Protective effect of nedocromil sodium on the IL1-induced release of GM-CSF from cultured human bronchial epithelial cells. Cultured human bronchial epithelial cells constitutively produce granulocyte-macrophage colony-stimulating factor (GM-CSF). The synthesis and release of GM-CSF is upregulated in bronchial epithelium of patients with symptomatic asthma and this may contribute to the local activation of inflammatory cells in their bronchial mucosa. The cause of this upregulation of GM-CSF expression is unknown, but an increased release of interleukin-1 (IL1) from other airway resident cells might be involved, as an increase in GM-CSF production can be induced in vitro in normal bronchial epithelial cells by IL1 and the airway secretions of asthmatics contain high amounts of this cytokine. In the present study, we have evaluated the effect of the anti-inflammatory and antiasthmatic drug, nedocromil sodium, on the spontaneous and IL1-induced expression of GM-CSF in cultured bronchial epithelial cells. This compound, at the concentration of 10(-5) M, reduced the IL1-induced increase in GM-CSF release from epithelial cells by more than 40%, but it did not affect the constitutive production of GM-CSF.


Evidence that MEHP inhibits rat granulosa cell function by a protein kinase C-independent mechanism. We have recently shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the reproductive toxicant di-(ethylhexyl) phthalate (DEHP), inhibited FSH- but not forskolin-, isoproterenol-, or cholera toxin-stimulated granulosa cell cAMP accumulation in vitro. In addition, MEHP also inhibited FSH-stimulated progesterone production, a cAMP-dependent process. Similar to MEHP, the protein kinase C (PKC) activator, 12-0-tetradecanoyl-phorbol 13-acetate (TPA) has been shown to inhibit rat granulosa cell cAMP accumulation in a FSH-specific manner, and decrease FSH-stimulated progesterone production. Due to the similarity with respect to inhibition of cAMP accumulation, we conducted studies to determine if the inhibitory actions of MEHP on granulosa cell function are mediated via activation of PKC. Treatment of granulosa cells for 48 h with 100 microM MEHP produced no effect on forskolin- or isoproterenol-stimulated progesterone production, indicating that MEHP does not have a post-cyclic AMP site of action with respect to progesterone inhibition. Unlike the FSH-specific effect seen with MEHP, treatment with 10 nM TPA inhibited FSH-, forskolin-, and isoproterenol-stimulated progesterone production. In addition, maximally inhibitory concentrations of TPA and MEHP caused significantly greater inhibition of FSH-stimulated cAMP accumulation than either compound alone. Finally, addition of the progesterone precursor, pregnenolone, reversed the FSH-stimulated progesterone production inhibition by MEHP, but not that by TPA. Taken together, these data indicate that the inhibitory effects of MEHP on granulosa cell function are independent of phorbol ester-sensitive PKC activation.


Oral administration of carvedilol and prazosin inhibits the prostaglandin F2 alpha- and noradrenaline-induced contraction of human hand veins in vivo. Carvedilol is a beta-blocker with additional vasodilating activity. This study was performed in order to determine whether the vasodilator action of orally administered carvedilol in man is based upon an alpha-adrenoceptor antagonism exclusively or if evidence for an additional mechanism could be confirmed. The influence of carvedilol (50 mg p.o.) and prazosin (2 mg p.o.) upon the vasoconstrictor effect of noradrenaline and prostaglandin F2 alpha, infused into superficial hand veins, was established in 8 healthy male volunteers. Increasing dosages of the vasoconstrictors below their threshold of systemic activity were employed in order to obtain dose-response curves of the hand veins congested at a venous occlusion pressure of 40 mmHg. These dose-response curves were repeated 1 and 3.5 h after oral administration of either carvedilol, prazosin, or placebo. The ex vivo, in vitro alpha 1-receptor occupancy in plasma was measured before and after each vasoconstrictor dose-response curve, using an alpha 1-radioreceptor binding assay. Washout periods of 48 h were kept between study days, investigating the influence of one orally administered drug upon one of the local vasoconstrictor dose-response curves at a time. In the alpha 1-radioreceptor assay, plasma concentrations from 0.9- to 1.7-fold the equilibrium dissociation constant (Ki) of carvedilol could be evaluated 1 as well as 3.5 h after medication, corresponding with a receptor occupancy of 44%-63%. After prazosin, 9-13 times the Ki values were determined, which amounts to an alpha 1-adrenoceptor occupation of about 90%-93%.(ABSTRACT TRUNCATED AT 250 WORDS)


Alcohol and drug abuse treatment at the University of Connecticut Health Center. This article describes a multidisciplinary team approach to the treatment of alcohol and drug dependence and comorbid disorders. The principal aim of this approach is the identification and treatment of patients' chemical dependence, as well as the medical and psychiatric disorders that contribute to and/or stem from the chronic use of alcohol and drugs. The focus of treatment is on the identification of high risk and other problem situations, training coping skills to handle these situations, developing insight, and enhancing patients' motivation for sobriety and ongoing treatment. In this article we describe the features of our program that address these various goals.


Engaging patients with alcohol problems in treatment: the first consultation. This paper considers the impact of the first consultation at a specialist alcohol clinic on patients' perceptions of their drink problem and on their expectations of help from three sources: an alcohol clinic, general practitioners and Alcoholics Anonymous. At intake, males had higher expectations than females of help from the alcohol clinic while people in manual occupations and those in the 'less heavy' drinking category had higher expectations of general practitioner help than other patients. Change was found to occur during the first clinic consultation so that by the end of the session patients had raised their rating of problem severity and their expectations of help from the three sources examined. Overall, patient perceptions of the clinic assessment session were positive. The study highlights the importance of obtaining a full understanding of the process of engagement in treatment and indicates the potential of a clinical assessment to effect change in patient attitudes.


The use of services for alcohol problems: general practitioner and specialist alcohol clinic. A sample of 40 patients referred to a specialist alcohol clinic were given a full assessment by a clinician and then randomly allocated to one of two groups. The first group received continuing care from the clinic. The second group were returned to the management of their general practitioners who were offered specialist support in caring for their patients. Patients and general practitioners were followed-up 6 months after the clinic assessment. This paper reports on the uptake of clinic and general practice services by patients and examines the possible factors associated with continuing attendance, including patient attributes, service activity, patients' self-assessments of their drink problem and their expectations of help from services. Qualitative data is used to examine patients' perceptions and experiences of clinic and general practice-based care for drinking problems. The findings highlight some of the difficulties reported by patients in receiving help for their problems particularly from general practitioners.


Are polioviruses a cause of schizophrenia? Pre-natal infection with polioviruses could contribute to the subsequent development of schizophrenia. The hypothesis draws support from the declining incidence of schizophrenia, the excess of schizophrenic winter births, and the increased rates of schizophrenia among West Indian immigrants. There are parallels with other late sequelae of poliovirus infections. These postulations generate a testable hypothesis of a genetic link between schizophrenia and susceptibility to poliomyelitis.


