cancer in China, were systematically analyzed for the presence of human papillomavirus (HPV) infections by light microscopy, electron microscopy (TEM), in situ DNA hybridization, Southern blot hybridization, and polymerase chain reaction (PCR) techniques. On light microscopy, HPV-suggestive lesions were found in a total of 49.0% (25 of 51) of the specimens, including the flat type (22 of 51) and, less frequently, an inverted one (2 of 51). Of the 51 formalin-fixed, paraffin-embedded specimens, 43.1% (22 of 51) contained HPV DNA sequences by in situ hybridization. Of the positive cases, HPV 6 was present in three (5.9%), HPV 11 in three (5.9%), HPV 16 in eight (15.7%), HPV 18 in six (11.8%), double infections with HPV 11/18 in one (2.0%), and HPV 16/18 in one. In most cases the HPV-positive signals were localized in the hyperplastic and/or dysplastic epithelium adjacent to invasive carcinomas. In two specimens, however, HPV DNA sequences were found in the frankly invasive lesions, one being HPV 6 and the other HPV 18. On TEM, HPV-like particles located in the nuclei of koilocytotic cells were demonstrated in two of the five specimens previously shown to be HPV-positive by in situ hybridization. By means of the PCR technique, all specimens positive for HPV by in situ hybridization also contained amplified HPV sequences. Moreover, three additional samples negative by in situ hybridization were found to contain HPV 11 DNA sequences. Of the 20 DNA samples extracted from the fresh carcinoma samples (containing some surrounding tissues as well) 9 were shown to contain HPV DNA sequences by Southern blot hybridization under low-stringency conditions. Of these, eight samples remained positive when hybridized with the probe cocktail of HPV 11, 16, 18, and 30 DNA under high-stringency conditions. HPV DNA sequences in these carcinoma specimens appeared to be present mainly in an integrated form. The present results confirm the HPV involvement in esophageal squamous cell lesions and suggest that HPV infection might be an important etiologic factor in the pathogenesis of esophageal cancer, most probably acting synergistically with other carcinogenic factors.


Two-step PCR in the retrospective diagnosis of enteroviral viraemia. Enteroviral infections are potentially serious in man causing a variety of syndromes and a viraemic phase is probable in most patients. However, virological diagnosis is usually indirect by isolation of the agent from throat or stools when possible, and/or slow due to the need to titrate antibodies in paired sera. Because of its high sensitivity the polymerase chain reaction (PCR) offers the possibility of rapid detection of even low-grade viraemia. We have developed a 2-step PCR procedure adapted for enteroviruses and applied it to stored sera from patients with enteroviral meningitis. The acute sera from 7/12 patients were positive but the 4 convalescence sera tested, 2 of which had been positive at the acute phase, were negative. The assay should now be tested prospectively on serum and cerebrospinal fluid and could be developed for routine diagnosis.


Foscarnet for treatment of cytomegalovirus infections in bone marrow transplant recipients. 42 episodes of verified or clinically suspected cytomegalovirus (CMV) infection in 40 bone marrow transplant (BMT) recipients were treated with foscarnet (trisodium phosphonophormate hexahydrate). CMV infection was verified in 31/42 treatment episodes. Symptoms treated were pneumonia (n = 17), pancytopenia with or without fever (n = 12), enteritis (n = 5), fever (n = 4), encephalitis (n = 2), retinitis (n = 1) and hepatitis (n = 1). Foscarnet was given as a continuous intravenous infusion. Side-effects observed were increase in serum creatinine (38%), decrease in serum calcium (19%), increase in serum bilirubin (12%), decrease in hemoglobin concentration (7%), increase in serum calcium (5%), increase in serum transaminase (5%), hypophosphatemia (2%) and tremor (2%). CMV was eradicated from blood and/or urine in 11/25 (44%) of assessable treatment episodes with infection verified by isolation. Overall clinical improvements including eradication of CMV, afebrility and/or improvements in laboratory abnormalities were seen in 14/31 (45%) episodes of verified infection. All 15 patients with CMV interstitial pneumonia (CMV IP) died. We conclude that foscarnet is nephrotoxic but otherwise well tolerated with moderate clinical and virostatic effects on CMV infection. The effect on CMV IP is discouraging.


Isolation of polioviruses from sewage and their characteristics: experience over two decades in Sweden. Indigenous polio ceased in Sweden in 1962 after 5 years' use of killed polio vaccine. In 1967, it was considered of interest to investigate whether poliovirus was present in the sewage. A method for selective isolation of poliovirus from sewage was developed. The method appeared to increase the yield. The studies were carried out at intervals up to 1990. In 1989-90, the virus isolates were characterized by the use of monoclonal antibodies differentiating between vaccine-like (Sabin-like) and non-vaccine-like strains. Polioviruses of both kinds were isolated throughout the period. Two periods were of special interest. The first was in 1977, when a single, paralytic, type-2 case occurred in Sweden in an unvaccinated sect. The second was in 1984-85 when a type-3 epidemic broke out in Finland, followed by vaccinations of the whole Finnish population with live oral polio vaccine. On both occasions the implicated viruses could be traced to a high degree in sewage in Sweden. The absence of poliovirus isolations from faecal specimens of patients and the isolation of live poliovirus vaccine virus, i.e. a vaccine not used in Sweden, indicate that the virus strains are imported.


Intraepithelial neoplasia of the lower female genital tract: etiology, investigation, and management. Lower genital tract neoplasia appears associated with certain types of the human papilloma virus. Surgical approaches using lasers and electricity are available to treat the clinical diseases and in turn provide good cosmetic and functional anatomy. The surgical method employed depends upon the anatomical and histological distribution of disease regardless of its site. Of the three areas (cervix, vagina and vulva), cervical intraepithelial neoplasia is the least difficult to cure. Vulvar intraepithelial neoplasia is the most difficult since recurrences can occur at any time and in some patients for many years.


Effect of piperacillin/tazobactam therapy on intestinal microflora. The effect of piperacillin/tazobactam treatment upon the intestinal microflora was studied in 20 patients with intraabdominal infections. The patients received piperacillin 4 g combined with tazobactam 500 mg q 8 h by intravenous injection for 4-8 days. Stool specimens were collected before, during and after therapy for cultivation of aerobic and anaerobic microorganisms. Six patients had measurable concentrations of piperacillin (1.2-276 mg/kg/faeces) and 4 patients tazobactam concentrations (0.8-22.2 mg/kg/faeces) in the faecal specimens during therapy. The number of enterobacteria and enterococci slightly decreased while there were no changes in the number of staphylococci and bacilli. The anaerobic microflora was also slightly affected. There was a minor decrease in the number of bifidobacteria, eubacteria, lactobacilli, clostridia and veillonella but the numbers of anaerobic Gram-positive cocci and bacteroides were not influenced by the treatment. After therapy, the aerobic and anaerobic microflora returned to normal levels in all patients. None of the patients had Clostridium difficile or cytotoxin in the stools or developed diarrhoea.


The association of serum hepatitis C virus RNA with serum aminotransferase activities. Serum samples from 71 patients with hepatitis C virus infection at various stages were studied to determine the clinical significance of the detection of hepatitis C virus RNA by the polymerase chain reaction. There was a significant correlation of serum HCV RNA with elevation of the serum aminotransferase levels. These data suggest that the detection of HCV RNA in the serum might be useful for evaluation of the extent of ongoing liver damage.


