Effect of protein kinase C and calcium on bradykinin-mediated contractions of rabbit vessels. Bradykinin caused graded contractions of rings of rabbit aorta and jugular vein with EC50 values of 1.3 microM and 2.2 nM. In denuded preparations, responses of bradykinin in jugular vein but not in aorta were potentiated 1,000-fold. Both preparations bathed in calcium-free solution showed markedly depressed responses to bradykinin, but addition of 1 mM EGTA further inhibited bradykinin responses only in aorta. Time-course experiments carried out in calcium-free solution plus EGTA revealed that bradykinin contractions in rabbit aorta were very sensitive to extracellular calcium, whereas responses of the jugular vein depended on both extracellular and intracellular calcium sources. Responses to bradykinin in both tissues were unaffected by nicardipine (1 microM) but were partially antagonized by NiCl2 (0.1-0.3 mM). Ryanodine (30 microM) incubated in calcium-free medium markedly inhibited jugular vein responses to bradykinin but had no effect on aortic responses. Phorbol ester (1 microM) caused a slow tonic contraction in jugular vein but not in aorta and inhibited bradykinin responses in the former preparation. Staurosporine (1-100 nM) and 1-(5-isoquinolinesulfonyl)-2-methylpiperizine (H-7, 3 and 10 microM) caused a dose-dependent inhibition of bradykinin-induced contractions in jugular vein but were less effective in aorta.(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelin vascular receptors and responses in deoxycorticosterone acetate-salt hypertensive rats. The vasoconstrictor effect, the binding, and the response of inositol phosphates to endothelin-1 (ET-1) were investigated in blood vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats within 2 weeks of development of hypertension and in uninephrectomized control rats. In DOCA-salt and uninephrectomized rats, plasma levels of endothelin were similar (1.2 +/- 0.1 fmol/ml). Thoracic aorta and mesenteric artery rings devoid of endothelium presented significantly decreased responses to increasing concentrations of ET-1. Binding of ET-1 to mesenteric artery membranes was significantly lower in DOCA-salt rats (106 +/- 22 fmol/mg protein) than in uninephrectomized rats (172 +/- 19 fmol/mg protein, p less than 0.05), whereas affinity was similar. Phosphoinositide metabolism was examined in aorta and mesenteric arteries after incubation with [3H]myoinositol. Inositol phosphates were separated by high-performance liquid chromatography. In response to 100 nmol/l ET-1, accumulation of inositol 1,4,5-trisphosphate after 20 seconds and of inositol monophosphate, inositol bisphosphate, and inositol 1,3,4-triphosphate after 30 minutes (in the presence of 25 mmol/l LiCl) were significantly lower in DOCA-salt hypertensive than in uninephrectomized control rats, in both aorta and mesenteric arteries. In conclusion, decreased density of ET-1 receptors in DOCA-salt hypertensive rats results in decreased activation of phospholipase C and, consequently, reduced vasoconstriction induced by ET-1. Because the decrease in vasoconstrictor effects of ET-1 is found in the absence of endothelium, it is likely that receptor downregulation rather than prior receptor occupancy underlies these findings.


Detection of antibodies to a linear epitope on the major coat protein (L1) of human papillomavirus type-16 (HPV-16) in sera from patients with cervical intraepithelial neoplasia and children. Antibodies to the major (L1) coat protein of human papillomavirus type 16 (HPV-16) in sera from patients with cervical intra-epithelial neoplasia (CIN) have been investigated by means of recombinant proteins and synthetic peptides. When L1-HPV-16 fusion proteins were used in immunoblot assays, no antibody reactivity was found in sera from 52 patients with CIN or from 21 unrelated children. Amino-acid sequence analyses indicated that L1-HPV-16 amino acids 473 to 492 may contain an HPV-16 type-restricted epitope since the greatest diversity occurs in this region. In the ELISA, seropositivity to peptides 473 to 492 was more common among CIN patients whose biopsies contained HPV-16 DNA (91%, 21 of 23) than among their children (24%, 5 of 21; p less than 0.001) or other CIN patients with HPV-16 DNA-negative biopsies (66%, 19 of 29; p less than 0.05), but was unrelated to the severity of the CIN lesion. Antibodies to L1-HPV-16 peptide 473 to 492 among seropositive CIN patients cross-reacted with the analogous L1-HPV-33, but not with the L1-HPV-6b peptide, and were predominantly IgM. In contrast, antibodies which recognized a less variable region of L1-HPV-16 (amino acids 279 to 293) showed no association with HPV-16 DNA status. Seropositivity to the L1-HPV-6b (amino acids 473-492) was less frequent (33%) among CIN patients and unassociated with HPV-16 DNA status (p greater than 0.1); however 51% (37 of 72) of patients with genital warts had antibodies to this peptide.


Expression and splicing patterns of human papillomavirus type-16 mRNAs in pre-cancerous lesions and carcinomas of the cervix, in human keratinocytes immortalized by HPV 16, and in cell lines established from cervical cancers. We have analysed the splicing patterns of human papillomavirus (HPV) type-16 mRNAs in a human epithelial cell line immortalized by HPV 16 (HPKII), in cell lines established from cervical carcinomas (SiHa and CaSki) and in pre-invasive and invasive carcinomas of the cervix. The presence of mRNA species previously described, which could encode the E6, E6I, E6II, E6III, E7, E2, E2C, E4, E5 and L1 proteins, was determined, using the RNA polymerase chain reaction (PCR) technique with primers that flank unique splice sites. The state of the viral DNA in the tumor biopsies was established by Southern blot analysis. The various HPV 16 transcripts could be detected in cell lines and in tumor biopsies. The size of the RNA PCR products were in agreement with the previously mapped splice sites. The full range of transcripts was revealed in the HPKII cell line and in a number of pre-invasive carcinomas. Messenger RNAs which could encode the E6III, E4 and E5 proteins were most prevalent in all types of tumor. The overall results of DNA and RNA analyses in cell lines and tumor specimens indicate that (1) expression of either of the early or late transcripts studied is not specifically related to (a) tumor stage or (b) the physical state of the viral genome; and (2) alterations in the splicing patterns of HPV 16 transcripts may not be involved in tumor progression.


Different fatty-acid profiles in phosphoinositides from human fibroblastic meningiomas with or without chromosome 22 monosomy. The fatty-acid composition of phosphoinositides derived from 13 human fibroblastic meningiomas, divided according to the presence or the absence of monosomy of chromosome 22, was analyzed. Phosphoinositides were separated into their 3 main components: phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate. In all the tumors, regardless of cytogenetic differences, remarkable differences were detected between phosphatidylinositol and phosphatidylinositol-4-phosphate or phosphatidylinositol-4,5-bisphosphate. In meningiomas with 22 monosomy, there was a significant decrease in the relative amount of arachidonate in all the phosphoinositide fractions; this situation might determine diminished down-regulation of the corresponding diacylglycerol, derived by the phospholipase-C-stimulated hydrolysis of phosphoinositides, and, consequently, different protein-kinase-C stimulation.


Increased phosphorylation of nuclear phosphoproteins in human lung-cancer cells resistant to cis-diamminedichloroplatinum (II). A novel non-phorbol-ester-like tumor promoter, okadaic acid (OA) has been shown to be an inhibitor of protein phosphatase I and IIA and, thus, to cause an "apparent activation" of protein kinase C (PKC). We previously showed that cis-diamminedichloroplatinum(II) (CDDP)-resistant cells, PC-9/CDDP, were cross-resistant to OA and that the cross-resistance was not due to the increased efflux of OA. We hypothesized that the phosphorylation status of some cellular proteins might be important in CDDP-resistance. No significant difference in PKC activity or total protein phosphatase activity measured in vitro was seen between PC-9 and PC-9/CDDP cells, nor in their sensitivity to inhibition by OA, nor in the amount of phosphorylation of whole cells or TCA-insoluble material. By SDS-PAGE after incubation of intact cells with 32P, we detected a marked increase, compared to PC-9 cells, in phosphorylation of the nuclear proteins of MW 32 and 20 kDa in CDDP-resistant PC-9/CDDP cells with no apparent difference in protein content. When phosphorylation of nuclear proteins observed in PC-9/CDDP cells was analyzed by 2-dimensional SDS-PAGE, the 32-kDa protein had a PI of about 4.5. The 32-kDa and 20-kDa bands were increased in a dose-dependent manner by CDDP treatment. On the other hand, no increase in phosphorylation of these proteins was observed in parental PC-9 cells. These results demonstrate a marked difference in the phosphorylation status of specific nuclear proteins between parental and CDDP-resistant cell lines, which may be related to CDDP-resistance.


Enhanced insulin-receptor tyrosine kinase activity associated with chromosomal translocation (1;19) in a pre-B-cell leukemia line. The gene for the insulin receptor has been assigned to chromosome 19 near the breakpoint of the translocation t(1;19) which occurs in 25% of pre-B-cell leukemias. Insulin receptors in a pre-B-cell leukemia cell line (ACV) with t(1;19) were found to have 2-fold higher affinity for insulin, 5-fold higher basal and insulin-stimulated beta sub-unit autophosphorylation, and 2-fold higher basal and 4-fold higher insulin-stimulated beta sub-unit kinase activity on the synthetic peptide poly(Glu,Tyr), compared to receptors in a B-cell line (ADD) with normal karyotype from the same patient. ACV cells had a novel 13-kb receptor mRNA species and expressed a DNA polymorphism localized to the tyrosine kinase domain of the receptor gene. These findings suggest that t(1;19) in the ACV cell may result in rearrangement of the insulin receptor gene and translation of a receptor with enhanced tyrosine kinase activity.


Varicella zoster and herpes simplex virus infections. There are six herpes viruses, three of which, the varicella-zoster virus and the herpes simplex viruses types 1 and 2, are of particular concern to patients and staff in critical care units. These viruses, especially in their reactivated states, may present atypically in critically ill and immune-suppressed patients, and, by the time the diagnosis is made, exposures of other patients and clinicians may have occurred. Pregnancy and immunosuppressed states can result in severe, even life-threatening varicella-zoster virus infections in susceptible adults. Similarly, nosocomial herpes simplex virus infections can have serious consequences for exposed patients and staff. An additional problem after herpes simplex virus infection is the potential of lifelong and possibly frequent recurrences. In this article, the manifestations, modes of transmission, and treatment will be discussed. Special emphasis will be placed on describing the types of patients who are at high risk of presenting with varicella-zoster virus or herpes simplex virus infection so that physicians and nurses can use appropriate preventive measures to avert nosocomial infections in patients and staff.