Psychophysiological investigations of patients with unilateral symptoms in the hyperventilation syndrome. Anxiety states sometimes lead to hyperventilation (HV) which may, in turn, give rise to a variety of physical symptoms. One way in which HV may present is with unilateral somatosensory symptoms, often left-sided. We report nine such cases. The mechanisms of lateralisation was examined using EEG and bilateral somatosensory evoked potentials which were carried out before and after HV. No difference in conduction velocity was found between affected and unaffected arms, but non-specific abnormalities were frequently noted in the EEGs. The results support the role of a central rather than a peripheral mechanism in the production of unilateral symptoms in HV.


Anterograde transport of HSV-1 and HSV-2 in the visual system. The anterograde spread of herpesvirus in the visual system subsequent to retinitis has been observed clinically. We compared the ability of two well-studied Herpes simplex virus (HSV) strains to be transported in the anterograde direction in the hamster visual system: strain McIntyre, representing HSV-1, and strain 186, representing HSV-2. Intravitreal injection of HSV-2 labeled more retinorecipient neurons than did HSV-1, suggesting important type differences in the ability of HSV to infect retinorecipient neurons after intravitreal injection. The most likely explanation for our results is that HSV-2 is more efficiently adsorbed than HSV-1 in the retinal ganglion cells. Our results also suggest that HSV may be useful as an anterograde transneuronal tracer for neuroanatomical studies of the visual system.


[An open study on the efficacy and tolerance of a low molecular weight heparin (CY 216) in the therapeutic treatment of deep venous thrombi in orthopedics]. A prospective clinical trial was performed to evaluate the efficacy and safety of a low molecular weight heparin (nadroparine, CY 216) in the treatment of patients with deep venous thrombosis following recent orthopaedic surgery or trauma. Forty-three patients received nadroparine subcutaneously, 100 IU AXa.kg-1 every 12 hours for a period of 10 days. A quantitative assessment of deep vein thrombosis was made according Marder's scoring after 10 days of treatment. A substantial lysis was observed in 73 per cent and a revascularisation in 52 per cent of the patients. These results are statistically significant, although not correlated with the clinical symptoms and the size of the thrombi. Tolerance was assessed on the rate of bleeding events: a minor bleeding occurred in 6 per cent of the patients. Laboratory adjustment was performed in 8 per cent of the cases. The results of this study suggest that nadroparine administered subcutaneously at a fixed daily dose of 200 IU AXa.kg-1 is as efficient and safe as unfractionated heparin in the treatment of early venous thrombosis following orthopaedic surgery or trauma.


The effects of equine rhinovirus, influenza virus and herpesvirus infection on tracheal clearance rate in horses. The response of horses exposed to three common respiratory viruses was studied by measuring tracheal mucociliary clearance rates in the trachea. Tracheal clearance rates (TCR) were determined before, during illness and following recovery in horses exposed to equine rhinovirus (ERhV-2), equine influenza virus (EIV) and equine herpesvirus (EHV-4) by means of lateral scintigraphs made following an injection of technetium-99m sulphide colloid into the tracheal lumen. In six horses exposed to ERhV-2, TCR remained within normal limits. Exposure to EIV resulted in reduced TCR in six of seven horses, with TCR remaining below the 95% confidence limits of normal values for each horse for up to 32 days despite the resolution of clinical signs. Moderate changes were observed in six horses exposed to EHV-4, but significant reductions in TCR were evident in three animals. Measurement of TCR was a useful, minimally-invasive technique which demonstrated that respiratory viruses may cause persistent changes in TCR, even though clinical signs are not evident.


Development and evaluation of an enzyme-linked immunosorbent assay for detection of bovine antibodies to epizootic hemorrhagic disease of deer viruses. An indirect enzyme-linked immunosorbent assay (I.ELISA) is described for detection of bovine serum antibody to epizootic hemorrhagic diseases of deer virus (EHDV). Serum samples, at a dilution of 1:200, were incubated with group-specific EHDV antigens, pre-adsorbed to microtiter plates. Bound antibodies were detected by a murine monoclonal antibody to bovine immunoglobulin (Ig)G1 (heavy-chain specific) conjugated with horseradish peroxidase. The performance of the I.ELISA in detecting antibodies to EHDV in sequential serum samples from calves experimentally infected with serotypes 1,2,3 and 4 was evaluated. The I.ELISA detected EHDV antibodies from 14 days postinfection when seroconversion by the standard agar gel immunodiffusion (AGID) test was also evident. The group-specific antibodies to EHDV increased exponentially during the first two to four weeks postinfection and remained relatively stable for about 12 months in some calves. Unlike observations with the AGID test, no reaction was seen in the I.ELISA between blue-tongue virus (BTV) antigen and sera from calves given a single dose of EHDV. The performance of the I.ELISA and AGID were compared using 3,135 AGID negative bovine field sera from herds in Ontario, Alberta and British Columbia and 130 AGID positive samples collected from cattle in 1987 and 1988 during and after outbreaks of EHD in the Okanagan Valley, British Columbia. The specificity and sensitivity of the assay relative to the AGID test were 99.3% and 91.5% respectively, with an overall agreement of 99.0% between the tests.(ABSTRACT TRUNCATED AT 250 WORDS)


A short incubation serum neutralization test for bovine viral diarrhea virus. A three day serum neutralization (SN) test for the detection of antibodies to bovine viral diarrhea virus (BVDV), which is an improvement on the existing five day test, is described. The improved test results in a more rapid viral cytopathic effect and utilizes Madin Darby kidney (MDBK) cells, and horse serum as a medium supplement. A comparison of tests utilizing the NADL and the Singer strains of BVDV and the use of either secondary bovine kidney cells with calf serum (BKCS) or continuous MDBK cells with horse serum (MDHS) was performed. Analysis of the SN results of 685 serum samples from 445 Quebec and Ontario cattle showed that there was no difference, as expected, in the means of the SN antibody titers when the NADL strain was used in either the BKCS or MDHS system but SN antibody titers were elevated (p less than 0.01) when the Singer strain was used in the MDHS system. The SN test with the Singer strain also yielded significantly higher titers for sera from 200 Alberta cattle.


Cytometric and electron microscopic studies of the direct interaction of divalent nickel with intact and chemically modified HuT-78 lymphoblasts. Cytometric and ultrastructural studies on 24 hr cultures of intact, 1.0 mM H5IO6, and 0.1 mM SeO2-oxidized HuT-78 lymphoblasts were performed after their direct, 30 min interaction with 1.0 mM NiCl2. Except for moderately depressed cell viability, divalent nickel did not alter the progression of intact and oxidized target cells through the phases of the cell cycle. Although the plasma membrane remained structurally intact, marked distortion of mitochondria structure and increased osmiophilia were an invariable attribute of all nickel-pulsed cells. Moreover, numerous electron-opaque, intracellular depositions were detected in SeO2-oxidized, nickel-pulsed cells. It is concluded that the initial state of plasma membrane, and the interaction of nickel with other trace elements, have jointly determined the response of HuT-78 cells to brief and direct, divalent nickel pulses.


Efficacy of antiserum produced in goats and pigs to passively protect piglets against virulent transmissible gastroenteritis virus. The protective effect of sera produced in swine and goats exposed to virulent transmissible gastroenteritis virus (TGEV) or modified-live TGEV was tested in hysterectomy-derived, colostrum-deprived three-day-old pigs. Pigs were given serum with their daily ration of milk, and their immunity to virulent TGEV was determined. The pigs were observed for ten days for clinical signs of TGEV infection. One of nine pigs receiving goat serum was protected whereas all three pigs receiving three doses of swine serum per day were protected. Because virus was not isolated from the goats after oral/intranasal vaccination, it is suggested the virus did not replicate in either the respiratory or digestive tract of the goat.