Pharmacodynamic effects of antibiotics. Studies on bacterial morphology, initial killing, postantibiotic effect and effective regrowth time. Pharmacodynamics of antibiotics deals with time course of drug activity and mechanisms of action of drugs on bacteria. In this thesis pharmacodynamic parameters have been studied after brief exposure of gram-positive bacteria to daptomycin, imipenem or vancomycin and after short exposure of gram-negative bacteria to amikacin, ampicillin, aztreonam, cefepime, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, imipenem, mecillinam, or piperacillin. The studies have been focused on morphological alterations, initial killing, postantibiotic effect (PAE) and effective regrowth time (ERT) and a method, based on bioluminescence assay of intracellular ATP has been used. The basic principle behind this technique is that ATP in living cells is present in a relatively constant amount, and hence affords a measure of the number of microbial cells. The PAE describes the delayed regrowth of bacteria after brief exposure to antibiotics. The number of cells measured after this antibiotic exposure describes the initial killing and is also the start value for calculating the PAE. PAEs of 2-3 h were obtained by bioluminescence for gram-positive bacteria exposed to imipenem or vancomycin. This is in agreement with results obtained by viable count and is probably due to similar weak initial decrease in cell density when assayed by both methods. Long (greater than 3 h) concentration dependent PAEs and moderate (less than or equal to 1 log10) initial decrease in intracellular ATP were in general seen for gram-positive bacteria exposed to daptomycin and for gram-negative bacteria exposed to imipenem or amikacin when assayed by bioluminescence. These very long PAEs and rather weak initial killing have to be compared with the shorter PAEs and stronger initial killing reported by us and others using viable count. Furthermore, this study showed that there was a relatively good concordance between microscopy and bioluminescence, which are direct methods, in determining the initial killing and PAE of imipenem on Escherichia coli. The ERT, defined as the time for bacterial density to increase 1 log10 from the pre-exposure inoculum, was independent of the method used for measuring regrowth of E. coli after brief exposure to imipenem. The combination of mecillinam with ampicillin, aztreonam, ceftazidime or piperacillin and the combination of amikacin with ceftazidime, ceftriaxone or piperacillin induced longer PAEs on gram-negative bacteria than the sum of PAEs of the individual antibiotics. A strong initial killing in combination with a long PAE cause a long ERT and may allow the antibiotic concentration to stay below MIC during long periods of time without any regrowth. This may, in clinical practice, have implications for long dosing intervals.


Phospholipase A2 inhibition decouples lung injury from gut ischemia-reperfusion. Phospholipase A2 (PLA2) has recently been implicated as a key enzyme of local inflammation after gut ischemia-reperfusion (I/R). The hypothesis of this study is that PLA2 inhibition decouples remote organ injury from gut I/R. Sprague-Dawley rats were pretreated with a PLA2 inhibitor, quinacrine (10 mg/kg, intravenously), before the induction of gut ischemia (45 minutes of superior mesenteric artery occlusion) followed by 6 hours of reperfusion. 125I-labeled albumin leak was employed as a marker of pulmonary endothelial permeability and myeloperoxidase as a monitor of neutrophil (PMN) traffic in the gut and lung. To further characterize the impact of PLA2 inhibition, PMNs were harvested at 6 hours of reperfusion and superoxide production was measured in the presence or absence of an activating stimulus, N-formyl-methionyl-leucyl-phenylalanine. Gut I/R increased gut PLA2 activity, elicited gut PMN influx, and produced lung leak; these events were prevented by PLA2 blockade. Gut I/R also markedly enhanced PMN superoxide production with N-formyl-methionyl-leucyl-phenylalanine, and this priming was ablated by PLA2 inhibition. These data suggest that PLA2 activation is a proximal step in the pathogenesis of distant organ injury after splanchnic hypoperfusion, a process that appears to involve PMN priming in the gut bed.


Ketoconazole inhibits alveolar macrophage production of inflammatory mediators involved in acute lung injury (adult respiratory distress syndrome). Acute inflammatory lung injury (adult respiratory distress syndrome [ARDS]) causes significant morbidity and death in surgical patients. The alveolar macrophage elaborates proinflammatory mediators implicated in acute pulmonary injury. The macrophage products, leukotriene B4 (LTB4), thromboxane A2 (TXA2), and procoagulant activity (PCA), initiate inflammatory cascades that lead to microvascular thrombosis and neutrophil infiltration, two common features of ARDS. One potential method of preventing or attenuating lung injury is to inhibit the production of inflammatory mediators. Preliminary studies indicate that ketoconazole, known primarily for its antifungal properties, may prevent ARDS. LTB4, TXB2, and PCA production by rabbit alveolar macrophages was measured after treatment with endotoxin or Ca ionophore and ketoconazole or selective 5-lipoxygenase (MK 886) and thromboxane synthetase (imidazole) inhibitors. Ketoconazole significantly inhibits alveolar macrophage production of LTB4, TXB2, and PCA. Ketoconazole inhibition of PCA is independent of effects on 5-lipoxygenase and thromboxane synthetase. Ketoconazole inhibition of alveolar macrophage proinflammatory mediators may be of benefit in preventing ARDS by minimizing neutrophil infiltration and microvascular thrombosis. Inhibition of 5-lipoxygenase and thromboxane synthetase, without affecting cyclooxygenase, may offer a selective advantage by allowing production of other homeostatic eicosanoids.


Human B-cell lymphoma in severe combined immunodeficient mice after active infection with Epstein-Barr virus. B-cell lymphomas (BCL) occur with increased frequency in immunosuppressed patients. BCL develop in severe combined immunodeficient (SCID) mice after engraftment with human peripheral blood leukocytes (PBL; hu-PBL-SCID mice) and infection with Epstein-Barr virus (EBV). The contributions of latent and active EBV infection to BCL development, the potential enhancing effects of immunosuppressive therapy, and inhibitory effects of antiviral therapy on the development of BCL in this model were studied. SCID mice were engrafted with PBL from EBV-seropositive donors (latent infection), PBL from EBV-seronegative donors followed by infection with EBV (active infection), PBL from EBV-seropositive donors followed by infection with EBV (latent plus active infection), or EBV-transformed B-lymphoblastoid cells and monitored for the development of BCL. Hu-PBL-SCID mice were treated with the immunosuppressive agents cyclosporine or methylprednisolone or the antiviral agents acyclovir or ganciclovir. Tumors developing in hu-PBL-SCID mice were high-grade lymphomas of human B-cell origin and contained EBV-DNA. BCL developed in 70% of mice 11 to 14 weeks after latent infection. BCL developed after 4 to 7 weeks in all hu-PBL-SCID mice after active infection. Treatment with cyclosporine or methylprednisolone had no effect on BCL development after active infection, but inhibited rather than enhanced the development of BCL in latently infected mice. Ganciclovir, but not acyclovir, inhibited BCL development after active infection. The hu-PBL-SCID mouse provides an in vivo model of BCL associated with immunosuppression. Active EBV infection results in the rapid development of BCL in this model even when latently infected B cells are present. Inhibition of BCL development in latently infected hu-PBL-SCID mice by immunosuppressive therapy may reflect inhibition of a T-cell/B-cell interaction necessary for B-cell activation. Inhibition of BCL development by granciclovir suggests a possible role for this agent in the management of BCL associated with immunosuppression.