Development of an ELISA for the detection of 2-nitroimidazole hypoxia markers bound to tumor tissue. Canine and rodent tumors covalently bind the fluorinated 2-nitroimidazole, CCI-103F, in a way that immunohistochemical analysis shows is consistent with the location of tumor hypoxia. We have now developed a rapid, quantitative, and non-radioactive enzyme linked immunosorbent assay for the binding of CCI-103F in biopsy samples of spontaneous canine tumors. Issues of antigen stability during tissue processing, calibration of the ELISA, and the use of biopsy samples for measuring tumor hypoxia by the ELISA approach are addressed.


Potentiation of radiation response in human carcinoma cells in vitro and murine fibrosarcoma in vivo by topotecan, an inhibitor of DNA topoisomerase I. DNA topoisomerase I, a nuclear enzyme important for solving topologic problems arising during DNA replication, has been identified as a principal target of a plant alkaloid, 20(s)-camptothecin. In view of the profound biochemical effects of camptothecin and its analogues on DNA replication and the differential cytotoxic effects on human tumors in xenografts, experiments were performed to determine whether topotecan, a camptothecin analogue, would potentiate the radiation effects on human carcinoma cells in culture and murine fibrosarcoma in mice. Cell culture studies showed that a dose dependent reduction in cell survival was obtained with a 4 hr exposure of the drug following irradiation of cells. No enhancement of cell killing was seen when cells were treated with the drug before irradiation. Preliminary in vivo tumor studies showed a significant radiosensitizing effect of topotecan that was both drug dose (20 mg/kg) and time sequence (4 hr before irradiation) dependent. There was no enhanced skin reaction following the combined treatments.


Interaction of buthionine sulfoximine and the stabilization of DNA-topoisomerase II complexes by doxorubicin. Although it has been shown previously that the depletion of cellular thiols increases doxorubicin cytotoxicity, the mechanism of sensitization is not clear. To study this question, the effect of D,L-buthionine-S,R-sulfoximine (BSO) on doxorubicin cytotoxicity and the stabilization of DNA-topoisomerase II complexes (cleavable complexes) was investigated in V79 cells. Incubations with BSO (10 mM) were for 5 hr beginning 4 hr prior to doxorubicin exposure since a 4 hr incubation with 10 mM BSO is known to decrease glutathione levels below 5% of control V79 cells. These BSO pre-treatment increased doxorubicin cytotoxicity. At doxorubicin concentrations of 5 micrograms/ml, BSO resulted in an 8-10 fold decrease in surviving cells, compared to cells exposed to doxorubicin alone. It was determined that BSO pre-treatments did not affect the accumulation of doxorubicin into the cell, the rate of cleavable complex stabilization by doxorubicin, or the rate of dissociation of stabilized cleavable complexes. These data suggest that BSO-induced doxorubicin sensitization occurs at a step following the stabilization of cleavable complexes or by an independent mechanism.


The efficacy of pharmacokinetic monitoring and dose modification of etanidazole on the incidence of neurotoxicity: results from a phase II trial of etanidazole and radiation therapy in locally advanced prostate cancer. Fifty-four patients have been entered on a Phase II trial to study the efficacy of etanidazole (ETA) for locally advanced prostate cancer. The primary goal was to study the incidence of and time to a complete response for patients receiving ETA and radiation therapy. The secondary goal was to prospectively evaluate the utility of pharmacokinetic monitoring and dose-modification of the incidence and severity of the dose-limiting peripheral neurotoxicity. Within a constant radiation therapy regimen, the dose of ETA was either (a) unmodified (2 g/m2, 3 times weekly for 17 doses); (b) altered by a schedule modification of either number of doses or dose adjustment; or (c) individualization of single dose size so that the total number of doses (19 doses) were maintained but the single dose size was adjusted to keep the total AUC of plasma concentration versus time to less than 40 mM-hr. Sufficient efficacy data are not yet available. The use of drug dose modification has reduced the incidence of neurotoxicity from (a) unmodified: 17/26 = 65% (1 grade II); (b) schedule adjustment: 5/9 = 55% (no grade II); and (c) individualized dose modification: 1/19 (no grade II) = 6%. The minimum number of time points needed to accurately assess the AUC will be determined. Pharmacokinetic monitoring will be important in the use of ETA so that drug underdosing can be avoided while minimizing the risk of serious neurotoxicity.


Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli. Nucleotide sequences called Chi (5'-GCTGGTGG-3') enhance homologous recombination near their location by the RecBCD enzyme in Escherichia coli (Chi activation). A partial inhibition of Chi activation measured in lambda red gam mutant crosses was observed after treatment of wild-type cells with DNA-damaging agents including UV, mitomycin, and nalidixic acid. Inhibition of Chi activation was not accompanied by an overall decrease of recombination. A lexA3 mutation which blocks induction of the SOS system prevented the inhibition of Chi activation, indicating that an SOS function could be responsible for the inhibition. Overproduction of the RecD subunit of the RecBCD enzyme from a multicopy plasmid carrying the recD gene prevented the induced inhibition of Chi activation, whereas overproduction of RecB or RecC subunits did not. It is proposed that in SOS-induced cells the RecBCD enzyme is modified into a Chi-independent recombination enzyme, with the RecD subunit being the regulatory switch key.


Characterization of two hypertransposing Tn5 mutants. Transposition of Tn5 in Escherichia coli is regulated by two transposon-encoded proteins: transposase (Tnp), promoting transposition preferentially in cis, and the trans-acting inhibitor (Inh). Two separate transposase mutants were isolated that replace glutamate with lysine at position 110 (EK110) and at position 345 (EK345). The EK transposase proteins increase the Tn5 transposition frequency 6- to 16-fold in cis and enhance the ability of transposase to act in trans. The purified mutant transposase proteins interact with transposon outside end DNA differently from the wild-type protein, resulting in the formation of a novel complex in gel retardation assays. During characterization of the transposase proteins in the absence of inhibitor, we found that wild-type transposase itself has a transposition-inhibiting function and that this inhibition is reduced for the mutant proteins. We present a model for the regulation of Tn5 transposition, which proposes the existence of two transposase species, one cis-activating and the other trans-inhibiting. The phenotype of the EK transposase mutants can be explained by a shift in the ratio of these two species.


Isolation and characterization of the Escherichia coli htrD gene, whose product is required for growth at high temperatures. Those genes in Escherichia coli defined by mutations which result in an inability to grow at high temperatures are designated htr, indicating a high temperature requirement. A new htr mutant of E. coli was isolated and characterized and is designated htrD. The htrD gene has been mapped to 19.3 min on the E. coli chromosome. Insertional inactivation of htrD with a mini-Tn10 element resulted in a pleiotropic phenotype characterized by a severe inhibition of growth at 42 degrees C and decreased survival at 50 degrees C in rich media. Furthermore, htrD cells were sensitive to H2O2. Growth rate analysis revealed that htrD cells grow very slowly in minimal media supplemented with amino acids. This inhibitory effect has been traced to the presence of cysteine in the growth medium. Further studies indicated that the rate of cysteine transport is higher in htrD cells relative to the wild type. All of these results, taken together, indicate that the htrD gene product may be required for proper regulation of intracellular cysteine levels and that an increased rate of cysteine transport greatly affects the growth characteristics of E. coli.


Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria. Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.


Characterization of the novel nisin-sucrose conjugative transposon Tn5276 and its insertion in Lactococcus lactis. A novel, chromosomally located conjugative transposon in Lactococcus lactis, Tn5276, was identified and characterized. It encodes the production of and immunity to nisin, a lanthionine-containing peptide with antimicrobial activity, and the capacity to utilize sucrose via a phosphotransferase system. Conjugal transfer of Tn5276 was demonstrated from L. lactis NIZO R5 to different L. lactis strains and a recombination-deficient mutant. The integration of Tn5276 into the plasmid-free strain MG1614 was analyzed by using probes based on the gene for the nisin precursor (nisA) and the gene for sucrose-6-phosphate hydrolase (sacA). The transposon inserted at various locations in the MG1614 chromosome and showed a preference for orientation-specific insertion into a single target site (designated site 1). By using restriction mapping in combination with field inversion gel electrophoresis and DNA cloning of various parts of the element including its left and right ends, a physical map of the 70-kb Tn5276 was constructed, and the nisA and sacA genes were located. The nucleotide sequences of Tn5276 junctions in donor strain NIZO R5 and in site 1 of an MG1614-derived transconjugant were determined and compared with that of site 1 in recipient strain MG1614. The results show that the A + T-rich ends of Tn5276 are flanked by a direct hexanucleotide repeat in both the donor and the transconjugant but that the element does not contain a clear inverted repeat.


DNA sequence of IS91 and identification of the transposase gene. IS91 is a 1,830-bp insertion sequence that inserts specifically at the sequence CAAG or GAAC of the target and does not duplicate any sequence upon insertion (23). By transposon mutagenesis, we have identified open reading frame 426 (ORF426; bp 454 to 1731) as the putative ORF for the transposase. It displays a cysteine-rich, potential metal-binding domain in its N-terminal region. Adjacent to ORF426, there is an ORF (ORF121) which precedes and terminally overlaps ORF426 by one amino acid. Tn1732 insertions in ORF121 do not affect the transposition frequency. IS91 has sequence similarities to IS801 from Pseudomonas syringae. Their putative transposases are 36% identical, including conservation of the cysteine-rich cluster. The information concerning IS801 insertion specificity and target duplication has been reevaluated in the light of our results.


Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity. Mutations in aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase, which selectively inhibit phosphatase activity have been isolated. These mutations yield amino acid substitutions within a 113-residue region of this 578-residue protein. These mutations may define a regulatory domain of this protein.


A 24-amino-acid polypeptide is essential for the biosynthesis of the coenzyme pyrrolo-quinoline-quinone. At least four genes are required for the biosynthesis of the coenzyme pyrrolo-quinoline-quinone (PQQ) in Acinetobacter calcoaceticus. The DNA region where one of these genes was mapped codes for a polypeptide of only 24 amino acids. Here we show that indeed this small peptide is essential for PQQ synthesis. Site-directed mutagenesis shows that at least one glutamate and one tyrosine residue of the polypeptide are essential for its function.