Transmissible gastroenteritis virus antibody production in vitro by porcine peripheral blood leukocytes. The purpose of this study was to demonstrate the in vitro production of transmissible gastroenteritis virus (TGEV)-specific antibodies by peripheral blood leukocytes (PBL) harvested from piglets infected with TGEV. Piglets were infected with the virulent Purdue strain of TGEV and at intervals postinfection their PBL were cultivated in the presence of TGEV antigen, control antigen or pokeweed mitogen (PWM). The culture supernatants were tested for TGEV antibodies by a fixed cell enzyme immunoassay. Antibodies were never found in the supernatants of unprimed PBL cultures from control piglets, nor in cultures stimulated with control antigen, and antibodies were produced more frequently in response to stimulation of primed PBL with viral antigen than with PWM. In PBL cultures stimulated with viral antigen, TGEV antibodies of the IgG class were produced more frequently than IgA class antibodies. Optimal antibody responses were produced by PBL harvested two weeks after infection and cultivated at a concentration of 10(7) cells/mL for five days.


Angiosarcoma of the breast after lumpectomy and radiation therapy for adenocarcinoma. Three cases of angiosarcoma of the breast after lumpectomy and radiation therapy for adenocarcinoma are presented. Only two similar cases have been documented. The role of radiation therapy and chronic lymphedema is discussed. Although the overall survival is usually less than 22 months, two of these patients are still alive after 2 years.


Recurrent adult nephroblastoma. Long-term remission after surgery plus adjuvant high-dose chemotherapy, radiation therapy, and allogeneic bone marrow transplantation. The authors report a case of Stage IV, unfavorable histologic type adult nephroblastoma. The patient was treated with multimodal therapy: combination chemotherapy consisting of cyclophosphamide, doxorubicin, cisplatin, and etoposide succeeded by nephrectomy and radiation therapy. After a disease-free period of 27 months, a pararectal relapse was treated by surgery, high-dose chemotherapy, and allogeneic bone marrow transplantation (BMT). The patient is alive and disease-free 3.5 years after BMT.


Chromatin structure, DNA methylation, and gene expression at sites of viral integration in human fibroblasts. Implications for chromosomal fragility. We analyzed the structural and functional properties of a chromosomal region in which a recombinant hybrid virus adenovirus 5/SV40 preferentially integrates. Our results demonstrated that the structure of the cellular targets for DNA and RNA viruses is very similar and that the cellular sequence flanking the integrated virus possesses, simultaneously, all the features postulated to be the molecular basis for chromosomal fragility.


t(6;12)(q23;q13) and t(10;16)(q22;p11) in a phyllodes tumor of breast. Cytogenetic analysis of short-term cultures from a phyllodes tumor showed clonal chromosome changes including t(6;12)(q23;q13) and t(10;16)(q22;p11). This is the first reported karyotype in this tumor type. We discuss the breakpoints of these translocations in relation to the involvement of possible candidate genes.


Complex karyotypic aberrations, including i(12p), in malignant mixed mullerian tumor of uterus. We report the cytogenetic findings in a malignant mixed mullerian tumor of the uterus in a 59-year-old woman. Karyotypic analysis of short-term cultures revealed two abnormal clones of cells characterized by extensive structural and numerical rearrangements. An i(12p) maker chromosome was present in addition to other changes in both clones. This marker, characteristically associated with testicular germ cell tumors in males, has recently been reported in ovarian germ cell tumors, a mediastinal germ cell tumor and in a mixed mullerian tumor of the ovary.


Stimulation of anchorage-independent cell growth by endothelin in NRK 49F cells. Endothelin (ET) is a vasoconstrictor peptide originally isolated from vascular endothelial cells. Recent studies have revealed that ET has many biological functions including growth factor-like activity. The present study aims to clarify whether ET-1 possesses the ability to stimulate anchorage-independent cellular growth, an indicator of factors with transforming activity. We found that NRK 49F cells possess a large number of high-affinity ET-1 receptors; labeled 125I-ET-1 binding was displaced by unlabeled ET-2 in a similar dose response, but in the case of ET-3, 100-fold more was required. Specific 125I-ET-3 binding was undetectable in NRK 49F cells, indicating that ET receptors in NRK 49F cells are ET-1/ET-2 selective. NRK 49F is a cell line which is most commonly used to assay for anchorage-independent cellular growth. Therefore, we explored whether ETs promote anchorage-independent cellular growth in this cell line. ET-1 and ET-2 stimulated NRK colony formation dose dependently in the presence of 1 nM epidermal growth factor (EGF). In contrast, ET-3 did not have colony-stimulating ability. In the presence of EGF, the maximal effect of ET-1 was approximately 90% of that of transforming growth factor-beta. Moreover, in the presence of maximal stimulating concentrations of EGF and transforming growth factor-beta, ET-1 additionally induced colony formation. These results indicate that ET-1 and -2 possess transforming growth factor-like activity for NRK 49F cells. Since ET-1 and -2 increased intracellular calcium levels, this ion may participate in signal transduction pathways by which ET-1 and -2 promote colony formation.


Herbal medicine use, Epstein-Barr virus, and risk of nasopharyngeal carcinoma. Herbal medicine use is thought to be linked to nasopharyngeal carcinoma (NPC) either through its ability to reactivate the Epstein-Barr virus (EBV) or through a direct promoting effect on EBV-transformed cells. To investigate this, 104 histologically confirmed NPC cases and 205 matched controls were studied in The Philippines. Blood was collected to assess antibody titers against EBV, and an interview was administered which elicited information concerning herbal medicine use and other risk factors for NPC. Subjects strongly positive for anti-EBV antibodies (Epstein-Barr nuclear antigen [EBNA]) (titers greater than or equal to 1:80) were at a 21-fold excess risk of disease (95% confidence interval, 8.4, 51.8). Herbal medicine use was also associated with NPC (relative risk, 2.5; 95% confidence interval, 1.4, 4.5). Associations persisted after adjustment for education, smoking, Chinese ancestry, and consumption of salted fish. Exposure to herbal medicines among subjects testing negative/weakly positive for anti-EBNA antibodies was not associated with an elevation in risk (relative risk, 0.6), strong positivity to anti-EBNA antibodies in the absence of herbal medicine use was associated with a significant 16-fold excess risk of disease, and exposure to herbal medicines among subjects testing strongly positive for anti-EBNA antibodies was associated with a significant 49-fold excess risk of NPC when cases were compared to controls. Similar results were obtained when other serological measures of EBV exposure were used. Anti-EBV antibody titers were elevated in herbal medicine users compared to nonusers among cases but not among control subjects. This suggests that, if herbal medicines interact with EBV in the development of NPC, they do not do so by reactivating EBV infection but rather through a direct proliferative effect on EBV-transformed cells. Although the interaction between EBV and herbal medicines is biologically plausible, larger, more detailed studies need to be conducted to validate this preliminary finding.


Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines. Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell lung cancer cell lines express the EGF receptor.