Detection of nitric oxide by electron paramagnetic resonance spectroscopy during rejection and graft-versus-host disease after small-bowel transplantation in the rat. Our previous observation that nitric oxide (NO) is synthesized during antigen-specific immune reactions in vitro led us to investigate whether NO is produced during the in vivo immune response to a vascularized organ allograft. Orthotopic small-bowel transplantation in the rat was performed by standard microsurgical techniques in the LBNF1 to Lewis (rejection alone), Lewis to LBNF1 (graft-versus-host disease [GVHD] alone), and a syngeneic strain combination with and without immunosuppressive therapy with FK 506. The recipient serum NO2-/NO3- levels (stable end products of NO metabolism) were measured and erythrocytes were evaluated for the presence of nitrosylferrohemoglobin (specific for NO bound to hemoglobin). Animals that acutely rejected small-bowel allografts or suffered from acute GVHD showed significantly elevated serum NO2-/NO3- levels on days 6 and 9, and nitrosylferrohemoglobin electron paramagnetic resonance signals of different intensity were detected on days 3, 6, and 9. FK 506-treated allograft recipients and recipients of syngeneic grafts showed normal serum NO2-/NO3- levels and lacked nitrosylferrohemoglobin signals at all time points. This study indicates that NO is produced early during the course of small-bowel allograft rejection and GVHD and might therefore serve as a simple marker to detect such immune reactions.


The possible inhibitory role of the leucine-zipper DNA binding protein c-fos in the regulation of hepatic gene expression after sepsis. The leucine-zipper c-fos has been implicated in the regulation of gene expression. We investigated the possible role of c-fos in the regulation of hepatic gene expression after sepsis. Based on previous data demonstrating that sepsis inhibits hepatic gene expression of carnitine palmitoyltransferase (CPT), we hypothesized that c-fos may play a role in the inhibition of CPT gene expression after sepsis. We studied c-fos gene expression after peritoneal sepsis induced by cecal ligation and puncture (CLP) or sham-CLP. To investigate the possible inhibitory role of c-fos on CPT gene transcription, we investigated the effect of c-fos on c-jun-driven CPT promoter-chloramphenicol acyltransferase reporter gene expression in a HepG2 hepatoma cell cotransfection model. To investigate the possible role of cyclic adenosine monophosphate (cAMP) in the regulation of c-fos in vivo, we treated either the sham-CLP group or the CLP group with either vehicle or cAMP. Peritoneal sepsis in the rat model resulted in a four-fold increase in hepatic c-fos mRNA and c-fos protein. In the cotransfection model, c-fos significantly inhibited c-jun-induced chloramphenicol acyltransferase activity. Treatment with cAMP resulted in a 50% decrease in c-fos protein in either the sham-CLP or CLP group. We conclude that (1) sepsis increases hepatic c-fos transcription and translation, (2) c-fos inhibits c-jun-induced CPT gene expression, and (3) cAMP probably does not directly mediate the increase in c-fos after sepsis.


Pharmacological properties of a low molecular weight butyryl heparin derivative (C4-CY 216) with long lasting effects. We have investigated the pharmacological properties of an O-acetylated butyryl derivative of the low molecular weight heparin CY 216 (C4-CY 216). In a purified system the ability of C4-CY 216 to catalyze thrombin and factor Xa inhibition was comparable to that of CY 216. The antithrombin and antifactor Xa catalytic efficiencies of C4-CY 216 were reduced 217 and 12 times respectively when albumin (10 mg ml-1) was added to the reagents, while those of CY 216 were essentially unchanged. In plasma, the antifactor Xa specific activity of C4-CY 216 was close to that of CY 216 but the antithrombin specific activity was 2 times lower. After bolus and continuous intravenous injection to rabbits, the clearances of the two activities of C4-CY 216 were on average half the corresponding values of CY 216. After subcutaneous injection, the bioavailability of C4-CY 216 was comparable to that of CY 216. C4-CY 216 was as potent as CY 216 in preventing venous thrombosis in the thromboplastin-Wessler model and the duration of the antithrombotic effect was longer than that of the parent compound. The chemical alteration of CY 216 did not enhance the prohaemorrhagic effect in the rat tail transection model. Therefore, the new concept of heparin derivative having a low clearance and long lasting effects that we have recently reported for unfractionated heparin may also be applied to a low molecular weight heparin.


The glycosaminoglycan of recombinant human soluble thrombomodulin affects antithrombotic activity in a rat model of tissue factor-induced disseminated intravascular coagulation. Previous studies on recombinant human soluble thrombomodulin (rsTM) from Chinese hamster ovary cells revealed that rsTM was expressed as two proteins that differed functionally in vitro due to the presence (rsTM beta) or absence (rsTM alpha) of chondroitin-4-sulfate. The current study evaluates the in vivo behavior of rsTM in rats and in a rat model of tissue factor-induced disseminated intravascular coagulation (DIC). rsTM beta was more potent than rsTM alpha for prolongation of the activated partial thromboplastin time (APTT) and their in vivo half-lives determined by ELISA were 20 min for rsTM beta and 5.0 h for rsTM alpha. Injection of a tissue factor suspension (5 mg/kg) resulted in DIC as judged by decreased platelet counts and fibrinogen concentrations, prolonged APTT, and increased fibrin and fibrinogen degradation products (FDP) levels. A bolus injection of either rsTM (0.2 mg/kg) 1 min before induction of DIC essentially neutralized effects on platelets, fibrinogen, and FDP levels, and had only a moderate effect on APTT prolongation. The dose of anticoagulant to inhibit the drop in platelet counts by 50% (ED50) was 0.2 mg/kg rsTM alpha, 0.07 mg/kg rsTM beta, and 7 U/kg heparin. The effect of increasing concentrations of rsTM and heparin on bleeding times were compared in experiments involving incision of the rat tail. Doubling of the bleeding times occurred at 5 mg/kg rsTM alpha, 3 mg/kg rsTM beta or 90 U/kg heparin. These values represent a 25-fold increase over the ED50 for rsTM alpha, 43-fold for rsTM beta and 13-fold for heparin.(ABSTRACT TRUNCATED AT 250 WORDS)


Assessment of toxicologic interactions resulting from acute inhalation exposure to sulfuric acid and ozone mixtures. Studies examining effects of air pollutants often use single compounds, while "real world" exposures are to more than one chemical. Thus, it is necessary to assess responses following inhalation of chemical mixtures. Rabbits were exposed for 3 hr to sulfuric acid aerosol at 0, 50, 75, or 125 micrograms/m3 in conjunction with ozone at 0, 0.1, 0.3, or 0.6 ppm, following which broncho-pulmonary lavage was performed. Various pulmonary response endpoints related to general cytotoxicity and macrophage function were examined. In addition, a goal of the study was to define an improved approach to the analysis of data sets involving binary pollutant mixtures. Results were evaluated using analysis of variance with multiple linear contrasts to determine the significance of any effect in the pollutant-exposed groups compared to sham control animals and to assess the type, and extent, of any toxicological interaction between acid and ozone. Interaction was considered to occur when the effects of combined exposure were either significantly greater or less than additive. Pollutant exposures had no effect on lavage fluid levels of lactate dehydrogenase, prostaglandins E2 and F2 alpha, nor on the numbers, viability, or types of immune cells recovered by lavage. Phagocytic activity of macrophages was depressed at the two highest acid levels and at all levels of ozone. Exposure to all mixtures showed significant antagonism. Superoxide production by stimulated macrophages was depressed by acid exposure at the two highest concentrations, while ozone alone had no effect. Significant antagonistic interaction was observed following exposure to mixtures of 75 or 125 micrograms/m3 acid with 0.1 or 0.3 ppm ozone. The activity of tumor necrosis factor elicited from stimulated macrophages was depressed by acid at 75 and 125 micrograms/m3 while ozone had no effect. Exposure to mixtures of 125 micrograms/m3 acid with 0.3 or 0.6 ppm ozone resulted in synergistic interaction. This study provided additional evidence for antagonism between two common air pollutants and demonstrated that the type of interaction between sulfuric acid and ozone depended upon the endpoint but that the magnitude of any interaction was not always related to the exposure concentrations of the constituent pollutants.