Effect of a novel class I antiarrhythmic agent, TYB-3823, on the calcium current in single guinea pig ventricular myocytes. The effect of TYB-3823 on Ca2+ current (ICa) of guinea pig ventricular myocytes was investigated by means of whole-cell patch-clamp technique. TYB-3823 (100-1,000 microM) caused a concentration-dependent decrease in ICa. Furthermore, a reduction of ICa induced by TYB-3823 (1,000 microM) was progressively accentuated by repetitive membrane depolarization, indicating a rate-dependent block of ICa. However, the inhibitory potency on ICa was approximately 1/1000 of a Ca2+ antagonistic agent, verapamil hydrochloride. Considering evidence that 3-30 microM TYB-3823 decreased the maximum upstroke velocity of the action potential of guinea pig ventricular muscles, it is indicated that the drug does not show its Ca2+ antagonistic property in the usual concentration range as a class I antiarrhythmic agent.


Serologic evidence of active Epstein-Barr virus infection in Epstein-Barr virus-associated lymphoproliferative disorders of children with acquired immunodeficiency syndrome. The DNA and nuclear antigens of Epstein-Barr virus (EBV) have been detected in specimens of tissue of non-Hodgkin lymphoma and lymphocytic interstitial pneumonitis from patients with acquired immunodeficiency syndrome. To determine whether there is serologic evidence of an active EBV infection in these disorders, we conducted a case-control study. The case patients were 10 children with acquired immunodeficiency syndrome and EBV genome-positive pneumonitis or lymphoma. We randomly selected one or, if available, two matched control patients with human immunodeficiency virus infection for each index patient and compared their EBV serologic profiles with those of the index case patient at the time of the biopsy. Ten case patients and 13 matched control patients were enrolled. All 10 case patients (100%) compared with 2 (15%) of 13 matched control patients had serologic evidence of either a primary or a reactivated EBV infection at the time the index patient had a biopsy performed (p less than 0.001). Therefore we found serologic and virologic evidence that EBV is etiologically related to EBV-associated lymphocytic interstitial pneumonitis and non-Hodgkin lymphoma in children with acquired immunodeficiency syndrome.


Determinants of response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in human lung cancer cell lines. Small-cell lung cancer (SCLC) is more sensitive to anticancer agents than non-small-cell lung cancer (NSCLC), but few studies have analyzed the mechanisms of natural drug resistance responsible for this difference. To elucidate these mechanisms, we determined drug sensitivity and evaluated the biochemical parameters affecting response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in both types of cancer cell lines, in particular the activity and content of DNA topoisomerase II, as well as etoposide uptake and cell doubling time. Drug sensitivity and cellular uptake of etoposide were determined by clonogenic assay and accumulation of radiolabeled drug, respectively. The topoisomerase II activity was assayed by decatenation of kinetoplast DNA to minicircle DNA using nuclear protein, and the content was determined by immunoblot analysis of nuclear extracts. We also compared the topoisomerase II content in parent cell lines with that in lines with cisplatin resistance acquired in vitro. Sensitivities to doxorubicin and etoposide were higher in SCLC cell lines than in NSCLC lines, and the difference was statistically significant. Etoposide uptake in SCLC cells was higher than in NSCLC cells; the difference was statistically significant, but this difference may not be sufficient to account for the variation in sensitivities of the cell lines. Topoisomerase II activities of nuclear protein from SCLC cell lines were reproducibly twofold higher than those for NSCLC cell lines. The topoisomerase II content in nuclear protein appeared to be higher in SCLC cell lines than in NSCLC cell lines and corresponded to the sensitivities to doxorubicin and etoposide. In the cisplatin-resistant NSCLC cell lines PC-7/CDDP and PC-14/CDDP, the topoisomerase II content was increased compared with that in the parent lines, but the topoisomerase II content in other cisplatin-sensitive parent lines was similar to that in resistant sublines. These findings suggest that the topoisomerase II activity and content may be major factors in determining sensitivity to topoisomerase II inhibitors.


Relationship of diet to risk of colorectal adenoma in men. Rates of colorectal cancer in various countries are strongly correlated with per-capita consumption of red meat and animal fat and inversely associated with fiber consumption. There have been few studies, however, of dietary risk factors for colorectal adenomas, which are precursors of cancer. Our purpose was to determine prospectively the relationship between dietary factors and risk of colorectal adenomas. Using data from the Health Professionals Follow-up Study, we documented 170 cases of adenomas of the left colon or rectum in 7284 male health professionals who completed a food-frequency questionnaire in 1986 and who had a colonoscopy or sigmoidoscopy between 1986 and 1988. Relative risk (RR) of adenoma was determined according to quintiles of nutrient intakes. After adjustment for total energy intake, saturated fat was positively associated with risk of colorectal adenoma (P for trend = .006); RR for the highest versus the lowest quintile of intake was 2.0 (95% confidence interval [CI] = 1.2-3.2). Dietary fiber was inversely associated with risk of adenoma (P for trend less than .0001); RR for men in the highest versus the lowest quintile was 0.36 (95% CI = 0.22-0.60). All sources of fiber (vegetables, fruits, and grains) were associated with decreased risk of adenoma. For subjects on a high-saturated fat, low-fiber diet, the RR was 3.7 (95% CI = 1.5-8.8) compared with those on a low-saturated fat, high-fiber diet. The ratio of the intake of red meat to the intake of chicken and fish was positively associated with risk of adenoma (P for trend = .02). These prospective data provide evidence for the hypothesis that a diet high in saturated fat and low in fiber increases the risk of colorectal adenoma. They also support existing recommendations to substitute chicken and fish for red meat in the diet and to increase intake of vegetables, fruits, and grains to reduce risk of colorectal cancer.


Health care workers infected with the human immunodeficiency virus. The next steps. The tragedy of five patients who contracted human immunodeficiency virus (HIV) infection from a seropositive dentist has alarmed the public. The Centers for Disease Control (CDC) recently revised its recommendations for preventing the transmission of HIV infection to patients during invasive procedures. The CDC abandoned a previous plan to list exposure-prone invasive procedures that HIV-infected health care workers should not perform. The CDC said "expert review panels" should decide on a case-by-case basis whether seropositive health care workers may perform invasive procedures. As of February 1992, the revised recommendations were under review by the US Department of Health and Human Services. Many issues remain to be clarified, such as how these panels will operate and whether decisions will be consistent in similar cases. Disregarding the CDC guidelines or infection-control precautions may further erode public trust and lead to draconian restrictions on HIV-infected health care workers. Physicians and dentists should respond more effectively to public fears about HIV transmission. The challenge is to protect patients while respecting the privacy and livelihood of health care workers.


Dynamic model of differentiation in Ewing's sarcoma cells. Comparative analysis of morphologic, immunocytochemical, and oncogene expression parameters. We report the establishment of a model of neural differentiation in four well-characterized Ewing's sarcoma cell lines. This process was induced by serum-depleted medium (1% fetal bovine serum) and agents such as dibutyryl cyclic AMP and retinoic acid. The morphologic changes were characterized predominantly by the presence of neurite-like elongated processes showing varicosities and branching along their course with numerous internal filaments and electron-dense granules. Immunocytochemically, differentiation was accompanied by a considerable increase in reactivity for neural markers of several types: neuroblastic, neuroepithelial, neuroendocrine, Schwannian and even glial. In contrast, the tumor promoter, phorbol 12-myristate 13-acetate inhibited differentiation. Several morphologic changes were observed in phorbol 12-myristate 13-acetate-treated cells: the cells became smaller and rounder, were poorly adherent to substrate, by electron microscopy lacked cytoplasmic organelles, electron-dense granules or neural processes, and showed decreased expression of neural markers. Northern blot analysis was performed to establish whether there was any relationship between neural differentiation and degree of N-myc, c-myc and dbl oncogene expression. There was no N-myc oncogene expression in the mRNA of Ewing's sarcoma cells, even after neural induced differentiation. The degree of c-myc and dbl oncogene expression appeared heterogeneous, and varied with the culture condition. Based on these results, it may be inferred that Ewing's sarcoma cells in vitro display a variable neural phenotype, there being a variety of biologic responses to diverse culture media and various differentiation agents, but with no consistent effect on N-myc, c-myc and dbl oncogene expression.


Ploidy of testicular carcinoma in situ.

Ploidy of testicular carcinoma in situ. The ploidy of carcinoma in situ and invasive germ cell tumors of the adult testis was compared by DNA flow cytometry. Irrespective of the tumor type with which it was associated, the median DNA index of carcinoma in situ was about the same as that of seminomas and higher than the DNA index of invasive nonseminomatous germ cell tumors. These data indicate that seminoma and carcinoma in situ cells are not only phenotypically similar but also have the same ploidy.


Comparative effects of tetrandrine and berbamine on production of the inflammatory cytokines interleukin-1 and tumor necrosis factor. Tetrandrine and berbamine are bisbenzylisoquinoline compounds which differ from each other in a minor way in terms of chemical structure, yet tetrandrine is 6-18 times more potent than berbamine in terms of inhibitory effects on production of interleukin-1 and tumor necrosis factor (TNF alpha) by monocytes and macrophages, and TNF beta production by lymphocytes. Moreover, tetrandrine significantly suppressed phosphoinositide turnover while berbamine did not. These findings may provide important insights into structure-activity relationships and the design of novel analogues and congeners useful in the therapy of chronic inflammatory and auto-immune diseases.


Long-term therapy with high-dose simvastatin does not affect adrenocortical and gonadal hormones in hypercholesterolemic patients. Simvastatin is an effective hypocholesterolemic drug that inhibits cholesterol synthesis selectively in the liver, but could have potential side effects on the adrenal gland, ovary, and testis, as these three glands use cholesterol for their hormonal biosynthesis. In this report, we examined adrenal and sex steroids in 10 type IIA hypercholesterolemic patients (three with familial [FH] and seven with polygenic hypercholesterolemia) over a period of 1 year on simvastatin therapy in order to confirm in vivo its selective action. Furthermore, we evaluated the adrenal reserve by a corticotropin rapid test, before starting treatment and again at the end of the third month on 20 mg of simvastatin per day, then at the sixth and 12th month on 40 mg/d. There was a significant lowering of total cholesterol (TC) (-31%), low-density lipoprotein cholesterol (LDL-C) (-39%), and apolipoprotein (apo) B (-39%); no statistically significant differences were seen in cortisol response to the corticotropin test between baseline and simvastatin-treated patients. No variation of any sex steroid was observed in patients of either gender. We conclude that long-term therapy with high-dose simvastatin does not interfere with either adrenocortical function or sex hormone production.