A third Wilms' tumor locus on chromosome 16q. Loss of heterozygosity studies have been used to identify chromosomal regions which are frequently deleted and thus indicate areas which may harbor tumor suppressor genes. As a result, both the WT1 gene located in chromosome 11p13 and an unidentified gene(s) within chromosome 11p15 have been implicated in Wilms' tumorigenesis. Cytogenetic and linkage studies suggest that additional non-chromosome 11 sites are involved in Wilms' tumor. Because these sites may also involve loss of heterozygosity, loci on 33 autosomal arms were screened for allele loss in a series of Wilms' tumors. We found that in addition to loss on chromosome 11p (11 of 25 informative tumors) there was significant loss on chromosome 16q (9 of 45 informative tumors), while the total frequency of allele loss excluding these loci was low (9 of 426 total informative loci). These data indicate that losses of both chromosome 11p and 16q alleles are nonrandom events and suggest that 16q is the location of a third tumor suppressor gene underlying Wilms' tumorigenesis. The parental origin of the lost chromosome 16q allele was determined in eight sporadic tumors. Alleles of paternal and of maternal origin were each lost in four sporadic tumors indicating that, unlike chromosome 11p, alleles of either parental origin are lost on 16q.


Differential effects of amsacrine and epipodophyllotoxins on topoisomerase II cleavage in the human c-myc protooncogene. Amsacrine and demethylepipodophyllotoxins (etoposide and teniposide) are potent topoisomerase II inhibitors which have optimum activity in different cancers. To investigate whether these differences are due to different activity on cellular oncogenes, drug-induced topoisomerase II cleavage sites were mapped and sequenced in the human c-myc protooncogene. In the presence of purified murine L1210 topoisomerase II, amsacrine induces prominent cleavage in the P2 promoter (site 2499/2502). Footprinting experiments indicate that topoisomerase II binds to the entire promoter region (approximately 20 base pairs on the sides of the P2 site). In the case of teniposide or etoposide, cleavage is more diffuse and markedly less at the P2 site. Mapping of cleavage sites in human small cell lung carcinoma cells (NCI N417) also shows that cleavage in the P2 promoter region is induced preferentially by amsacrine but not by demethylepipodophyllotoxins. Thus, selective gene damage among topoisomerase II inhibitors may contribute to differential anticancer activity.


Tumor necrosis factor as an autocrine growth factor for neuroblastoma. Recombinant tumor necrosis factor (TNF) stimulates the proliferation of two neuroblastoma cell lines, SKNFI and SKNBE, in both serum-free medium and fetal calf serum-supplemented medium but has no effect in medium without insulin. This effect is very similar with TNF doses ranging from 5 to 500 ng/ml but depends on the duration of treatment; when cells are treated for 168 h with TNF, the maximal index of proliferation is observed between 120 and 144 h of treatment. The two neuroblastoma cell lines express type A and type B TNF receptors and contain TNF protein; however, TNF is undetectable in culture supernatants. Treatment of the two neuroblastoma cell lines with a rabbit polyclonal antibody to TNF for 96 h fully inhibits [3H]thymidine incorporation; less than 5% viable cells are left in the samples after treatment. A combination of two monoclonal antibodies against type A and type B TNF receptors also inhibits over 85% of the [3H]thymidine incorporation by the two cell lines after 96 h of treatment; the use of a single antibody has a partial effect, suggesting that both receptors are functional on the neuroblastoma cell lines. Taken together, these results show that TNF is an autocrine growth factor for the two neuroblastoma cell lines SKNFI and SKNBE. The results described above have been confirmed on two other neuroblastoma cell lines, IRM32 and CLB-PE.


Platelet-derived growth factor and its receptors in human glioma tissue: expression of messenger RNA and protein suggests the presence of autocrine and paracrine loops. The expression of platelet-derived growth factor (PDGF) and its receptors was analyzed in 14 gliomas of various degrees of malignancy and compared with three gliosis cases by in situ hybridization and immunohistochemistry techniques. Expression of both PDGF A- and B-chains was higher in glioblastomas than in astrocytomas. The PDGF A-chain mRNA was predominantly found in cell-rich areas in glioblastomas. The cognate PDGF-alpha receptor (PDGFR-alpha) mRNA was heterogeneously distributed in gliomas of all grades, and PDGFR-alpha expression was higher in gliomas than in gliosis. Within some glioblastomas probed with PDGFR-alpha complementary RNA, cells heavily loaded with grains were intermingled with others containing low or moderate signals. The heavily labeled cells were often found in the vicinity of proliferating capillaries. Immunostaining with an anti-PDGF antibody and an affinity-purified antiserum against the PDGFR-alpha showed strong staining of most tumor cells with both antibodies in glioblastoma. In addition, the PDGFR-alpha antibodies yielded a strong staining of scattered cells, and the anti-PDGF antibody yielded staining of a few cells within the astrocytoma. Furthermore, high levels of the PDGF-beta receptor (PDGFR-beta) and PDGF B-chain mRNA as well as the beta receptor protein were found in hyperplastic capillaries. These results suggest the presence of autocrine and paracrine loops in glioma, activating the PDGFR-alpha in glioma cells and the PDGFR-beta in endothelial cells.


p53 Mutations in human hepatocellular carcinomas from Germany. Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined 13 cases of human hepatocellular carcinomas from Germany for the presence of p53 aberrations in exons 4 to 8 of the gene by single-strand conformation polymorphism and restriction fragment-length polymorphism analyses and by sequencing of polymerase chain reaction products. Single base substitutions occurred in two human hepatocellular carcinomas: a C:G----T:A transition at a CpG site in codon 257, and a T:A----A:T transversion at codon 273. One of these point-mutated tumors and two additional tumors without point mutations demonstrated a loss of one p53 allele. None of the tumors was mutated in codons 12 or 61 of the c-Ha-ras gene.


Radiosensitization of human tumor cells with levamisole. Levamisole in combination with radiation and chemotherapeutic agents is being studied in clinical trials. The mechanism of interaction of levamisole with these modalities is unknown. In order to determine if there is direct interaction between radiation and levamisole, a series of colony-formation assays was performed with the use of two human tumor cell lines. Cells were exposed to 0-10 Gy of radiation, with or without the addition of 0-1000 microM levamisole. Exposure to levamisole alone had no effect on cell survival; however, the combination of continuous exposure to levamisole at concentrations approaching 1000 microM and radiation revealed a potentiation of radiation-induced cell killing.


The APC gene, responsible for familial adenomatous polyposis, is mutated in human gastric cancer. Although gastric cancer is the most common cancer in the world, genetic changes during its carcinogenesis are not well understood. Since some gastric cancers are considered to originate from the intestinal metaplasia, it is likely that the adenomatous polyposis coli (APC) gene, the mutation of which causes adenomatous polyps in the colon, is associated with carcinogenesis of gastric cancer. Based on this idea, DNAs isolated from gastric cancers were examined by means of a RNase protection analysis coupled with polymerase chain reaction followed by sequencing of the polymerase chain reaction products. By screening nearly one-half of the coding region of the APC gene in 44 tumors, somatic mutations were detected in three tumors: a missense mutation, a nonsense mutation, and a 5-base pair deletion resulting in a frame shift which causes truncation of the gene product. These results suggest that the mutation of the APC gene also plays an important role during the carcinogenesis of at least some gastric cancers.