The interaction of L-triiodothyronine and 2,3,7,8-tetrachlorodibenzo-p-dioxin on Ah-receptor-mediated hepatic Phase I and Phase II enzymes and iodothyronine 5'-deiodinase in thyroidectomized rats. Across all levels of L-triiodothyronine (L-T3) treatment, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in increased hepatic cytochrome P-450-associated activities of 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-dealkylase (EROD) and aryl hydrocarbon hydroxylase (AHH). The treatment of thyroidectomized rats with L-T3 at physiologic replacement levels in concert with TCDD produced an increase in ECOD, EROD and AHH activity above that seen with only TCDD. TCDD as well as L-T3 enhanced the activity of hepatic 1-naphthol glucuronyl transferase (NGT). In addition, the combined effect of L-T3 and TCDD resulted in similar levels of induction of NGT at both physiologic and supraphysiologic doses of L-T3. TCDD treatment resulted in elevated serum T3 levels at both physiologic and supraphysiologic levels of L-T3. One TCDD dose inhibited hepatic microsomal 3,3',5'-triiodothyronine (reverse T3) 5'-deiodinase activity by 61% in thyroidectomized, T3-untreated rats. The inhibition of 5'-deiodinase activity was partially overcome by increasing the T3 dose.


Factors affecting the toxicity of dioxin-like toxicants: a molecular approach to risk assessment of dioxins. The numerous toxic responses of dioxin-like compounds are mediated by the intracellular Ah (aryl hydrocarbon) receptor. It has been suggested that the regulation of dioxins and similar substances could be placed on a molecular foundation by considering the proportion of Ah-receptor sites occupied by toxicant molecules. The present work has shown that the following formation not yet available would be needed in order to develop this approach: correlation between dioxin exposure and human tissue levels; accurate determination of the association constants for human Ah-receptor with toxicant, and for human receptor-ligand complex with DNA; and knowledge of the intracellular concentrations of both receptor binding sites and DNA binding sites. Furthermore, since not all dioxin-like substances behave identically, this information would need to be gathered for a wide variety of substances.


Growth inhibitory and antimitogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in T47D human breast cancer cells. The T47D human breast cancer cells used in this study express relative high levels of the progesterone receptor (637 +/- 118 fmol/mg protein) and lower levels of the estrogen and aryl hydrocarbon (Ah) receptors (81 +/- 3.4 and 55 +/- 8.2 fmol/mg protein, respectively). Treatment of these cells with 0.1, 1.0 and 10 nM concentrations of 17 beta-estradiol, transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) resulted in concentration-dependent increase in cell proliferation and the ratios of mitogen-treated/control cell numbers were 2.46, 2.00 and 1.90, respectively. In contrast, insulin did not significantly stimulate T47D cell proliferation and insulin-like growth factor-I (IGF-I) was active only at a concentration of 10 nM. In parallel studies, the proliferative agents also stimulated the uptake of [3H]thymidine into cellular DNA. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) did not affect T47D cell growth at concentrations of 0.1 and 1.0 nM, whereas at a concentration of 10 nM a 44% decrease in cell numbers was observed. In cells cotreated with TCDD plus 100 ng/ml insulin or 10 nM 17 beta-estradiol, EGF, TGF-alpha and IGF-I, TCDD caused a concentration-dependent decrease in cell proliferation and [3H]thymidine uptake. For example, at a 10 nM concentration of TCDD there was a 32, 45, 29, 25 and 32% decrease in the 17 beta-estradiol, TGF-alpha, EGF, IGF-I and insulin-induced cell growth, respectively. These results confirm the antiproliferative activity of TCDD in T47D cells and this was similar to results previously reported in MCF-7 human breast cancer cells for the interaction of TCDD and 17 beta-estradiol. In addition, the data also show that TCDD inhibits the growth stimulatory effects of other polypeptide growth factors in T47D cells.


[Chromosomal restructurings and the distribution of chromosome fragile sites in the peripheral blood lymphocytes in a breast cancer patient after cytostatic therapy]. Cytogenetic analysis was performed repeatedly on a breast cancer patient since the beginning of the antitumor treatment. Double minute chromosomes (DMS, 2-10 per cell) were found in less than 2% of peripheral blood lymphocytes besides other chromosomal abnormalities after radiation therapy and 8 months after chemotherapy. The level of structural chromosomal aberrations two years after the therapeutic treatment was 0.13-0.14 aberrations per cell, but DMS were not observed. Estimation of the fragile site (FS) frequency and distribution at this time revealed a significant expression of the common FS FRAGF (9q1.2) after the treatment of blood culture with 5-bromo-2-deoxyuridine at dose levels of 7 and 50 g/l and enhanced fragility in chromosome band 1p35-36.1 (FRA1A) in folate-deprived conditions. Rare FS were not found. The presented data are discussed.


[Detection of antibodies to infectious bronchitis of fowl using the ELISA, hemagglutination-inhibition test and agar-gel precipitation test]. Chicks of a conventional poultry flock, Shaver Starcross 288 hybrid, were vaccinated with infectious bronchitis (IB) virus H 120 at the age of 21 days. Three weeks later, the chicks were divided into three groups and separate groups were infected with infectious bronchitis viruses M 41 and D 274 or revaccinated with virus H 120. The content of specific antibodies to antigens prepared from homologous and heterologous viruses of infectious bronchitis used for chick vaccination and infection was investigated at regular intervals in the separate groups of chicks by means of an ELISA technique and haemagglutination-inhibition test (HIT). Serotype specificity of haemagglutination-inhibition test was documented by the results; the specificity was obvious mainly after the first vaccination and two weeks after infection, or after chick revaccination (Fig. 1). The dynamics of postinfective or postvaccinal antibodies, recorded by the ELISA technique, had analogical patterns in the separate groups of chicks, and there were no larger differences in the values determined on the basis of different antigens during the investigation (Fig. 2). A total of 52 group samples of fowl serum was examined by the ELISA technique and agar-gel precipitin test (AGPT) in another part of this study. Ten serums of identical origin represented the separate groups. The result of this examination was evaluated from the percentage of samples with precipitin activity in the group, or from the average value of ELISA. Mutual comparison of the mentioned values indicated that the precipitin activity was limited by the positivity degree of ELISA reaction (Fig. 3).(ABSTRACT TRUNCATED AT 250 WORDS)


Sequence and transcript analysis of the bovine herpesvirus 1 thymidine kinase locus. A detailed sequence and transcript analysis of the thymidine kinase (tk) gene of bovine herpesvirus 1 (BHV1, Cooper strain) was carried out. A tk open reading frame (ORF) of 1077 bp was identified and compared with tk ORFs previously published for other strains of BHV1. The Cooper sequence was in good agreement with that of strain Q3932 but differed significantly from strains 6660 and LA. Reanalysis of the LA tk sequence, however, failed to confirm this difference. Except for five single base substitutions, our results indicate that the Cooper, Q3932, and LA strains share the same tk sequence. The size of the tk mRNA was determined by Northern blot analysis. In contrast to the size of other herpesvirus tk mRNAs (approximately 1.5 kb), the BHV1 tk transcript was 4.3 kb. Northern blot analysis indicated that the tk transcript was 3' coterminal with the downstream 3.1-kb transcript which encodes a BHV1 homologue of the HSV1 H glycoprotein (gH). The 5' ends of the tk and gH mRNAs were mapped by S1 analysis to positions 135 and 91 bp upstream of their respective translation start sites. The 5' end of the tk transcript was found to overlap a 5.2-kb transcript with opposite polarity to the tk mRNA.