Erythrocyte sodium-potassium ATPase activity and thiol metabolism in genetically hyperglycemic mice. Erythrocyte sodium pump activity, osmotic fragility, and thiol status were measured in genetically hyperglycemic (db/db) mice and compared with their nondiabetic littermates (db/m). The data showed no major differences in these parameters. However, erythrocytes from streptozotocin (Stz)-induced diabetic rats had significantly lower activity of sodium pump and thiols with an almost fourfold increase in osmotic fragility as compared with erythrocytes from nondiabetic rats. Sorbinil (an aldose reductase inhibitor) treatment of Stz-diabetic rats normalized all these lesions, suggesting a key role for polyol pathway. However, sorbitol levels in erythrocytes from db/db and db/m mice were undetectable. The data suggest that in db/db mice, the relative lack of polyol pathway, a potential consumer of NADPH, may provide erythrocytes with optimal NADPH for glutathione reductase system, thus maintaining normal GSH levels even at the height of hyperglycemia. Thus, the genetically hyperglycemic mice may serve as a useful model to study diabetes related complications without involving polyol pathway.


Gene targeting in Arabidopsis thaliana.

Gene targeting in Arabidopsis thaliana. Gene targeting of a chromosomally integrated transgene in Arabidopsis thaliana is reported. A chimeric gene consisting of the promoter of the 35S RNA of CaMV, the polyadenylation signal of the octopine synthase gene and the coding region of the bacterial hygromycin phosphotransferase gene (hpt), which was rendered non-functional by deletion of 19 bp, was introduced into the genome of A. thaliana using Agrobacterium-mediated gene transfer. A total of 3.46 x 10(8) protoplasts isolated from 17 independent transgenic Arabidopsis lines harbouring the defective chimeric hpt gene were transformed via direct gene transfer using various DNA forms containing only the intact coding region of the hpt gene. Out of 150 hygromycin-resistant colonies appearing in the course of these experiments, four were the result of targeted recombination of the incoming DNA with the defective chromosomal locus as revealed by PCR and Southern blot analysis. Comparison with the number of transformants obtained when an hpt gene controlled by a promoter and terminator from the nopaline synthase gene was employed results in a maximal ratio of homologous to non-homologous transformation in A. thaliana of 1 x 10(-4).


Lethal overproduction of the Escherichia coli nucleoid protein H-NS: ultramicroscopic and molecular autopsy. The Escherichia coli hns gene, which encodes the nucleoid protein H-NS, was deprived of its natural promoter and placed under the control of the inducible lambda PL promoter. An hns mutant yielding a protein (H-NS delta 12) with a deletion of four amino acids (Gly112-Arg-Thr-Pro115) was also obtained. Overproduction of wild-type (wt) H-NS, but not of H-NS delta 12, resulted in a drastic loss of cell viability. The molecular events and the morphological alterations eventually leading to cell death were investigated. A strong and nearly immediate inhibition of both RNA and protein synthesis were among the main effects of overproduction of wt H-NS, while synthesis of DNA and cell wall material was inhibited to a lesser extent and at a later time. Upon cryofixation of the cells, part of the overproduced protein was found in inclusion bodies, while the rest was localized by immunoelectron microscopy to the nucleoids. The nucleoids appeared condensed in cells expressing both forms of H-NS, but the morphological alterations were particularly dramatic in those overproducing wt H-NS; their nucleoids appeared very dense, compact and almost perfectly spherical. These results provide direct evidence for involvement of H-NS in control of the organization and compaction of the bacterial nucleoid in vivo and suggest that it may function, either directly or indirectly, as transcriptional repressor and translational inhibitor.


Cytochrome c1 from potato: a protein with a presequence for targeting to the mitochondrial intermembrane space. Here we report the primary structure of potato cytochrome c1, a nuclear-encoded subunit of complex III. Using heterologous antibodies directed against cytochrome c1 from yeast two types of clones were isolated from an expression library, suggesting that at least two different genes are present and expressed in the genome. Northern blot analysis reveals that slightly varying levels of cytochrome c1 transcripts are present in all potato tissues analysed. A 1304 bp insert of one of the cDNA clones (pC13II) encodes the entire 320 amino acids of the precursor protein corresponding to a molecular weight of 35.2 kDa. As revealed by direct amino acid sequence determination of the cytochrome c1 protein another cDNA clone (pC18I) encodes the major form of cytochrome c1 present in potato tuber mitochondria. Western blots of subfractionated potato mitochondria show that the mature protein present in the membrane fraction is smaller than the pC13II encoded protein synthesized in Escherichia coli. The transient presequence of the protein is 77 amino acids long and has a bipartite polarity profile characteristic of presequences involved in targeting to the intermembrane space of fungal mitochondria. It consists of a positively charged NH2-terminal part which resembles "matrix targeting domains" and an adjacent hydrophobic region showing sequence similarities to "intramitochondrial sorting domains". The amino-terminal region of potato cytochrome c1 is the first presequence of a plant protein of the mitochondrial intermembrane space to be determined and may be useful in the study of intramitochondrial sorting in plants.


Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity. The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage lambda and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10(3)- to 10(4)-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage lambda. Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of lambda was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of lambda Ral-, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.


Positive regulation of the LPD1 gene of Saccharomyces cerevisiae by the HAP2/HAP3/HAP4 activation system. The LPD1 gene of Saccharomyces cerevisiae, encoding lipoamide dehydrogenase (LPDH), is subject to catabolite repression. The promoter of this gene contains a number of motifs for DNA-binding transcriptional activators, including three which show strong sequence homology to the core HAP2/HAP3/HAP4 binding motif. Here we report that transcription of LPD1 requires HAP2, HAP3 and HAP4 for release from glucose repression. In the wild-type strain, specific activity of LPDH was increased 12-fold by growth on lactate, 10-fold on glycerol and four- to five-fold on galactose or raffinose, compared to growth on glucose. In hap2, hap3 and hap4 null mutants, the specific activities of LPDH in cultures grown on galactose and raffinose showed only slight induction above the basal level on glucose medium. Similar results were obtained upon assaying for beta-galactosidase production in wild-type, or hap2, hap3 or hap4 mutant strains carrying a single copy of the LPD1 promoter fused in frame to the lacZ gene of Escherichia coli and integrated at the URA3 locus. Transcript analysis in wild-type and hap2 mutants confirmed that the HAP2 protein regulates LPD1 expression at the level of transcription in the same way as it does for the CYC1 gene. Site-directed mutagenesis of the putative HAP2/HAP3/HAP4 binding site at -204 relative to the ATG start codon showed that this element was required for full derepression of the LPD1 gene on non-fermentable substrates.


Cloning and nucleotide sequence of the Escherichia coli K-12 ppsA gene, encoding PEP synthase. We have cloned and sequenced the Escherichia coli K-12 ppsA gene. The ppsA gene codes for PEP synthase, which converts pyruvate into phosphoenolpyruvate (PEP), an essential step in gluconeogenesis when pyruvate or lactate are used as a carbon source. The open reading frame consists of 792 amino acids and shows homology with other phosphohistidine-containing enzymes that catalyze the conversion between pyruvate and PEP. These enzymes include pyruvate, orthophosphate dikinases from plants and Bacteroides symbiosus and Enzyme I of the bacterial PEP:carbohydrate phosphotransferase system.


The outcome of congenital cytomegalovirus infection in relation to maternal antibody status. Intrauterine transmission of cytomegalovirus (CMV) can occur whether a mother has prior immunity or acquires CMV for the first time during pregnancy. The degree of protection afforded an infected infant by the presence of antibody in the mother before conception is uncertain. We compared the outcomes of CMV-infected infants born to mothers who acquired primary CMV infection during pregnancy (primary-infection group) with those of CMV-infected infants born to mothers with immunity (recurrent-infection group). Screening for viruria identified 197 newborns with congenital CMV infection. Stored serum samples were used to categorize maternal infection as either primary or recurrent. We followed 125 infants from the primary-infection group and 64 from the recurrent-infection group. Serial medical, audiologic, psychometric, and eye examinations were used to identify sequelae of CMV infection. Only infants in the primary-infection group had symptomatic CMV infection at birth (18 percent). After a mean follow-up of 4.7 years, one or more sequelae were seen in 25 percent of the primary-infection group and in 8 percent of the recurrent-infection group. Thirteen percent of infants whose mothers had primary infection during pregnancy had mental impairment (IQ less than or equal to 70), as compared with none of those whose mothers had recurrent CMV infections. Sensorineural hearing loss was found in 15 percent of those in the primary-infection group and in only 5 percent of those in the recurrent-infection group. Bilateral hearing loss was identified only among children in the primary-infection group (8 percent). The presence of maternal antibody to CMV before conception provides substantial protection against damaging congenital CMV infection in the newborn. Primary maternal infection during pregnancy is associated with more severe sequelae of congenital CMV infection.


[Progressive multifocal leukoencephalopathy in a female patient with chronic lymphatic leukemia]. This case report describes a 78-year old female patient with chronic lymphocytic leukaemia and neurological symptoms due to progressive multifocal leukoencephalopathy (PML). At autopsy the histopathology was characteristic, the involvement of JC virus was established. PML occurs in immunocompromised patients and is caused by infection with JC papova virus. Epidemiology, clinical course, diagnostic procedures with special reference to the use of the polymerase chain reaction, and therapy are briefly reviewed.


Demyelinating disease after neurologically complicated primary Epstein-Barr virus infection. This report describes five patients who, following a neurologically complicated primary Epstein-Barr virus infection, developed progressive or relapsing neurologic deficits. The sequelae in four patients followed 4 to 12 years led to the diagnosis of multiple sclerosis (MS). The fifth patient presented with acute disseminated sclerosis and exhibits diffuse neurologic deficits that have persisted for 2 years. We suggest that the diagnosis of an unexplained acute neurologic or psychiatric syndrome should raise the question of a primary EBV etiology. A precisely timed serologic and hematologic study of the blood is imperative to capture the essential evidence. The data presented represent a clinical association between a neurologically complicated primary EBV infection and both chronic and acute demyelinating disease. The evidence does not justify a conclusion that EBV virus causes MS.