E.s.r. study of the alkaline degradation of disaccharides in methyl sulfoxide. The degradation of a series of deoxy sugars and disaccharides in methyl sulfoxide-tetrabutylammonium hydroxide has been studied by e.s.r. spectroscopy. The generation of radicals and the types of radical formed are dependent on the anomeric configuration of non-reducing moieties and on the positions of the linkages and deoxy groups. Mechanisms for the pathways of degradation are proposed.


Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. We have generated mice carrying a mutation of the gene encoding the low affinity NGF receptor p75NGFR by targeted mutation in embryonic stem cells. Mice homozygous for the mutation were viable and fertile. Immunohistochemical analyses of the footpad skin of mutant mice revealed markedly decreased sensory innervation by calcitonin gene-related peptide- and substance P-immunoreactive fibers. The defective innervation was correlated with loss of heat sensitivity and associated with the development of ulcers in the distal extremities. Complicated by secondary bacterial infection, the ulcers progressed to toenail and hair loss. Crossing a human transgene encoding p75NGFR into the mutant animals rescued the absent heat sensitivity and the occurrence of skin ulcers and increased the density of neuropeptide-immunoreactive sensory innervation of footpad skin. The mutation in the gene encoding p75NGFR did not decrease the size of sympathetic ganglia or the density of sympathetic innervation of the iris or salivary gland. Our results suggest that p75NGFR has an important role in the development and function of sensory neurons.


Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection. Integration of retroviral DNA can serve as a paradigm for cellular functions that are affected by the packaging of DNA into chromatin. We have used a novel polymerase chain reaction-based assay to survey DNA and chromatin for the precise distribution of many integration sites. Integration into naked DNA targets is non-uniform, implying a nucleotide sequence bias. In chromatin, integration occurs preferentially at positions where the major groove is on the exposed face of the nucleosomal DNA helix, generating a 10 bp periodic spacing of preferred sites. Chromatin assembly enhances the reactivity of many sites, so that integration occurs most frequently at sites in nucleosomal, rather than nucleosome-free, regions of minichromosomes. In contrast, integration is prevented in a region occupied by a site-specific DNA-binding protein. Comparisons of integration events mediated by viral nucleoprotein complexes or by two different retroviral integrases show that the integration machinery also affects target site selection.


[Effects of ANF and ACTH on aldosterone secretion in cultured human adrenal tissue]. We studied the effects of various concentrations of ANF and ACTH on aldosterone secretion in human normal adrenal tissue over different response times in vitro. We found that ANF 10(-8) mol/L at first stimulated but 24 h later inhibited basal aldosterone secretion. ANF 10(-8) mol/L also inhibited the ACTH-stimulated secretion of aldosterone. ACTH 10(-8) mol/L did not stimulate aldosterone secretion, while ACTH concentrations of 3 x 10(-8) and 5 x 10(-8) mol/L did. When ACTH and ANF were added simultaneously, the ANF-induced inhibition of ACTH-stimulated aldosterone secretion could be overcome by increasing the concentration of ACTH. Our results show that ANF caused a dose-dependent and time-dependent inhibition of basal and ACTH-stimulated increases in aldosterone secretion.


[Ca2+ channels and the abnormal electrical activity of demyelinated nerve]. Peripheral nerve demyelination was produced in adult rats by placing loosely-constrictive ligatures around the common sciatic nerve. The postoperative behavior of these rats indicated that hyperalgesia, allodynia and possible spontaneous pain were produced. In the meantime, abnormal spontaneous afferent activities (ectopic firings)originating from the demyelinated region were recorded. Evidence showed that the application of Ca2+ and Ca2+ channel blockers modulated the abnormal activity of the injured nerve. Ca2+ facilitation was dependent on its concentration (in the range of 10-20 mol/L), while 40mol/L Ca2+ always abolished firing. Verapamil, as well as La3+, applied locally or i. v. (for verapamil) not only strongly inhibited the spontaneous ectopic firings, but also blocked discharges elicited by tetraethylammonium. It is suggested that newly formed Ca2+ channels on the naked axolemma are largely responsible for the abnormal afferent activities following demyelination of the nerve.


[Establishment and characteristics of a lymphoblastoid cell line (CKM) resistant to 6-TG and ouabain]. Three lymphoblastoid cell lines (CKM-c, CKM-1 and CKM-8) have been established by EBV-transformation from cord blood. They have been maintained for 1 year after 120 passages with rapid growth. The population doubling times were 24.5h (CKM-1), 30 h (CKM-8) and 36 h (CKM-c). The B lymphocyte features were identified under an electron microscope. EBV antigens including EBNA, VCA-Ig A, MA and EA were negative in all the cell lines. The median chromosome number was 48-50. Interferon (IFN) was induced by NDV treatment of the CKM-c cell line. CKM-8 has been modified to be resistant to both thioguanine (30 micrograms/ml) and Ouabain (10(-5)) for the production of human-human hybridomas.


Accentuated vagal antagonism of beta-adrenergic effects on ventricular repolarization. Evidence of weaker antagonism in hostile type A men. Prior research has suggested a weaker parasympathetic antagonism of sympathetic effects on the heart in type A (coronary-prone) men. To confirm this phenomenon and extend our understanding of it, we investigated the effects of prior muscarinic blockade on the electrocardiogram T wave and other cardiovascular and neuroendocrine responses to isoproterenol in type A and type B (non-coronary-prone) men. Responses to two 5-minute intravenous isoproterenol infusions (0.01 micrograms/kg/min and 0.02 micrograms/kg/min) were evaluated in six type A and six type B men after pretreatment with either dextrose placebo or atropine (1.2 mg). Atropine significantly potentiated T wave attenuation in the recovery period after isoproterenol infusion (0.30 +/- 0.07 mV) compared with placebo (0.54 +/- 0.09 mV, p less than 0.001). Atropine also potentiated the heart rate increase to isoproterenol (39 +/- 3 beats per minute versus 20 +/- 2 beats per minute after placebo). Atropine enhanced decreases in systolic, diastolic, and mean arterial pressures as well as pulse pressure to isoproterenol. Atropine enhancement of many of these responses was increased among subjects with high scores on various hostility/anger scales. Isoproterenol alone produced greater T wave attenuation in type A than in type B men. However, atropine enhancement of T wave attenuation and blood pressure falls by isoproterenol was present only in type B men. These findings indicate that there is accentuated parasympathetic antagonism of T wave attenuation and blood pressure responses induced by beta-adrenergic stimulation. Relative weakness of this antagonism of sympathetic effects on the heart in hostile type A individuals may contribute to their higher coronary disease risk.