Rotavirus VP6 modified for expression on the plasma membrane forms arrays and exhibits enhanced immunogenicity. The major inner capsid protein of rotavirus is VP6, a 42-kDa polypeptide that forms the icosahedral surface of the rotavirus single-shelled particle. A chimeric form of VP6 (VP6sc) was constructed containing an upstream leader sequence derived from the influenza virus hemagglutinin and a downstream membrane-spanning (anchor) domain from a mouse immunoglobulin gene. When VP6sc was expressed in cells using a recombinant vaccinia virus, the protein was transported, glycosylated, and anchored in the plasma membrane as a trimer with the major domains of the protein orientated externally. Immunofluorescence and immunolabeling with colloidal gold indicated that VP6sc also localized in patches on the cell surface; electron microscopy revealed that the protein assembled into two-dimensional arrays which exhibited the same periodicity as the paracrystalline arrays formed by purified (viral) VP6. Mice inoculated with a recombinant vaccinia virus that expressed VP6sc produced rotavirus-specific antibodies at a titer 10 times higher than that achieved when wild-type, intracellular VP6 was delivered in the same way. Presentation at the cell surface therefore may represent a general method for enhancing the immunogenicity of rotavirus proteins.


Carcinogen-induced activation of SV40 gene expression in a semi-permissive environment. Carcinogen-induced expression of the integrated viral genome was examined on SV40-transformed Chinese hamster cells. Carcinogen treatment markedly increased the transcription rate and the steady state mRNA level of both early and late viral transcripts. Carcinogen-induced transcription was mediated by RNA polymerase II. The increase in viral gene expression was also detected at the protein level, although at a reduced amplitude. Enhanced transcription was apparent as early as 12 hr postexposure and was considerably elevated after 24-36 hr. The increased gene expression depended on the existence of a functional replication machinery, as indicated by two lines of evidence. First, a cell line that harbors origin-deleted SV40 failed to respond to carcinogen treatment by increasing transcription and expression of T antigen. Furthermore, carcinogen-induced overtranscription was inhibited by aphidicolin, an inhibitor of DNA polymerase alpha. The involvement of the replication apparatus in the enhanced expression points to mechanistic similarities between the carcinogen-induced viral gene expression in the drug-treated semipermissive cells and the SV40 lytic pathway under permissive conditions. It is therefore suggested that cellular permissivity to viral development is enhanced following exposure to carcinogens. The implications of these findings for the nature of cellular permissivity to viral infection and the synergistic effects of carcinogens and tumor viruses are discussed.


Characterization of a stable eukaryotic cell line expressing the Rous sarcoma virus integrase. The Rous sarcoma virus integration protein (IN) is required for efficient integration of viral DNA into the host genome. IN was expressed in mouse C127 cells using a bovine papillomavirus vector. This system utilizes the mouse metallothionein promoter and the SV40 late polyadenylation signal for efficient expression of IN. A stable cell line derived from a single hygromycin-resistant colony was characterized. The expression of IN increased significantly upon Zn2+ induction of the metallothionein promoter, but did not respond to "superinduction" protocols. Full-length nonphosphorylated IN was the major product of expression. A minor product resulting from initiation of translation at an internal Met codon was also produced. The expressed IN did not exhibit the polypeptide heterogeneity at its COOH-terminus nor phosphorylation as is seen when IN is immunoprecipitated from virions. Using subcellular fractionation and indirect immunofluorescence, IN was primarily localized to nuclei and in some cells appeared to concentrate at discrete loci within the nuclei.


The complete nucleotide sequence of cell fusing agent (CFA): homology between the nonstructural proteins encoded by CFA and the nonstructural proteins encoded by arthropod-borne flaviviruses. Cell fusing agent (CFA) is an RNA virus originally isolated from a line of Aedes aegypti mosquito cells. Although our characterization of the virus many years ago showed that it resembled the flaviviruses, there was no detectable serological cross-reaction with members of the genus flavivirus. Furthermore, unlike the well-studied members of the genus flavivirus, CFA did not replicate in any of several vertebrate cell lines tested. We have now determined the nucleotide sequence of the CFA genome. Comparison of the predicted amino acid sequence of the CFA polyprotein with viral protein sequences in Genbank, has made it apparent that CFA should now be assigned to the family Flaviviridae, genus flavivirus. The homology between CFA proteins and those of other flaviviruses was highest for NS5 (45%) and NS3 (34%). Little homology was found for the structural proteins. Thus, CFA is only distantly related to the other flaviviruses for which there is sequence information; nevertheless, with respect to their hydrophobicity plots, the CFA polyprotein and the polyproteins of other flaviviruses are remarkably similar. We suggest that CFA is an insect virus, which was present in the embryos from which the Ae. aegypti cell line was established. Thus, CFA seems to be the first member of the family Flaviviridae, genus flavivirus, to be identified as an insect virus.


Analysis of a human immunodeficiency virus type 1 isolate carrying a truncated transmembrane glycoprotein. We have recently reported the isolation of a human immunodeficiency virus type 1 (HIV-1), KB-1gp32 carrying a shorter size (32 kDa) of transmembrane glycoprotein (TMP) from TALL-1 cells persistently infected with KB-1gp41 virus strain (Shimizu et al., 1990a). Endoglycosidase treatments showed that the different size of the TMP between the two strains was due to a truncation of 9 kDa of polypeptide in the KB-1gp32 TMP coding region. Sequence analysis revealed the substitution of a CAG codon to a TAG stop codon just downstream of the putative membrane-spanning domain of the TMP of KB-1gp32. This resulted in a truncation of some 133 amino acids of the cytoplasmic domain of TMP. The data indicate that a premature stop codon in KB-1gp32 has been introduced during adaption of the parental virus to TALL-1 cells. We have constructed two chimeric clones between the env region of a clone pKB-1, derived from KB-1gp32, and an infectious molecular clone pNL-432. We have also constructed a site-directed mutant of pNL-432 carrying a premature stop codon at the same position as the env stop codon of pKB-1. Among the three clones carrying a premature stop codon in env, only one chimeric clone was infectious to TALL-1 but not MT-2 cells. This clone contained the entire tat, rev, vpu, and env genes of pKB-1. The pNL-432 mutant was not infectious. The results suggest that some sequences of pKB-1 might compensate for the truncation of the TMP during replication in TALL-1 cells.


Genetic analysis of an NTP-binding motif in poliovirus polypeptide 2C. Poliovirus polypeptide 2C is a nonstructural protein involved in replication of the viral genome. Analysis of the primary amino acid sequence of 2C shows homology to a family of proteins which contain a nucleoside-triphosphate (NTP)-binding motif. This motif consists of elements "A" (2/5 hydrophobic stretch) G/AXXGXGKS/T, where X stands for any amino acid, and "B" (3/5 hydrophobic stretch) D or DD/E. To assess the significance of the consensus sequence in 2C, we have engineered point mutations into the most conserved residues in the A and B sites and tested their effect on viral RNA replication in vivo and translation in vitro. Whereas in vitro translation of synthetic RNAs carrying mutations in the NTP-binding motif showed efficient processing of all viral proteins, indistinguishable from that of the parental strain, transfection of the RNAs into HeLa cells did not give rise to infectious virus. No viral RNA replication could be detected in cells transfected with mutant RNAs. However, revertants to the wild-type genotype in the A and B sites were obtained which gave rise to wild-type RNA synthesis, but pseudorevertants or second-site suppressors were not observed. Thus, viral RNA synthesis is greatly reduced but not entirely abolished in cells transfected with mutant RNAs. These results strongly suggest a functional role for the proposed NTP-binding motif of 2C in RNA replication and proliferation of poliovirus.