Clinical and electrophysiologic findings in chronic neuropathy of Lyme disease. We evaluated 25 patients with Lyme disease and chronic peripheral neuropathy. All had immunologic evidence of exposure to Borrelia burgdorferi and no other identifiable cause of neuropathy. Neuropathic symptoms began a median of 8 months (range, 0 to 165) after erythema migrans and had been present for a median of 12 months (range, 2 to 168) prior to evaluation. Twelve patients (48%) had generally symmetric distal, nonpainful paresthesia, and another 12 (48%) had generally asymmetric radicular pain. One patient (4%) had asymptomatic neuropathy. The most common physical finding was multimodal sensory loss, which was observed in 13 patients (52%); weakness and hyporeflexia were less common. Motor or sensory nerve conduction was slightly slow in 16 patients (64%). The paresthesia group more often had abnormalities on physical examination and on nerve conduction testing than did the radicular group. In 75% to 80% of patients from both groups, however, needle examination showed denervation in paraspinal and limb muscles. Among 20 patients who underwent lumbar puncture, only one had a slight spinal fluid pleocytosis. Six months after treatment with intravenous ceftriaxone, 19 patients (76%) were clinically improved. We conclude that Lyme disease can be associated with a reversible, mild chronic axonal sensorimotor polyradiculoneuropathy or polyradiculopathy.


Cholesterol emboli neuropathy.

Cholesterol emboli neuropathy. We report the clinical and pathologic features of a patient with peripheral neuropathy that was the first clinical expression of cholesterol emboli syndrome (CES). Biopsy of skeletal muscle and peripheral nerve revealed cholesterol clefts in lumens of small arteries, necrotizing arteritis, and severe degeneration of peripheral and intramuscular nerves. At autopsy, the peripheral nervous system was extensively affected by similar changes. We conclude that (1) peripheral neuropathy may be the initial manifestation of CES. Presumably, deposition of cholesterol leads to arteritis. (2) The underlying pathology of CES neuropathy is chronic axonal degeneration, possibly due to chronic ischemia of epineurial arteries. (3) Muscle biopsy is important in the antemortem diagnosis of CES. Nerve biopsy may show involvement of epineurial vessels. (4) CES may resemble polyarteritis nodosa clinically and pathologically. (5) CES may be under-recognized and should be included in the differential diagnosis of any neuropathy of uncertain cause, particularly when there is a history of vascular catheterization, or severe aortic atherosclerosis.


Linkage of atypical myotonia congenita to a sodium channel locus. We performed linkage analysis in a pedigree segregating an allele for autosomal dominant, painful myotonia that is potassium sensitive and responsive to acetazolamide. This allele was tightly linked to a skeletal-muscle, sodium channel locus which is now a candidate for the site of the mutational defect in acetazolamide-responsive myotonia congenita. Since this sodium channel locus is completely linked to the disease allele in all hyperkalemic periodic paralysis and paramyotonia congenita pedigrees studied, the molecular alteration causing acetazolamide-responsive myotonia congenita is likely an allelic defect in this human, skeletal-muscle, sodium channel gene.


Renal tumors in childhood.

Renal tumors in childhood. A great variety of tumors and tumor-like lesions affect the kidneys in childhood. We have discussed the practical aspects of the examination of tumors because the pathologist's role is becoming more critical to the staging and management of these patients. The use of newer techniques shows promise in renal tumors, and flow cytometry with DNA ploidy analysis may prove useful in more precisely identifying high-risk patients. Invaluable data continue to be generated from the large cooperative groups, particularly the National Wilms' Tumor Study, which helps refine our understanding of the renal tumors and their proper management. Close collaboration between the oncologist, the radiologist, the surgeon, the pathologist, and the radiation oncologist is the mainstay in providing the best possible outcome for these patients.


Role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of agonist-evoked calcium elevation in human platelets. Most platelet agonists activate and elevate the cytosolic free calcium concentration in human platelets through receptor-dependent mechanisms that are antagonized by cAMP- and cGMP-elevating agents. Nitrovasodilators such as nitroprusside and endothelium-derived relaxing factor are potent cGMP-elevating platelet inhibitors. In the present study, the role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of ADP- and thrombin-evoked calcium elevation and activation of human platelets was investigated. Preincubation of platelets with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP; a membrane-permeant selective activator of the cGMP-dependent protein kinase that does not significantly affect cGMP-regulated phosphodiesterases) inhibited the thrombin-induced phosphorylation mediated by myosin light chain kinase and protein kinase C. Nitrovasodilator-induced protein phosphorylation in human platelets was distinct from that induced by cAMP-elevating prostaglandins and could be mimicked by 8-pCPT-cGMP. Preincubation of human platelets with nitrovasodilators or 8-pCPT-cGMP inhibited the ADP- and thrombin-evoked calcium elevation in the presence and absence of external calcium. Nitrovasodilators and 8-pCPT-cGMP also inhibited the agonist-induced Mn2+ influx, but stopped-flow experiments indicated that the ADP receptor-operated cation channel was not significantly inhibited. These results suggest that in human platelets nitrovasodilators inhibit the agonist-induced calcium mobilization from intracellular stores and the secondary store-related calcium influx but not the ADP receptor-operated cation channel. The results also suggest that these nitrovasodilator effects are mediated by cGMP and the cGMP-dependent protein kinase.


c-Jun represses the human insulin promoter activity that depends on multiple cAMP response elements. Glucose is known to increase the cAMP concentration in pancreatic beta cells. To determine the mechanism by which cAMP augments insulin gene expression, we first identified the cAMP response elements (CREs) of the human insulin gene. In DNase I footprint analysis, the bacterially synthesized CRE-binding protein, CRE-BP1, protected four sites: two sites in the region upstream from the insulin core promoter, one site in the first exon, and one site in the first intron. To examine the roles of those four sites, we constructed a series of DNA plasmids in which the wild-type and mutant insulin promoters were linked to the chloramphenicol acetyl-transferase gene. Studies of the transcriptional activity of these plasmids after transfection into hamster insulinoma (HIT) cells showed that these four sites contributed additively to the cAMP inducibility of the insulin promoter. Surprisingly, the c-jun protooncogene product (c-Jun) repressed the cAMP-induced activity of the insulin promoter in a cotransfection assay with the c-Jun expression plasmid. Northern blot analysis demonstrated that the level of c-jun mRNA was dramatically increased by glucose deprivation in HIT cells. These results suggest that glucose may regulate expression of the human insulin gene through multiple CREs and c-Jun.


Enzymatic catalysis and dynamics in low-water environments. Enzymes suspended in organic solvents represent a versatile system for studying the involvement of water in enzyme structure and function. Addition of less than 1% (vol/vol) water to tetrahydrofuran containing 1 M 1-propanol leads to a substantial increase in the transesterification activity of subtilisin Carlsberg (from Bacillus licheniformis) that correlates with a sharp increase in the active-site polarity and a 90% decrease in the rotational correlation time (i.e., increase in mobility) of a nitroxide spin label within the active site. Water in excess of 1% has little additional effect on active-site polarity and coincides with a further increase in spin-label mobility, yet the transesterification activity decreases dramatically. Thus, transesterification activity increases and then decreases with increasing enzyme hydration and flexibility (which are presumably coupled through dielectric screening), suggesting that the conformation of partially hydrated subtilisin is different from that of the nearly dry enzyme--i.e., enzyme containing less than 9% (wt/wt) water.


Overexpression of an Aplysia shaker K+ channel gene modifies the electrical properties and synaptic efficacy of identified Aplysia neurons. Although potassium channels play a variety of roles in shaping the electrical properties of neurons, little is known about how these channels are constituted in neurons. To examine the assembly and physiological function of A-type K+ channels in mature differentiated neurons, we have developed a highly efficient gene transfer method for Aplysia neurons that has allowed us to express about 10(7) copies of the cloned Aplysia Shaker (Sh) K+ channel (AK01a) in single identified cells. We find that expression of AK01a phenocopies one of the native transient K+ currents (IAdepol), suggesting that the native channel carrying IAdepol is assembled as a homooligomer of AK01a. Overexpression of AK01a has substantial effect on the action potential, shortening its duration, enhancing its hyperpolarizing afterpotential, and depressing by more than half the amount of transmitter release by the action potential from the terminals. Thus, the AK01a channel not only contributes to the firing properties within a given neuron but also can regulate the signaling between interconnected cells.


Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation. DNA primase-dependent synthesis of oligoribonucleotides 10-15 nucleotides long was observed in the presence of ATP, UTP, GTP, and CTP by using the purified components of the simian virus 40 (SV40) DNA replication system. The DNA primase-catalyzed reaction required the SV40 large tumor antigen (T antigen), DNA polymerase alpha (pol-alpha), the three-subunit human single-stranded DNA binding protein (HSSB), and topoisomerase I. The synthesis of small RNAs was unaffected by the addition of activator 1, proliferating cell nuclear antigen, and DNA polymerase delta, proteins that can support extensive leading-strand synthesis. The RNA primers were derived predominantly from transcription of the lagging-strand template, even after prolonged incubation, indicating that the leading strand did not serve as a template. When the four dNTPs were added after oligoribonucleotide synthesis, pol-alpha extended the RNA primers hybridized to SV40 DNA. Pulse-chase experiments revealed that the small RNA chains were elongated to Okazaki-sized products. T7 DNA polymerase was also shown to rapidly extend oligoribonucleotide primers in the presence of aphidicolin or antibodies against pol-alpha, conditions under which pol-alpha was markedly inhibited. These findings suggest that interactions between T antigen, pol-alpha-primase, and HSSB position the pol-alpha-primase complex on the lagging-strand template for RNA primer synthesis.


Analogues of luteinizing hormone-releasing hormone containing cytotoxic groups. In an attempt to produce better cytotoxic analogues, chemotherapeutic antineoplastic radicals including an alkylating nitrogen mustard derivative of D-phenylalanine (D-melphalan), reactive cyclopropane, anthraquinone derivatives [2-(hydroxymethyl)anthraquinone and the anticancer antibiotic doxorubicin], and an antimetabolite (methotrexate) were coupled to suitably modified agonists and antagonists of luteinizing hormone-releasing hormone (LH-RH). Analogues with D-lysine6 and D-ornithine6 or N epsilon-(2,3-diaminopropionyl)-D-lysine and N delta-(2,3-diaminopropionyl)-D-ornithine were used as carriers for one or two cytotoxic moieties. The enhanced biological activities produced by the incorporation of D amino acids into position 6 of the agonistic analogues were further increased by the attachment of hydrophobic cytotoxic groups, resulting in compounds with 10-50 times higher activity than LH-RH. Most of the monosubstituted agonistic analogues showed high affinities for the membrane receptors of human breast cancer cells, while the receptor binding affinities of peptides containing two cytotoxic side chains were lower. Antagonistic carriers [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Trp3,Arg5,D-Lys6,D-Ala10] LH-RH [where Nal(2) is 3-(2-naphthyl)alanine], [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Trp3,Arg5,N epsilon-(2,3-diaminopropionyl)-D-Lys6,D-Ala10]LH-RH, and their D-Pal(3)3 homologs [Pal(3) is 3-(3-pyridyl)alanine] as well as [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Pal(3)3,Tyr5,N epsilon-(2,3-diamino-propionyl)-D-Lys6,D-Ala10]LH-RH were linked to cytotoxic compounds. The hybrid molecules inhibited ovulation in rats at doses of 10 micrograms and suppressed LH release in vitro. The receptor binding of cytotoxic analogues was decreased compared to the precursor peptides, although analogues with 2-(hydroxymethyl)anthraquinone hemiglutarate had high affinities. All of the cytotoxic analogues tested inhibited [3H]thymidine incorporation into DNA in cultures of human breast and prostate cancer cell lines. Some cytotoxic analogues also significantly suppressed the growth of mammary and prostate cancers in vivo in animal models.