Suppression of longitudinal versus transverse conduction by sodium channel block. Effects of sodium bolus. Arrhythmias resulting from treatment with sodium channel-blocking antiarrhythmic drugs have been successfully treated with sodium infusion, although the mechanism underlying this effect is uncertain. In this study, we used a multielectrode array to examine the effects of O-desmethyl encainide (ODE), a potent sodium channel-blocking metabolite of encainide, on conduction in canine ventricle. ODE depressed both longitudinal and transverse conduction velocities in a plasma concentration-related fashion (r = -0.74, -0.60; p less than 0.001). At ODE concentrations less than or equal to 300 ng/ml (n = 34), conduction velocity was depressed to the same extent in the longitudinal (-21.9 +/- 8.4%, SD) and transverse orientations (-22.0 +/- 8.8%). However, at concentrations greater than 300 ng/ml (n = 17), conduction was significantly more impaired longitudinally than transversely (-44.5 +/- 11.7% versus -34.4 +/- 13.7%, p less than 0.02). In 12 animals with high concentrations (mean, 432 +/- 32 ng/ml), a 5-meq/kg bolus of sodium chloride over 1 minute immediately increased conduction velocity; this effect was significantly greater and longer lasting in the longitudinal orientation. In two animals, conduction block in the longitudinal orientation was documented at high plasma ODE and was immediately reversed by sodium bolus. We conclude that the major effect of sodium in animals with excess sodium channel block is improvement of longitudinal propagation; this effect may underlie the antiarrhythmic action of sodium in the analogous clinical setting.


Early afterdepolarizations and triggered activity induced by cocaine. A possible mechanism of cocaine arrhythmogenesis. Cocaine may produce life-threatening cardiac arrhythmias, but it is not clear whether this is an indirect effect of coronary vasoconstriction and ischemia or a direct myocardial effect of the substance. Except for its effects on the Na+ current as a local anesthetic, little is known about the direct electrophysiological actions on cardiac cells. Therefore, we studied the effects of cocaine on action potentials and membrane currents in isolated feline ventricular myocytes to test the hypothesis that cocaine-induced arrhythmogenesis may be based on cellular and ionic mechanisms. Action potentials and membrane currents were recorded using the patch clamp technique. Single cells were isolated from feline left ventricles by enzymatic digestion. Exposure to cocaine (10 or 50 microM) depressed the plateau phase of the action potential and prolonged action potential duration. Action potential duration measured at 90% repolarization (APD90) was increased from 280 +/- 12 msec to 325 +/- 17 msec (p less than 0.01) by 5-minute exposure to 10 mumol cocaine, when the cells were stimulated at 1 Hz. During exposure to 50 mumol cocaine, APD90 was markedly increased from 298 +/- 13 msec to 437 +/- 35 msec (p less than 0.01) in seven of 16 cells, and early afterdepolarizations (EADs) developed in these cells. The take-off potential and the amplitude of EADs were -28.3 +/- 2.3 mV and 16.8 +/- 1.2 mV, respectively. Triggered activity arising from EADs was induced in four of the seven cells. Addition of 1 nmol isoproterenol augmented EADs and induced sustained triggered activity, whereas they were suppressed by exposure to 2 microM verapamil. Whole-cell voltage clamp experiments revealed that cocaine (50 microM) reduced the peak L-type Ca2+ current from 1.03 +/- 0.13 nA to 0.79 +/- 0.11 nA (23% reduction, p less than 0.05). Cocaine also reduced the peak delayed rectifier K+ current from 362 +/- 51 pA to 113 +/- 32 pA (69% reduction, p less than 0.01). However, cocaine did not affect activation and inactivation kinetics of these channels. Cocaine had no effect on the inward rectifier K+ current. We conclude that cocaine can prolong action potential duration and induce EADs and triggered activity by blocking the delayed rectifier K+ current, and that cocaine-induced abnormalities of repolarization, modulated by its inhibitory effects on catecholamine reuptake, may play a role in the potential of cocaine for induction of acute fatal arrhythmias.


A quantitative analysis of use-dependent ventricular conduction slowing by procainamide in anesthetized dogs. Use-dependent effects of antiarrhythmic drugs on phase 0 sodium current result in rate-dependent conduction slowing with important potential clinical consequences. The purpose of the present study was to determine whether state-dependent interactions of procainamide with sodium channels can be analyzed based on conduction changes in vivo. Procainamide infusions were used to produce stable drug concentrations causing greater than or equal to 25% conduction slowing at a basic cycle length (BCL) of 300 msec in morphine/chloralose-anesthetized dogs with formalin-induced atrioventricular block. Computer-based epicardial activation mapping was applied to assess the time course and pattern of conduction over a wide range of BCLs before and after drug administration. Action potential duration was measured from recordings of monophasic action potentials. The onset and steady-state values of fractional sodium channel block estimated from conduction changes were fitted to equations obtained from a stepwise exponential analysis. The rate constant for the onset of block (lambda *) decreased, as predicted, with decreasing cycle length. The slope of the relation between lambda * and recovery time at each BCL averaged 0.29 +/- 0.03 sec-1, resulting in a calculated recovery time constant (3.4 seconds) similar to values previously obtained by direct measurement. Estimates of binding and unbinding rate constants for the sodium channel during the action potential plateau and after repolarization were of the same order as previous results obtained using microelectrode methods in vitro. Use-dependent conduction changes produced by procainamide in vivo closely follow the predictions of mathematical models of drug-channel interactions, and underlying kinetic interactions with the sodium channel inferred from conduction changes agree with previous, more direct observations. These results support the relevance of basic concepts about antiarrhythmic drug actions on sodium channels for understanding drug effects on conduction in vivo and advance analytical tools that can be used to explore the latter in humans.


Endothelin-1 stimulates mitotic activity in the zona glomerulosa of the rat adrenal cortex. Subcutaneous infusion with endothelin-1 (ET-1; 30 pM min-1) for 24 h induced a 9-fold increase in the mitotic index (% of metaphase-arrested cells) of rat adrenal zona glomerulosa (ZG). Infusions with adrenocorticotrophic hormone (ACTH) angiotensin-II (ANG-II) or arginine vasopressin (AVP) (30 pM min-1/24 h) raised the ZG mitotic index 13-fold, 9-fold and 10-fold, respectively. Combined infusion with ET-1 and ACTH increased the ZG mitotic index 20-fold, while the effects of ET-1 and ANG-II or AVP were not additive. These findings suggest that ET-1 exerts a strong proliferogenic effect on the rat ZG, by a mechanism probably similar to that underlying the adrenoglomerulotrophic actions of ANG-II and AVP.


Comparison of azoles against aspergilli in vitro and in an experimental model of pulmonary aspergillosis. Current treatment modalities for bronchopulmonary aspergillosis are not very satisfying. We determined the in vitro activity of recently available azoles against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Subsequently, these agents were evaluated in an animal model of bronchopulmonary aspergillosis using A. fumigatus as test organism. In vitro, detectable activity was only found for itraconazole (all minimal inhibitory concentrations, MICs, less than or equal to 3.2 micrograms/ml). The MICs for SCH39304 were greater than or equal to 12.8 micrograms/ml and greater than or equal to 25.6 micrograms/ml for ketoconazole and fluconazole. In vivo, amphotericin B was the most active agent tested, and SCH39304 was the most active azole in terms of survival and reduction in lung weight, followed by itraconazole. Ketoconazole and fluconazole did not improve survival nor reduce the lung weight of infected animals. We conclude, (1) that in vitro activity of azoles against aspergilli does not always correlate with in vivo activity; (2) that in vivo, SCH39304 was the most active azole tested, followed by itraconazole; (3) that for those agents for which data about effectiveness in human pulmonary aspergillosis are available (amphotericin B, ketoconazole, itraconazole) antifungal activity in our model corresponds to activity as seen in human beings, and (4) that SCH39304 and itraconazole are rational choices for clinical trials in human pulmonary aspergillosis.