Small deletion in v-src SH3 domain of a transformation defective mutant of Rous sarcoma virus restores wild type transforming properties. RSV mutant virus PA101T was obtained while assaying the tumorigenicity of parental PA101 virus in chickens. PA101 is a transformation defective mutant of RSV which has a low src kinase activity. However, PA101 retained a temperature-sensitive ability to induce sustained proliferation of neuroretina cells. PA101T appeared as a wild-type phenotype revertant of PA101. Molecular cloning and sequencing of PA101T showed that this reversion is due to additional mutations in PA101 src gene. These mutations are a deletion eliminating three amino acids in the N-terminal region of SH3 domain and mutation of Ala 426 to Val. Analysis of the properties of chimeric src genes associating either half of PA101T with the complementary regions of PA101 or wild-type virus showed that the N-terminal moiety of PA101T src, which contains the deletion, confers wild-type transforming properties, whereas its C-terminal moiety, which contains single amino acid mutation, confers a partially temperature-sensitive phenotype. These results are consistent with other reports showing that mutations or deletions in this region of SH3 activate the transforming potential of c-src. They support the hypothesis that the N-terminal region of SH3 interacts with a cellular negative regulator of src activity.


Temperature-sensitive polioviruses containing mutations in RNA polymerase. Site-directed mutagenesis was performed to change the wild-type residue (asparagine) to aspartate, histidine, or tyrosine at amino acid 424 of the poliovirus RNA polymerase, 3Dpol. The mutations were introduced into plasmids containing full-length viral cDNA and plasmids which direct the expression of 3Dpol in Escherichia coli. Mutant viruses, recovered after transfection of HeLa cells with RNA transcripts of the full-length clones, produced small plaques at 32 degrees. In addition, the plaquing efficiency was decreased for all three mutants at 37 degrees, compared to 32 degrees. The polyprotein processing of all mutant viruses was normal at the temperatures tested, suggesting that the mutant plaque phenotypes were not due to incorrect processing of viral proteins. Analyses of viral RNA synthesis in infected cells and of the polymerase activities of mutant enzymes produced in E. coli suggested the following: (1) The his424 mutant enzyme appeared to be defective in the initiation of plus-strand RNA synthesis in HeLa cells. (2) The asp424 mutant enzyme appeared unable to assume proper conformation for active polymerase function when synthesized at 37 degrees. (3) The tyr424 mutant enzyme was totally inactive when synthesized in E. coli at 37 degrees.


Characterization of the stage(s) in the virus replication cycle at which the host-cell specificity of the feline parvovirus subgroup is regulated in canine cells. Feline panleukopenia virus (FPLV), mink enteritis virus (MEV), and canine parvovirus (CPV) are classified as a host-range variants. They show different host-range specificity in vivo and host-cell specificity in vitro. For instance, FPLV and MEV cannot grow or can grow only inefficiently in canine cell lines such as MDCK and the canine fibroma cell line A72. Here we have studied the mechanism(s) by which the different cell tropism is mediated in vitro. When FPLV or MEV was inoculated to A72 cells, viral DNA replicated slightly, few viral-antigen-positive cells were detected, and the culture fluid contained the threshold level of infectivity. On the other hand, when an infectious molecular clone of MEV (pMEV) was introduced into A72 cells, viral DNA replicated efficiently, and the culture fluid of pMEV-transfected cells contained much higher infectivities than that of MEV-infected cells. In spite of the restrictive growth in A72 cells, MEV could bind to A72 cells as efficiently as CPV. No detectable viral RNA was produced in MEV-infected A72 cells. In contrast, efficient viral transcription occurred in pMEV-transfected A72 cells. These results suggest that the restrictive infections of MEV and FPLV in A72 cells are not mediated by the attachment of the virus to the cells or by the events occurring after the viral transcription. It appears to be caused by the stage(s) in the virus replication cycle, which exists between a postadsorptional step required for virus penetration and the initiation of viral transcription.


An in vivo study of a glycoprotein gIII-negative bovine herpesvirus 1 (BHV-1) mutant expressing beta-galactosidase: evaluation of the role of gIII in virus infectivity and its use as a vector for mucosal immunization. We constructed a recombinant BHV-1 in which the glycoprotein gIII gene was replaced by the Escherichia coli lacZ gene. The resultant virus mimics the simple gIII deletion mutant in its growth characteristics in cell culture; however, it expresses beta-galactosidase in virus-infected cells. Further characterization of its virulence and the immune responses elicited by it was conducted in cattle. The mutant virus retained the ability to establish an infection when administered intranasally. Infected animals were also capable of transmitting virus to sentinel penmates. However, the mutant virus showed a reduced replication efficiency in the respiratory tract of cattle, as manifested by significantly lower virus shedding and a shorter duration of shedding when compared to wild-type (wt) BHV-1 infections. The mutant virus induced an efficient anti-BHV-1 antibody response and convalescent cattle were fully protected from subsequent wt virus challenge. In addition, cattle infected with the lacZ-expressing virus developed antibodies to beta-galactosidase. Our results demonstrate that the presence of gIII is not a prerequisite for BHV-1 infection; however, gIII does play an important role in maintaining virus replication efficacy in its natural host. With respect to developing BHV-1 as a vaccine vector, our results indicate that deletion of the gIII gene, which partially attenuates the virus and serves as a vaccine virus marker, does not compromise immunogenicity to BHV-1. Most importantly, this vector is effective in delivering foreign antigens to mucosal surfaces of the respiratory tract.


The Sendai virus nonstructural C proteins specifically inhibit viral mRNA synthesis. An in vitro transcription system for paramyxoviruses is described, in which polymerase-free templates are combined with cell extracts containing polymerase made in vivo via transfected plasmids. Both P and L are required for polymerase activity, and both must be coexpressed for optimum activity. mRNA synthesis here was found to be inversely proportional to the level of C expression, whereas defective interfering genome replication was largely unaffected by the level of C in the extract. The inhibition of transcription appeared to be due to the C' and C, but not the Y1 and Y2 proteins, and only occurred when C'/C was coexpressed with P and L. C'/C appears to intervene during polymerase formation, possibly by forming polymerase complexes which are inactive for transcription, but still competent for genome replication.


Complete nucleotide sequence of the Marek's disease virus ICP4 gene. The Marek's disease virus (MDV) gene encoding a homologue to the ICP4 protein of herpes simplex virus has been mapped to BamHl fragment A based on the physical map of the MDV genome (Fukuchi et al., 1984). The gene lies completely within the inverted repeat flanking the unique short region of the genome. The complete nucleotide sequence of the MDV ICP4 gene has been determined. The coding region is 4245 nucleotides long and has an overall G+C content of 52%. The MDV ICP4 protein is predicted to have a structure similar to that of ICP4-like proteins of other herpesviruses in that it has five distinct regions, the second and fourth of which are highly conserved. In addition, the protein contains the characteristic run of serine residues located toward its amino terminus. The MDV ICP4 gene is expressed in MDV-infected chicken embryo fibroblasts.