Metabolism of glyceryl trinitrate to nitric oxide by endothelial cells and smooth muscle cells and its induction by Escherichia coli lipopolysaccharide. Here, we demonstrate that the metabolism of glyceryl trinitrate (GTN) to nitric oxide (NO) occurs not only in bovine aortic smooth muscle cells (SMCs) but also in endothelial cells (ECs) and that this biotransformation is enhanced by pretreatment with Escherichia coli lipopolysaccharide (LPS). Two bioassay systems were used: inhibition of platelet aggregation and measurement of cGMP after stimulation by NO of guanylate cyclase in SMCs or ECs. In addition, NO produced from GTN by cells was measured as nitrite (NO2-), one of its breakdown products. Indomethacin (10 microM)-treated SMCs or ECs enhanced the platelet inhibitory activity of GTN. This effect was abrogated by coincubation with oxyhemoglobin (oxyHb; 10 microM), indicating release of NO from GTN. LPS (0.5 microgram/ml; 18 h) enhanced at least 2- to 3-fold the capacity of SMCs or ECs to form NO from GTN, and this enhancement was attenuated when cycloheximide (10 micrograms/ml) was incubated together with LPS. Furthermore, when incubated with GTN (200 microM) SMCs or ECs treated with LPS (0.5 microgram/ml; 18 h) released more NO from GTN than nontreated cells as indicated by a much higher (8- to 9-fold) increase in the levels of cGMP. Exposure of SMCs to GTN (600 microM) for 30 min led to an increase in the levels of NO2- dependent on cell numbers, which was enhanced when SMCs were treated with LPS. Incubation of nontreated or LPS-treated cells with NG-monomethyl-L-arginine (300 microM; 60 min) did not influence the metabolism of GTN to NO. SMCs failed to enhance the antiplatelet activity of sodium nitroprusside. Anesthetized rats treated with an intraperitoneal injection of LPS (20 mg/kg) 18 h beforehand showed enhanced hypotensive responses to GTN (0.25-1 mg/kg). These effects were blocked by methylene blue (10 mg/kg) but not by indomethacin (3 mg/kg). LPS did not alter the hypotensive responses induced by phentolamine, verapamil, or SIN-1. Thus, both in vitro and in vivo, LPS induces the enzyme(s) metabolizing GTN to NO.


Retrovirus insertion into herpesvirus in vitro and in vivo. Retroviruses and herpesviruses are naturally occurring pathogens of humans and animals. Coinfection of the same host with both these viruses is common. We report here that a retrovirus can integrate directly into a herpesvirus genome. Specifically, we demonstrate insertion of a nonacute retrovirus, reticuloendotheliosis virus (REV), into a herpesvirus, Marek disease virus (MDV). Both viruses are capable of inducing T lymphomas in chickens and often coexist in the same animal. REV DNA integration into MDV occurred in a recently attenuated strain of MDV and in a short-term coinfection experiment in vitro. We also provide suggestive evidence that REV has inserted into pathogenic strains of MDV in the past. Sequences homologous to the REV long terminal repeat are found in oncogenic MDV but not in nononcogenic strains. These results raise the possibility that retroviral information may be transmitted by herpesvirus and that herpesvirus expression can be modulated by retroviral elements. In addition, retrovirus may provide a useful tool to characterize herpesviral function by insertional mutagenesis.


The free radical in pyruvate formate-lyase is located on glycine-734. Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherichia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central 13C nucleus (A parallel = 4.9 mT and A perpendicular = 0.1 mT) and to 13C nuclei in alpha and beta positions agree with literature data for glycine radical models. N-coupling was verified through uniform 15N-labeling. The large 1H hyperfine splitting (1.5 mT) dominating the EPR spectrum was assigned to the alpha proton, which in the enzyme radical is readily solvent-exchangeable. Oxygen destruction of the radical produced two unique fragments (82 and 3 kDa) of the constituent polypeptide chain. The N-terminal block on the small fragment was identified by mass spectrometry as an oxalyl residue that derives from Gly-734, thus assigning the primary structural glycyl radical position. The carbon-centered radical is probably resonance-stabilized through the adjacent carboxamide groups in the polypeptide main chain and could be comparable energetically with other known protein radicals carrying the unpaired electron in tyrosine or tryptophan residues.


Sexually transmitted viruses other than HIV and papillomavirus. This review has focused on a select group of viruses that can be sexually transmitted. The viruses include the herpesviruses, hepatitis A virus, hepatitis B virus, delta virus, non-A, non-B hepatitis virus(es), and molluscum contagiosum. Their impact on the population alone or in association with HIV disease necessitates a clear understanding of their ability to cause infection and of the manifestations of these infections. Characterization of these particular pathogens and treatment have been discussed with respect to the most current data available. Despite the growing sophistication in the field, we are still limited in our endeavors to identify and manage many viral infections. Therefore, measures to prevent transmission are continually being evaluated in an attempt to minimize exposure to these pathogens.


Biology of genital tract human papillomaviruses. A large number of HPV types infect humans. They infect squamous epithelia of the skin and mucous membranes. Infections are widespread in sexually active populations. Some HPV types have oncogenic potential. Infections with HPV-16, HPV-18, and some additional types are risk factors for cervical cancer. The HPV-6 and HPV-11 types are responsible for most of the genital condylomas and nearly all respiratory papillomas. The immunologic responses to HPVs are not well understood.


Human papillomavirus--related diseases in the female patient. Human papillomavirus (HPV) has been strongly associated with malignancy in the female lower genital tract. Because squamous-cell carcinoma of the cervix is preceded by a spectrum of easily detectable and treatable premalignant changes, it is very preventable. The management of the patient with an abnormal Papanicolaou smear and the treatment of cervical, vaginal, and vulvar disease are outlined.


Human papillomavirus in the male patient. Human papillomavirus is a double-stranded DNA virus associated with a broad spectrum of clinical states including condylomata acuminata, latent and subclinical infection (acetowhitening), Bowen's disease, and carcinoma of the penis and anus. The virus is transmitted by direct contact with site-subtype specificity; additional studies are needed to elucidate the exact transmissibility and disease course of HPV infection. The association of HPV-16 and HPV-18 with anogenital malignancy increases the importance of treating such infections and raises questions about the role of HPV in oncogenesis. Treatment modalities for HPV include cytotoxic agents, surgical excision, immunotherapy, and laser ablation. Success rates appear best for laser ablation of evident disease. No therapeutic modality appears superior for treating latent disease. The public health ramifications of HPV are vast and warrant investigation to further our scientific and clinical understanding of oncogenesis and its prevention.


Dietary fibre: the effectiveness of a high bran intake in reducing renal calcium excretion. Fifteen healthy women were given a standardized calcium-rich diet (1800 mg calcium/day) with or without 36 g bran for 5 days. A similar study was also carried out with rice, soy and wheat bran. Urine samples were also collected 24 h. With all brans renal calcium excretion decreased and renal oxalic acid excretion increased. However, influence of rice bran was statistically significant. After 5 days of consuming 36 g rice bran/day 14 of 15 subjects showed decreased calcium excretion, but increased oxalic acid excretion. Relative supersaturation with calcium oxalate, as a measure for the risk of calcium stone formation, increased after addition of all brans.


RP-30A: new tracer for detection of changes in testicular blood flow in rat torsion model. RP-30A is a radioactive tracer being evaluated for the detection of regional myocardial blood flow. This study compares RP-30A to technetium 99m pertechnetate as radioactive tracers for the detection of testicular blood flow changes in early testicular torsion. The left testis of adult male Sprague-Dawley rats was subjected to either thirty or sixty minutes of 720 degrees torsion. Injections of RP-30A or 99mTc-pertechnetate followed by sacrifice and scintillation counting of the testes was performed. No significant difference was detected between the torted testes and the right control testes in both groups receiving 99mTc-pertechnetate and the thirty-minute group receiving RP-30A. The torted testes of the sixty-minute group receiving RP-30A revealed a significant difference (decrease) in uptake indicating that RP-30A may be a more sensitive tracer in detecting testicular blood flow changes in early testicular torsion.


The E7 functions of human papillomaviruses in rat 3Y1 cells. Among more than 60 human papillomavirus (HPV) genotypes, several HPVs are believed to be high risk because they are found in close association with cervical carcinoma. We compared the E7 genes from HPVs 1, 6b, 16, 18, and 33 for their transactivating, transforming, and mitogenic functions in a single cell line rat 3Y1. Whereas both the low-risk (1 and 6b) and the high-risk (16, 18, and 33) HPVs were transactivating for the adenovirus E2 promoter, only the high-risk HPVs were capable of focal transformation as assayed by an efficient method using the SR alpha-promoter and in conjunction with the HPV 16 E6 gene. The putative oncogenicity of HPVs appears to be reflected in vitro by the focal transformation, but not by the transactivation. Transient expression of the E7 genes controlled by the dexamethasone-responsive MMTV-LTR showed that the HPV 16 mutant E7s only with residual transforming activity were not mitogenic, but that, although the low-risk HPV E7s were less efficient, both the low-risk and high-risk HPV E7s were capable of inducing cellular DNA synthesis. Probably, the capability to induce cell DNA synthesis is necessary but not sufficient for the E7-mediated focal transformation.