Antibacterial activity of the new quinolone CI-960 (PD 127391) against clinical isolates at a major tertiary care center in Saudi Arabia. The antibacterial activity of the new fluoroquinolone CI-960 (PD 127391) was evaluated against 1,162 clinical isolates and compared with other quinolones and various commonly used antibiotics. CI-960 was highly effective against members of Enterobacteriaceae inhibiting 626 of the 629 isolates at a less than or equal to 0.03 to 0.12 microgram/ml concentration. All the 305 isolates of Pseudomonas aeruginosa, Xanthomona maltophilia, and Acinetobacter were susceptible to CI-960. It was the only effective drug against 10 multi-resistant isolates of pseudomonads and Acinetobacter. All staphylococci, including methicillin-resistant Staphylococcus aureus, were inhibited by less than or equal to 0.03-0.5 microgram/ml of CI-960. Like other drugs of its class, it had little activity against enterococci.


In vitro activity of sparfloxacin (CI-978), a new broad-spectrum fluoroquinolone. The in vitro activity of sparfloxacin (CI-978, AT-4140), a new fluoroquinolone, was compared with ciprofloxacin, norfloxacin and other commonly used antimicrobial agents against 650 strains of Enterobacteriaceae, 237 isolates of other gram-negative bacilli and 318 strains of gram-positive cocci. The MICs of sparfloxacin against 90% of the members of Enterobacteriaceae were between 0.12 and 0.5 microgram/ml. All the 48 isolates of notoriously drug-resistant Serratia marcescens were inhibited by less than 0.03-4.0 micrograms/ml of sparfloxacin. All the 90 isolates of Acinetobacter, 80 of the 88 strains of Pseudomonas aeruginosa and all the 28 isolates of Xanthomonas maltophilia were susceptible to sparfloxacin. The MIC90 for all the cocci tested ranged between 0.5 and 4.0 micrograms/ml of sparfloxacin. It inhibited 92% of enterococci as compared with 27% for ciprofloxacin and 22% for norfloxacin. It was better or comparable in activity to other fluoroquinolones and superior to penicillins, cephalosporins and aminoglycosides tested. There was cross-resistance between ciprofloxacin, norfloxacin and sparfloxacin.


[Infantile spasms. A retrospective study of 105 cases]. This paper is a summary of our observations on 105 cases of infantile spasms. The age of onset was around six months after birth, but the patients came for treatment mainly about one year after onset. Fever of unknown cause, asphyxia, birth injury, infection of the central nervous system, tuberous sclerosis, phenylketonuria and recent immunization etc. were possible etiology. Clinically, it is characterized by head nodding, mental retardation, myoclonic jerks and various neurologic deficits. EEG findings showed classical or modified arrhythmia or other epileptiform patterns. About one third of 22 cases examined had abnormal brain stem auditory evoked potentials. Among 42 patients who underwent CT scanning before ACTH treatment, 18 were normal and 7 abnormal; during ACTH treatment 3 normal and 4 abnormal; after completion of treatment, 4 normal and 6 abnormal, suggesting no further atrophy of the brain. Examination of trace elements of the hair by proton-induced X-ray emission (PIXE) method in 23 patients revealed a significant difference in lead, calcium and zinc contents between patients and 101 controls, but no statistical difference in iron and copper contents between the two groups. Sodium valproate, prednisone and ACTH appear to be effective in the treatment of infantile spasms. Eight patients fully recovered, and they can go to school without difficulty. Many patients derived various degrees of improvement of the satisfaction of their parents. Two patients were still amended and often attacked by myoclonus. The effects, side effects of these drugs, and the possible pathogenesis were discussed.


[Observation on the changes in the frequency of end-plate potentials evoked by sera with different antibodies from patients with myasthenia gravis in nerve-muscle preparation of rats]. Using glass microelectrode recording the effect of sera with presynaptic membrane (PrM) antibody and acetylcholine receptor (AchR) antibody on the frequency of miniature end-plate potentials (MEPPs) was tested in the phrenic nerve-diaphragm preparation of rats. Sera from 5 health control persons did not affect the MEPP frequency. Twenty tested patient sera were identified to include 3 cases with AChR antibody, 6 cases with PrM antibody, 5 cases with both and 6 cases without both antibodies. The results showed that 2 cases with PrM antibody first increased for 1 to 2 minutes and then decreased the MEPP frequency. The MEPP frequency was decreased in total 11 cases, including 2 cases with AChR antibody, 3 cases with PrM antibody, 4 cases with both and 2 cases without both antibodies. The MEPP frequency was not changed in total 7 cases, including 4 cases without both antibodies and 1 case from each other 3 kinds of sera. The results demonstrated that serum from some patients with myasthenia gravis, especially from those with PrM antibody has some factors which act on the motor presynaptic ending.


[Extracellular calcium in the rat brains and the effects of nifedipine in experimental cerebral infarction using calcium ion selective microelectrodes in vivo]. Extracellular Ca2+ (Ca2+ e) changes in the cerebral infarction areas of the rats with the double-tubulus calcium ion-selective microelectrodes made of calcium active material, di-2-isooctylphenyl calcium phosphate to observe the effects of two concentrations of nifedipine given before the middle cerebral artery (MCA) occlusion. Twenty-four rats were divided into three groups: group A: with just the left MCA occlusion, Group B: with intramuscular injection of nifedipine 1mg/kg one hour before the MCA occlusion, and Group C with intramuscular injection of nifedipine 5mg/kg one hour before the MCA occlusion. The results indicated that the mean concentration of Ca2+ e in the normal brain Group A was 1.119 mM. The Ca2+ e concentrations became 0.4613 mM, 0.191 mM and 0.0654 mM, 2, 5 and 30 minutes after the MCA occlusion respectively. The other groups, Group B and Group C, receiving two kinds of dosages of nifedipine respectively showed the Ca2+ e concentrations to be 0.5262 mM and 0.5588 mM, 0.3456 mM and 0.3058 mM, 0.2156 mM and 0.1989 mM, 2, 5 and 30 minutes after the MCA occlusion respectively. There is no statistic significance between group B and group c (P greater than 0.25) By comparing Group A with Group B or C, it was found that the Ca2+ e level of Group B or C is about 3 times greater than that of Group A (P less than 0.0025) and (P less than 0.005) 30 minutes after the MCA occlusion.


Validity of patients' self-reported drug use as a function of treatment status. Recent reviews conclude that there is some evidence that drug abusers' self-reports are reliable and valid. However, there are wide variations among studies depending upon the samples and procedures used to obtain the data. The current study was conducted to extend the findings in this area. An examination of the intake interviews and same day urinalyses on 150 patients enrolling for outpatient opioid detoxification or maintenance revealed a fairly high agreement between drug use self-reports and urinalyses. A second study (N = 70) looking at the validity of self-reported drug use at intake and at 4 weeks follow-up revealed some noticeable changes in validity measures, suggesting that contingencies on positive urine results influence self reported drug use of addicts.