Transformation by human papillomavirus type 16 (HPV16) DNA but not HPV6b DNA is enhanced by addition of the human cytomegalovirus enhancer. Primary human cervical epithelial cells immortalized by human papillomavirus type 16 (HPV16) DNA exhibit altered morphology and differentiation characteristic of transformation, but show a lack of transformed phenotype relative to HPV18 DNA immortalized cells in terms of anchorage-independent growth (Pecoraro, Lee, Morgan, and Defendi, 1991, Am. J. Pathol. 138, 1-8). This is completely corrected by inserting a strong heterologous enhancer derived from human cytomegalovirus DNA upstream from the HPV16 long control region. The cells immortalized by this DNA form colonies in agar comparable to those formed by HPV18 DNA immortalized cells. The enhanced transformation capability correlates with increased levels of HPV16 E6-E7 and E5 transcripts. The HPV16 DNA containing this strong enhancer also transforms C127 mouse cells with increased efficiency and strength relative to the natural HPV16 DNA, as measured by the numbers and size of the colonies in agar. The positive effects of this strong enhancer appear specific for HPVs associated with genital malignancies such as HPV16, since HPV6b DNA (primarily in benign tumors) with or without the strong cytomegalovirus enhancer is incapable of immortalizing primary human cervical epithelial cells or allowing efficient growth of C127 mouse cells in agar. These results suggest that the diminished oncogenic properties of HPV16 versus HPV18 DNA in cultured cells and in human malignancies may reside in the long control regions of these viruses and, additionally, may define another difference in the oncogenic properties of HPVs associated with benign or malignant genital neoplasia.


Stalling by RNA polymerase II in the polyomavirus intergenic region is dependent on functional large T antigen. RNA polymerase II encounters an elongation block and stalls in vivo during transcription of the late strand of polyomavirus DNA. In this study, we performed transcriptional run-on assays and localized the stalling site to a 164-nucleotide region (nt 11-175) that contains specific binding sites for polyomavirus large T antigen. The effect of large T antigen on elongation by RNA polymerase II through this region was examined in cells infected with a mutant polyomavirus (AT3-ts25E) which encodes a thermolabile large T antigen. Removal of functional large T antigen by shifting to the nonpermissive temperature (39 degrees) eliminated stalling by RNA polymerase in this region, although RNA polymerases transcribing other regions of the viral genome were unaffected. RNA polymerase resumed stalling when functional large T antigen was again allowed to accumulate by shifting back to the permissive temperature (32 degrees). We conclude that stalling by RNA polymerase II in vivo is dependent on the presence of functional large T antigen.


Identification and subcellular localization of the Q gene product of visna virus. The genome of the sheep visna lentivirus contains an open reading frame, Q, which has a coding potential of 230 amino acid residues. This paper reports the identification and the subcellular localization of the Q ORF-encoded protein detected in lysates of visna virus-infected sheep choroid plexus cells. Sera from sheep either experimentally or naturally infected with visna virus reacted with the bacterially synthesized Q protein indicating that the in vivo expressed Q product is immunogenic. Antibodies raised against a synthetic N-terminal peptide, reacted with either the bacterial Q or the in vitro translated Q protein as well as with the Q protein expressed during cellular infection. This 29 kDa protein is detectable late in the lytic viral cycle, i.e., 72 hr postinfection, and this expression correlates with the late transcription of its 4.8-kb mRNA. These results provide evidence for the first time that the Q ORF is a late gene of visna virus and that the Q protein is located in the cytosol compartment, without evidence of accumulation at the cell membrane, or in cell-free virion particles.


Detection of enhancer repeats in the long terminal repeats of feline leukemia viruses from cats with spontaneous neoplastic and nonneoplastic diseases. Enhancer duplication in the long terminal repeat of feline leukemia virus (FeLV) was examined in primary cells from naturally FeLV-infected cats with various neoplastic and nonneoplastic diseases using the polymerase chain reaction. In all cases, a 170-bp band, corresponding to a standard exogenous FeLV with one copy of enhancer, was detected. Repeated enhancer sequences were found in all 8 cases of thymic-form lymphosarcoma, in some cases of lymphosarcoma of other forms (3/8) and myeloid tumors (2/3), and in only 1 of 6 cases with nonneoplastic diseases. The copy number of FeLV proviruses with a repeated enhancer seemed higher than that of those with one copy of enhancer in 3 cases of thymic form lymphosarcoma. In 5 cases of thymic form lymphosarcoma and in 1 case of erythroleukemia, coexistent FeLVs with double and triple enhancers of different sizes were found. Of the enhancer elements, only the SV40 core binding site was found in all the enhancer direct repeats of these FeLVs. All the provirus clones with single and duplicated enhancer sequences from a single tumor showed mutations or deletions characteristic to that tumor, indicating that enhancer repeats may arise in individual animals after infection with a single virus clone. The present findings indicate that FeLV with enhancer repeats generated in the cat is associated with the induction of neoplastic diseases in natural conditions.


HSV-1-inducible proteins bind to NF-kappa B-like sites in the HSV-1 genome. Several putative NF-kappa B-binding sites in the ICP0 and Vmw65 herpes simplex virus type-1 (HSV-1) genes have been identified. Oligonucleotides encoding some of these sites bind specifically to purified NF-kappa B protein and an NF-kappa B-like protein in nuclear extracts of phorbol ester- or cycloheximide-induced human embryonic lung (HEL) cells. HSV-1 infection of HEL cells induced a nuclear factor that binds specifically to kappa B sites in the ICP0 and Vmw65 gene regions and comigrates with complexes formed by purified NF-kappa B. The HSV-1-inducible nuclear factor bound to the authentic immunoglobulin (Ig) kappa B site. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing two copies of the Ig kappa B site upstream of the c-fos promoter (kappa B2-CAT) showed activity in HEL cells. HSV-1 infection of kappa B2-CAT-transfected HEL cells, however, induced a dramatic increase in CAT activity; mutation in the NF-kappa B-binding site of kappa B2-CAT abolished the inducibility of CAT gene expression. Our results demonstrate that the HSV-1 ICP0 and Vmw65 gene regions contain binding sites for NF-kappa B, and that HSV-1-inducible proteins bind to NF-kappa B-like sites in the HSV-1 genome.


The expressed VP4 protein of bluetongue virus binds GTP and is the candidate guanylyl transferase of the virus. A minor core protein, VP4, of bluetongue virus serotype 10 (BTV-10) has been synthesized in insect cells infected with a genetically manipulated recombinant baculovirus. When insect cells were coinfected by this recombinant virus and a recombinant baculovirus expressing the two major core proteins (VP3 and VP7) of the virus, core-like particles (CLPs) consisting of all three proteins were formed. Purified CLPs reacted with [32P]GTP which was covalently bound to VP4 only. Similarly reconstituted CLPs with VP1 or VP6 did not form covalent complexes with [32P]GTP. The virion-derived VP4 was also shown to have GTP-binding activity. The covalent binding of GTP indicates that expressed VP4 not only is biologically active but also is the candidate guanylyl transferase of the virus. The optimum reaction conditions for GTP binding by VP4 have been investigated.


Proteolysis in the maturation of avian retroviruses does not require calcium. After budding from the plasma membrane, retrovirus particles undergo a process of maturation, which includes changes in morphology caused by several proteolytic cleavages of the precursor of the internal structural proteins, products of the gag gene. Cleavage is mediated by the viral protease, PR. The fact that in most systems cleavage appears to occur only after assembly is complete, suggests that PR may become enzymatically active as a consequence of release of the virion from the cell. Using avian leukosis virus as a model system, we tested the hypothesis that leakage of calcium ions into newly budded virions plays a role in their maturation. We found that in both quail Qt35 cells and monkey COS-1 cells, maturation occurred normally in calcium-free medium and in the presence of EGTA. A calcium ionophore also did not affect maturation. We conclude that calcium influx does not act as a trigger for PR-mediated maturation.