A mutation in the catalytic domain of pp60v-src is responsible for the host- and temperature-dependent phenotype of the Rous sarcoma virus mutant tsLA33-1. We have analyzed a host- and temperature-dependent mutant of Rous sarcoma virus in order to learn more about the nature of mutations which lead to a host range phenotype. We have cloned and sequenced the v-src genes from this mutant, tsLA33-1, and from its presumed parent, tsLA33. Both the tsLA33 and the tsLA33-1 pp60v-src proteins contain multiple mutations. The tsLA33 v-src gene product has amino acid alterations at four positions. In the tsLA33-1 v-src gene product, two of these four mutations have reverted to wild type. We have constructed chimeras between the two mutant v-src gene products and between each mutant and the Prague A v-src gene product. To assess the contribution of each amino acid change to the transformation phenotypes of tsLA33 and tsLA33-1, we expressed the hybrid proteins in both chicken embryo fibroblasts and Rat-3 fibroblasts. Additionally, we have measured the protein tyrosine kinase activity of chimeras constructed between the tsLA33 and tsLA33-1 pp60v-src proteins. Our results indicate that mutations in the catalytic domain of each protein are the principal determinants of the transforming ability and protein tyrosine kinase activity of the tsLA33 and tsLA33-1 pp60v-src proteins.


Regulation of gene expression directed by the long terminal repeat of the feline immunodeficiency virus. The long terminal repeat (LTR) of a retrovirus contains sequence elements that constitute a promoter for controlling viral gene expression in infected cells. We have examined regulation of LTR-directed gene expression in feline immunodeficiency virus (FIV), a T-lymphocytopathic lentivirus associated with a fatal AIDS-like disease in domestic cats. Two independent virus isolates, designated FIV-Petaluma and FIV-PPR, have been molecularly cloned and show greater than 85% sequence homology. Both clones (termed pF34 and pPPR) produce infectious virus after transfection of permissive feline cells. Basal promoter activity of the LTRs was measured in various cell lines in transient expression assays using plasmids containing the viral LTR linked to the bacterial chloramphenicol acetyltransferase gene. Both LTRs were strong promoters in several cell lines, although in some cell lines the pF34 LTR had four- to fivefold higher basal activity than the pPPR LTR. FIV LTR mutations affecting the first AP4 site, AP1 site, ATF site, or NF-kappa B site resulted in decreased basal activity of the FIV promoter. Mutational analysis also revealed a negative regulatory element. In cotransfection experiments, both pF34 proviral DNA and pPPR proviral DNA appeared to transactivate either the pF34 LTR or the pPPR LTR; however, levels of transactivation were very low. Cotransfection of both LTRs with FIV subgenomic clones containing various viral open reading frames resulted in low level or no transactivation. The LTRs of both FIV clones responded to cell activation signals in human T-lymphoid cells (Jurkat) treated with phytohemagglutinin and phorbol-12-myristate-13-acetate. Promoter function of both FIV LTRs was also enhanced in cells treated with either forskolin, an inducer of intracellular cyclic-AMP (c-AMP), or dibutyryl c-AMP. Analysis of site-specific mutants showed that a potential AP1 site in the U3 domain of the LTR was required for T-cell activation responses mediated by protein kinase C, whereas a putative ATF site was the target for c-AMP-induced responses mediated by protein kinase A. These studies revealed that cellular transcription factors play a significant role in regulation of FIV gene expression.


The V5A13.1 envelope glycoprotein deletion mutant of mouse hepatitis virus type-4 is neuroattenuated by its reduced rate of spread in the central nervous system. Following intracerebral inoculation of adult Balb/c Byj mice, the MHV-4 strain of mouse hepatitis virus (MHV) had an LD50 of less than 0.1 PFU, whereas its monoclonal antibody resistant variant V5A13.1 had an LD50 of 10(4.2) PFU. To determine the basis for this difference in neurovirulence we have studied the acute central nervous system (CNS) infection of these two viruses by in situ hybridization. Both viruses infected the same, specific neuroanatomical areas, predominantly neurons, and spread via the cerebrospinal fluid, along neuronal pathways and between adjacent cells. The neuronal nuclei infected and the spread of virus within the brain are described. The main difference between the parental and variant viruses was the rate at which the infection spread. MHV-4 spread rapidly, destroying large numbers of neurons and the animals died within 4 days of infection. The variant virus spread to the same areas of the brain but at a slower rate. This difference in the rate of virus spread was also apparent from the brain virus titers. The slower rate of spread of the variant virus appears to allow intervention by the immune response. Consistent with this, the variant virus spread slowly in athymic nu/nu mice, but in the absence of an intact immune response, infection and destruction of neurons eventually reached the same extent as that of the parental virus and the mice died within 6 days of infection. We conclude that the V5A13.1 variant of MHV-4 is neuroattenuated by its slower rate of spread in the CNS.


Three transcriptionally distinct forms of Epstein-Barr virus latency in somatic cell hybrids: cell phenotype dependence of virus promoter usage. Phenotypically distinct human B cell lines display two transcriptionally distinct forms of Epstein-Barr virus (EBV) latency. Latency I (Lat I) in group I Burkitt's lymphoma (BL) cell lines is characterized by selective expression of the virus-coded nuclear antigen EBNA 1 from a uniquely spliced mRNA driven by the Fp promoter. Latency III (Lat III) in group III BL and EBV-transformed lymphoblastoid cell lines (LCLs) is characterized by expression of EBNAs 1, 2, 3a, 3b, 3c, and -LP from mRNAs driven by the Cp or Wp promoter and of the latent membrane proteins (LMPs 1, 2A, and 2B) from mRNAs driven by the LMP promoters. Here we have altered the group I BL and LCL phenotypes by cell hybridization and screened for attendant changes in EBV latency by PCR analysis of viral mRNAs and immunoblotting of viral proteins. Fusion of group I BL cells with LCLs activated the BL virus genome from a Lat I to Lat III pattern of gene expression. Fusion of LCLs with nonlymphoid lines repressed virus gene expression from Lat III either to Lat I or to another form of latency (Lat II) hitherto not seen in vitro and characterized by selective expression of the Fp-driven EBNA 1 mRNA and of the LMP 1, 2A, and 2B transcripts. There are therefore three forms of EBV latency which can be interconverted by altering cellular phenotype and thereby virus promoter usage.


Cleavage of concatemeric DNA at the internal junction of "translocation" mutants of pseudorabies virus and inversion of their L component appear to be linked. When pseudorabies virus (PrV) strains are grown in chicken embryo fibroblasts (CEF), variants ("translocation" mutants) arise in which there is a duplication of the leftmost sequences of the genome and their translocation in inverted orientation next to the internal inverted repeat bracketing the S component. In these variants, the UL becomes bracketed by inverted repeats and is found in two orientations relative to the Us. To study the cis-functions involved in cleavage of concatemeric DNA as well as those involved in inversion of the L component and to ascertain whether the two events are linked in the "translocation" mutants, a viral mutant (vLD68) was constructed in which the terminal 64 bp of the L component (that include sequences with homology to the pac 2 site of HSV) and the 4 terminal bp of the S component were deleted from the internal junction. Although revertants that have acquired the 68 bp at the internal junction emerge rapidly in populations of vLD68, analysis of the characteristics of this mutant revealed that: (1) the termini derived from both orientations of the L component include the 64 bp that have been deleted from the internal junction of vLD68; (2) in contrast to other "translocation" mutants, the internal junction of the vLD68 genome is not a good substrate for cleavage; (3) inversion of the L component of true vLD68 DNA does not occur or is rare; a good correlation exists in the populations of vLD68 between the proportion of revertants that have acquired an intact internal junction and the proportion of genomes with an L component that inverts. These results show that an intact internal junction in "translocation" mutants is necessary for both inversion of their L components and cleavage at their alternative internal junction. Since cleavage at the alternative junction will result in inversion of the L component, we conclude that inversion of the L component of "translocation" mutants of PrV can be attributed to cleavage of concatemeric DNA at the internal alternative junction.


SV40 large T antigen trans-activates the long terminal repeats of a large family of human endogenous retrovirus-like sequences. The Simian Virus 40 (SV40) large T antigen (T) is required for the initiation of viral replication, the autoregulation of early gene expression, and the activation of late gene expression in productively infected cells. In addition to these roles, T has been implicated in the transcriptional activation of a variety of viral and cellular promoters. We have used the chloramphenicol acetyltransferase (CAT) reporter gene system to study the effect of T on the long terminal repeats (LTRs) of a large family of human endogenous retrovirus-like sequences, RTVL-H. Here we show that T can activate expression from certain RTVL-H LTRs 5- to 30-fold. Competition experiments in which an excess of plasmid containing only an RTVL-H LTR was cotransfected with an LTR-CAT reporter gene construct confirmed that this effect is specific for RTVL-H sequences. Restriction enzyme analysis using methylation-sensitive enzymes has shown that this activation is not due to plasmid replication. We have also observed this trans-activation effect in two CV-1 cells lines containing stably integrated LTR-CAT constructs. These results demonstrate that a known transforming protein can alter the transcriptional capabilities of RTVL-H LTRs. As there are approximately 3000 related LTRs in the genomes of humans and other primates, these findings suggest that a large number of these promoters and their associated transcripts may be transcriptionally stimulated by this and other oncogens.


Replication, latent infection, and reactivation in neuronal culture with a herpes simplex virus thymidine kinase-negative mutant. Herpes simplex virus type 1 (HSV-1) mutant viruses lacking functional viral thymidine kinase activity are reported to be incapable of replication in neurons. To investigate the role of viral thymidine kinase (TK) activity in the HSV-1 infection of the neuron, we studied a thymidine kinase-negative (TK-) mutant virus engineered to eliminate TK function without affecting the other known transcripts encoded in this region of the genome. Studies using the mouse eye model demonstrated that the mutant behaved as is reported for other TK- viruses: DNA of the mutant virus was detected in the ganglia during the latent infection by polymerase chain reaction, but virus did not reactivate after explantation of the ganglia. Utilizing the neuronal cultures, we investigated the ability of the mutant virus to replicate in neurons and the capacity of the mutant virus to establish latency and reactivate. With a low multiplicity of infection (m.o.i.), replication of the TK- mutant virus in sensory neurons in culture was significantly delayed compared to that of the wild-type virus. However, when a high m.o.i. was used, the mutant and the wild-type viruses replicated with similar kinetics. The TK- mutant virus was capable of establishment of latency and reactivation from the latent infection in sensory neurons in culture. These data suggest that HSV-1 thymidine kinase activity facilitates viral replication, but that TK activity is not essential for either replication or reactivation from latent infections in neurons in vitro.


Serum levels of intact parathyroid hormone in elderly patients with hip fracture living at home. The serum levels of intact parathyroid hormone and cholecalciferol metabolites have been measured in patients with hip fracture above 70 years of age admitted to hospital from home-living conditions and compared with serum levels in age- and sex-matched home-living control subjects. It was found that patients with hip fracture had significantly lower levels of calcidiol (29.7 +/- 15.9 vs 46.0 +/- 27.8 nmol/l) and calcitriol (63.6 +/- 25.0 vs 91.1 +/- 39.5 pmol/l) with no difference in serum levels of intact parathyroid hormone (4.7 +/- 2.1 vs 5.3 +/- 3.3 pmol/l). The data suggest that secondary hyperparathyroidism is not an important risk factor in our population of patients with hip fracture.


Sex-related responses of beta-endorphin, ACTH, GH and PRL to cold exposure in humans. To establish a possible different reaction between the male and the female to short-term exposure to cold, thermal, cardiovascular and pituitary hormonal responses to cold stress were measured in eight normal men and eight women (ages 19-24). The women were eumenorrheic and were tested in the follicular phase. Each subject, lightly clad, was required to remain for 30 min in a room at an ambient temperature of 25 degrees C followed by a 30 min period in a cold room at 4 degrees C. A month later, control tests were carried out at a constant 25 degrees C temperature for 1 h in the same subjects. Skin temperature, heart rate, blood pressure and plasma levels of beta-endorphin, ACTH, cortisol, GH and PRL were measured before and after cold exposure in the two groups. Before the test, all examined parameters were similar in the two groups. During cooling, blood pressure rose and pulse rate decreased significantly in the men, but not in the women, whereas skin temperature dropped in both groups. However, after cold exposure skin temperature was significantly lower in the women than in the men. A slight, but not significant increase in beta-endorphin, ACTH, cortisol and GH levels was observed after cooling in the men, whereas the women showed significant increments of these hormones. When values of skin temperature were combined with the differential (after minus before cold test) hormonal values, significant negative correlations were found for beta-endorphin, ACTH, cortisol and GH.(ABSTRACT TRUNCATED AT 250 WORDS)


Neonatal ovariectomy and pituitary-adrenal responsiveness in the adult rat. Litters of female rat pups were handled daily from birth to weaning. When 12 days old the pups were ovariectomized or received sham surgery. At 70 days sham and ovariectomized animals were decapitated immediately or placed within a novel environment for 10, 20, or 40 min before decapitation. Basal plasma levels of androstenedione in ovariectomized animals were approximately half that of intact sham controls, and stress caused only a small rise in androstenedione in this group. Androstenedione levels in ovariectomized animals were consistently at or below the lower sensitivity of the radioimmunoassay, indicating that circulating levels of androstenedione in the female rat are primarily of ovarian origin. Basal ACTH and corticosterone levels did not differ significantly between the two groups. However, stress-induced levels of ACTH and corticosterone were significantly depressed in ovariectomized subjects compared to controls following exposure to the novel environment. Furthermore, while ACTH levels started to return to baseline by 40 min in controls, this effect was not observed in ovariectomized animals. These findings demonstrate that prepubertal removal of ovarian humoral factors can have a long-term impact on the responsiveness of the hypothalamic-pituitary-adrenal axis to stress.


Thyrotropin action is impaired in the thyroid gland of old rats. Aging in rats is characterized by low plasma concentrations of thyroid hormones with unchanged levels of TSH, suggesting an altered TSH action in addition to the impaired regulation of TSH secretion. To evaluate TSH action we determined TSH binding to thyroid membranes of young and old male rats (3-4 and 24-26 months of age), as well as the activity of adenylate cyclase in basal and stimulated conditions. Saturation analyses of [125I]-bTSH to thyroid membranes in the presence of increasing quantities of unlabelled bTSH (0.03-100 mU) show two types of binding sites, one of high affinity (Ka 1.5 10(9) mol l-1) the other of lower affinity (Ka 1.2 10(8) mol l-1), which are similar in both age groups. The number of TSH binding sites of high affinity is less in old rats than in young rats (7.6 +/- 0.9 vs 14.8 +/- 1.1 TSH mU/mg protein, N = 11 and 10 respectively, p less than 0.001), whereas the number of binding sites of low affinity is not significantly different (76.0 +/- 8.2 vs 99.1 +/- 9.0 TSH mU/mg protein). The activity of adenylate cyclase determined in basal conditions is similar in both old and young rats (1.11 +/- 0.12 vs 1.04 +/- 0.9 nmol cAMP/2 h x mg/protein). TSH (10 mU) induced a significant increase in cAMP formation with the thyroid membranes from young rats but not with those from old rats. In contrast, the stimulation of cAMP formation by GTP (2 mmol/l) or forskolin (10 mmol/l), two direct stimulators of adenylate cyclase, is similar in both groups of rats (200% and 250%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of cell growth, c-myc mRNA, and 1,25-(OH)2 vitamin D3 receptor in C3H/10T1/2 mouse embryo fibroblasts by calcipotriol and 1,25-(OH)2 vitamin D3. Calcipotriol is a synthetic 1,25-(OH)2D3 analogue with high affinity for the 1,25-(OH)2D3 receptor, but with a lower affinity than 1,25-(OH)2D3 for vitamin D binding protein in serum. The inhibitory action of calcipotriol and 1,25-(OH)2D3 on proliferation of C3H/10T1/2 mouse embryo fibroblasts was examined in the non-transformed cell line Cl 8 and in the two transformed, tumorigenic cell lines Cl 16 and TPA 482. Upon exposure to 10 nmol/l calcipotriol or 1,25-(OH)2D3, the proliferation of Cl 8 cell line was almost completely suppressed, whereas both hormones had no effect on the cell lines Cl 16 and TPA 482. Calcipotriol was at least as effective as 1,25-(OH)2D3 in inducing up-regulation of the 1,25-(OH)2D3 receptor. Displacement studies showed no difference between calcipotriol and 1,25-(OH)2D3 in the affinity for the receptor present in Cl 8 or Cl 16 cell extracts. Furthermore, the inhibition of cell growth in Cl 8 cells by calcipotriol was not accompanied by any consistent change in the steady-state expression of c-myc mRNA. In conclusion, calcipotriol had potent growth inhibitory effect on the non-transformed cell line similar to 1,25-(OH)2D3. In the transformed cell lines, calcipotriol did not inhibit proliferation despite potent up-regulation of the 1,25-(OH)2D3 receptor.


Ultrastructural alterations of the conduction system in mice exhibiting sinus arrest or heart block during Coxsackievirus B3 acute myocarditis. By light and electron microscopy we studied the sinus nodes and atrioventricular (AV) conducting tissue of six C3H/He mice having coxsackievirus B3 acute myocarditis. Sinus arrest was documented in all six mice, and second- or third-degree AV block was documented in three of the six mice. Although myocarditic changes in the conduction system, especially in the sinus node, were less than those in atrial and ventricular working myocardium, there were distinct abnormalities within both the sinus node and AV conducting tissue in all six hearts. Important ultrastructural alterations were inflammatory cell infiltrates and significant injury of specialized cells and of neural tissue. Specialized cells showed various features of degeneration and necrosis. Neural tissue damage included degeneration of axons and Schwann cells and disorganization of the neuromuscular junctions. Inflammatory cells, particularly macrophages, were often in intimate contact with injured specialized cells and neural tissue. Interstitial edema and bleeding and lymphatic vessel dilatation were also observed. These pathologic changes are considered to play an important role in the development of the documented disturbances of rhythm and conduction.


Role of water-soluble dietary fiber in the management of elevated plasma cholesterol in healthy subjects. Guidelines for the use of water-soluble dietary fibers (WSDF) in the dietary management of elevated plasma cholesterol are not well-established. Consequently, 4 studies were conducted to explore the plasma lipid-lowering effects of a variety of WSDF. Studies were randomized, double-blind, placebo-controlled trials involving healthy men and women (plasma cholesterol greater than 5.17 mmol/liter; greater than 200 mg/dl). Study duration ranged from 4 to 12 weeks. The WSDF acacia gum yields a low viscosity, palatable beverage when mixed in water. However, despite its WSDF classification, acacia gum consumed for 4 weeks as the sole WSDF source (15 g of WSDF/day) or primary source in a WSDF mixture (17.2 g of WSDF/day; 56% derived from acacia gum) did not produce a significant lipid-lowering effect versus placebo. When 15 g of WSDF/day consisting of psyllium hust, pectin, and guar and locust bean gums (medium viscosity) was consumed for 4 weeks, significant reductions in cholesterol resulted (total cholesterol 8.3%, low-density lipoprotein cholesterol 12.4%; p less than 0.001) that were comparable to changes achieved with 10 g of WSDF/day from high-viscosity guar gum. The magnitude of the lipid-lowering effect was related to intake of WSDF ranging from 5 to 15 g/day (low-density lipoprotein cholesterol +0.8% [placebo], -5.6% [5 g/day], -6.8% [10 g/day], -14.9% [15 g/day]; p less than 0.01 for trend). The effects of WSDF on plasma lipids were similar for men and women. A diet rich in selected WSDF may be a useful adjunct to the dietary management of elevated plasma cholesterol.


Youth at risk. Sex, drugs, and human immunodeficiency virus. Adolescents and young adults are at risk for human immunodeficiency virus type 1 infection due to unprotected sexual intercourse and drug use. In 1988 and 1989, blinded surveys were conducted in 84 sexually transmitted disease clinics, 115 women's health clinics, and 19 drug treatment centers in 38 metropolitan areas. Blood specimens from 153,242 clients, aged 15 to 24 years, were tested for human immunodeficiency virus type 1 antibodies after all client identifiers were removed. In sexually transmitted disease clinics, the median rate was 0.4% among 15- to 19-year-olds, compared with 1.4% among 20- to 24-year-olds. Among heterosexual adolescents, rates in females were significantly higher than in males (Wilcoxon signed rank test). Rates in heterosexuals were highest in the northeastern and southeastern United States and in Puerto Rico. In 20- to 24-year-old male clients in sexually transmitted disease clinics who had sex with males, rates ranged from 9.7% to 55.6%. In drug treatment centers, the median rate among 20- to 24-year-old men and women was 8.3% (range, 0% to 33.3%). Rates in women's health clinics were much lower (median, 0.1%). The high rates of infection in certain groups of adolescents and young adults indicate the need for improved care, education, and outreach targeted toward those at high risk.