In vivo transfection of bovine leukemia provirus into sheep. Bovine leukemia virus is horizontally transmitted mainly through infected cells by direct blood transfer. In this report, a cloned bovine leukemia virus (BLV) provirus was examined for its infectivity by direct inoculation into sheep. One hundred micrograms of plasmid DNA containing a complete provirus was mixed with a cationic liposome solution and injected intradermally into five sheep at three different locations. Seroconversion occurred 1 to 2 months after injection as demonstrated by immunodiffusion, indirect ELISA (for the gp51 envelope protein), and blocking ELISA (for gp51 and the major capsid protein, p24). These results demonstrate that BLV infection can be efficiently initiated by direct transfection into sheep. This approach should thus facilitate investigation of the involvement of BLV genetic determinants in the induction of leukemia in ruminants.


Identification of a generalised packaging sequence for D-type retroviruses and generation of a D-type retroviral vector. In order to construct vectors based upon D-type, rather than C-type, retroviruses, we have identified a 624-bp fragment of Mason-Pfizer monkey virus (MPMV) which constitutes a packaging sequence for at least two D-type retroviruses. When this fragment was included in an extensively deleted D-type vector genome, the D-type viruses MPMV and SRV-5, but not the C-type viruses MLV-A or MLV-E, rescued the vector RNA from HeLa cells. The recombinant virus stocks have the host range of the rescuing D-type virus as shown by expression of an internal (SV40-puromycin) cassette replacing the retroviral structural genes. The recombinant MPMV was specifically neutralized by anti-MPMV serum and receptor interference was demonstrated when it was plated on cells productively infected with wild type MPMV. When the putative D-type packaging sequence was removed from the vector genome, even though the other sequence elements required for efficient reverse transcription remained, the vector was no longer rescued from HeLa cells. These results complement the recent demonstration of broad specificity of rescue of a C-type vector (carrying only the packaging sequence of Mo-MLV) by several different C-type, but not D-type, viruses. Replacement of the D-type packaging sequence by most of the extended packaging sequence of Mo-MLV prevented the otherwise D-type vector from being rescued by D-type viruses and did not allow it to be rescued by C-type viruses. This was probably because of the incompatibility of the D-type vector sequences with the C-type retroviral proteins involved in viral reverse transcription and integration. Hence, we have localized a packaging sequence that is recognized by D-type, but not by C-type, retroviruses and have constructed a D-type vector which may be useful in gene transfer experiments.


Neuraminidase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes. The interaction of infectious bronchitis virus (IBV) with erythrocytes was analyzed. The binding activity of IBV was not sufficient to agglutinate chicken erythrocytes. However, it acquired hemagglutinating activity after treatment with neuraminidase to remove alpha 2,3-linked N-acetylneuraminic acid from the surface of the virion. Pretreatment of erythrocytes with neuraminidase rendered the cells resistant to agglutination by IBV. Susceptibility to agglutination was restored by resialylation of asialo-erythrocytes to contain alpha 2,3-linked sialic acid. These results indicate that IBV attaches to receptors on erythrocytes, the crucial determinant of which is sialic acid alpha 2,3-linked to galactose. In contrast to other enveloped viruses with such a binding specificity (influenza viruses and paramyxoviruses) IBV lacks a receptor-destroying enzyme.


Selective inhibition of the 3'-to-5' exonuclease activity associated with Epstein-Barr virus DNA polymerase by ribonucleoside 5'-monophosphates. Epstein-Barr virus (EBV) DNA polymerase possesses a proofreading 3'-to-5' exonuclease activity (Tsurumi, T. (1991) Virology 182, 376-381). The 3'-to-5' exonuclease activity can be selectively inhibited by ribonucleoside 5'-monophosphates, while no inhibition of the DNA polymerase activity can be observed even when the template/primer concentrations are rate-limiting. Deoxynucleoside monophosphates except 5'dGMP have almost no effect on the exonuclease activity. Of the four ribonucleoside monophosphates, 5'GMP is the most potent (62% inhibition at 5 mM). The kinetic study shows that 5'-GMP inhibits the exonuclease activity competitively with respect to DNA template/primer. During DNA polymerization process the EBV DNA polymerase catalyzes the DNA-dependent conversion of complementary deoxynucleoside triphosphate to monophosphate form. With poly(dT).oligo(rA) as a template primer, selective inhibition of the exonuclease activity by 5'-GMP results in a decrease in the amount of free dAMP generated which is complementary to the template DNA, suggesting the functional relationship between the editing exonuclease activity and the chain elongation activity of the EBV DNA polymerase molecule.


Constitutive synthesis of polyoma antisense RNA renders cells immune to virus infection. Mouse fibroblasts were stably transfected with expression plasmids in which sequences of the early region of polyomavirus were inserted both in sense and antisense orientation. The cell lines that synthesize in the antisense orientation, a 1195-bp viral genome fragment covering the Ori, Cap, ATG, and all of the early mRNA splicing sites acquire resistance to viral infection. Smaller fragments covering Ori, Cap, and ATG sites or the splicing sites, as well as fragments cloned in sense orientation, failed to confer cell immunity to polyoma infection. The resistance proved to be directly dependent upon the specific antisense RNA and to be inversely proportional to the multiplicity of infecting polyoma.


The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1. A nuclear localization signal (NLS) has been identified in the N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.


Calcium chelation induces a conformational change in recombinant herpes simplex virus-1-expressed rotavirus VP7. Rotavirus, strain SA11, glycoprotein VP7 that was expressed by a recombinant herpes simplex virus-1 or contained in purified rotavirus particles lost reactivity with the neutralizing monoclonal antibody (mAb) 159, but not with nonneutralizing mAbs, upon chelation of calcium by EGTA. Exposing VP7, but not the neutralizing mAbs, to a transient excess of EGTA over calcium eliminated VP7 neutralizing epitopes. Therefore, a calcium chelation-induced conformational change in VP7, not in the neutralizing mAbs, caused the epitope loss. Addition of excess calcium or strontium, but not magnesium or barium, to EGTA-treated VP7 restored its 159 epitope. These results suggest that VP7 binds calcium in the absence of other rotavirus proteins and that the calcium chelation-induced conformational change in VP7 may mediate uncoating of double-shelled rotavirus particles.


A bovine rotavirus serotype 1: serologic characterization of the virus and nucleotide sequence determination of the structural glycoprotein VP7 gene. Bovine rotavirus T449 was isolated from feces of a calf with diarrhea. Serological characterization by serotype-specific monoclonal antibodies showed that the T449 virus belonged to serotype 1. This is the first report of a bovine rotavirus that does not belong to serotype 6, 8, or 10. The serotype 1 designation was confirmed by using an immunoperoxidase focus neutralization assay. The gene encoding the major neutralization antigen (VP7) was cloned and its nucleotide sequence was determined. The sequence obtained was 1062 bp in length and contained an open reading frame corresponding to 326 amino acid residues. Comparative analysis of the deduced amino acid sequence with the corresponding sequence of the human serotype 1 rotavirus strain, Wa, revealed a 90% identity. When compared to the predicted amino acid sequence of VP7 protein of the other serotypes an overall divergence of 20 to 25% was detected. These data show that the serological typing agrees with the result of the genetic analysis.