Characteristics of attenders to community based alcohol treatment centre with special reference to sex difference. Alcohol misusers attending a London community day treatment centre were interviewed about their drinking habits and the expectations they had of the treatment they were receiving. The results show that the women experienced twice as many alcohol related problems as the men and were significantly more dependent upon alcohol. The men reported that they hoped to improve their health and confidence by coming for treatment.


Effects of an oil enriched in gamma linolenic acid on locomotor activity and behaviour in the Morris Maze, following in utero ethanol exposure in rats. Ethanol is known to decrease the quantity of polyunsaturated fatty acids in brain membranes possibly as the repercussion of an inhibition of delta-5- and delta-6-desaturases. Consequently behavioural changes may occur. Evening Primrose Oil (EPO) which is rich in gamma linolenic acid (10% 8:3 (n-6)) was proposed to try to circumvent these deleterious effects of ethanol. An animal model of the foetal alcohol syndrome was used in which ethanol was administered throughout gestation and into the weaning period, in a milk diet. EPO was administered concurrently with ethanol to establish the effect of this essential fatty acid and ethanol on the animal's behaviour. Animals were tested at approximately 60 days of age for their responses in two different behavioural paradigms, i.e. the stressful memory task of the Morris Maze and the non-stressful activity monitor. In this study we report an increase in learning ability in male (Control-EPO and alcohol-EPO versus their control: P less than 0.00001 and P less than 0.01, respectively) and female rats (Control-EPO and alcohol-EPO versus control: P less than 0.0001 and P less than 0.00001, respectively) after administration of EPO. It was also found that ethanol plus EPO administration consistently raised the activity scores of the rats in the activity monitor (daytime activity scores for male and female rats were P less than 0.00001 and P less than 0.0001, respectively), while ethanol alone decreased the scores (male and female rats P less than 0.00001 and P less than 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)


Mad, bad and dangerous to know: dimensions and measurement of attitudes toward injecting drug users. Attitudes toward injecting drug users were assessed using a 53-item questionnaire which was administered to 143 workers at a drug and alcohol research unit and an AIDS treatment facility. Factor analysis revealed three interpretable dimensions: intravenous drug use as a matter of both public concern and personal inadequacy; intravenous drug users as criminals who should be removed from society; and social avoidance of, and personal distaste for, intravenous drug users. The scale and subscales had good test-retest reliability and internal consistency. Its potential use as a measure of attitudes toward injecting drug users for both treatment research and AIDS research is discussed.


Basic fibroblast growth factor stimulates the sustained proliferation of mouse epidermal melanoblasts in a serum-free medium in the presence of dibutyryl cyclic AMP and keratinocytes. Basic fibroblast growth factor (bFGF) stimulated the sustained proliferation of mouse epidermal melanoblasts derived from epidermal cell suspensions in a serum-free medium supplemented with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP). The melanoblasts could be subcultured in the serum-free medium supplemented with the two factors in the presence of keratinocytes, but not in the absence of keratinocytes. In these conditions, some melanoblasts proliferated without differentiating for more than 20 days including a subculture. This is the first report of a successful culture of melanoblasts from mammalian skin. This culture system is expected to clarify further markers for melanoblasts and requirements for their proliferation and differentiation.


Metalloproteinases mediate extracellular matrix degradation by cells from mouse blastocyst outgrowths. The maintenance and developmental remodeling of extracellular matrix is crucial to such processes as uterine implantation and the cell migratory events of morphogenesis. When mouse blastocysts are placed in culture they adhere to extracellular matrix, and trophoblast giant cells migrate out onto the matrix and degrade it. The secretion of functional proteinases by developing mouse embryos increases dramatically at the time of implantation. By zymography we identified the major secreted gelatin-degrading proteinase, also known as type IV collagenase, as one migrating at 92 x 10(3) Mr. Several casein-degrading proteinases were also secreted. The tissue inhibitor of metalloproteinases (TIMP) inhibited all of the embryo-derived proteinases detected by gelatin gel zymography, indicating that they are metalloproteinases, whereas TIMP did not inhibit all of the caseinases. Urokinase was also secreted. Addition of TIMP at 5-500 nM effectively inhibited the degradation of matrix by the trophoblast outgrowths. Blocking antibodies directed against 92 x 10(3) Mr gelatinase abolished matrix degradation by the trophoblast cells. These observations suggest that several metalloproteinases are regulated in early development and that 92 x 10(3) Mr gelatinase, in particular, has a rate-limiting function in degradation of the maternal extracellular matrix by trophoblast cells.


Disruption of pioneer growth cone guidance in vivo by removal of glycosyl-phosphatidylinositol-anchored cell surface proteins. Cell surface proteins anchored to membranes via covalently attached glycosyl-phosphatidylinositol (GPI) have been implicated in neuronal adhesion, promotion of neurite outgrowth and directed cell migration. Treatment of grasshopper embryos with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that cleaves the GPI anchor, often induced disruptions in the highly stereotyped migrations of peripheral pioneer growth cones and afferent neuron cell bodies. In distal limb regions of embryos treated with PI-PLC at early stages of pioneer axon outgrowth, growth cones lost their proximal orientation toward the central nervous system (CNS) and turned distally. Pioneer growth cones in treated limbs also failed to make a characteristic ventral turn along the trochanter-coxa (Tr-Cx) segment boundary, and instead continued to grow proximally across the boundary. Treatment at an earlier stage of development caused pre-axonogenesis Cx1 neurons to abandon their normal circumferential migration and reorient toward the CNS. None of these abnormal phenotypes were observed in limbs of untreated embryos or embryos exposed to other phospholipases that do not release GPI-anchored proteins. Incubation of embryos with PI-PLC effectively removed immunoreactivity for fasciclin I, a GPI-anchored protein expressed on a subset of neuronal surfaces. These results suggest that cell surface GPI-anchored proteins are involved in pioneer growth cone guidance and in pre-axonogenesis migration of neurons in the grasshopper limb bud in vivo.


In situ alkylation of cysteine residues in a hydrophobic membrane protein immobilized on polyvinylidene difluoride membranes by electroblotting prior to microsequence and amino acid analysis. For identification of cysteine residues on microsequence analysis it is crucial to derivatize the sulfhydryl groups. This reaction requires a desalting step which often represents a major obstacle, especially if the sample consists of limited amounts of a hydrophobic membrane protein. An alkylation procedure is described, allowing efficient derivatization (greater than 90%) of cysteines and cystines even in low microgram quantities, as revealed by test analyses with lysozyme and a hydrophobic membrane protein. The modified protein is recovered in high yields in a form suitable for both microsequence analysis and amino acid analysis. The method involves electrophoretic desalting by miniaturized Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ alkylation after electro-transfer onto polyvinylidene difluoride membranes. Precautions against NH2-terminal blocking during sample preparations are provided. The general applicability of the method is illustrated by the structural characterization of the low abundance membrane receptor for human urokinase plasminogen activator.


A study to compare oral sumatriptan with oral aspirin plus oral metoclopramide in the acute treatment of migraine. The Oral Sumatriptan and Aspirin plus Metoclopramide Comparative Study Group. In a double-blind, placebo-controlled study, the efficacy, safety and tolerability of 100 mg oral sumatriptan, given as a dispersible tablet, was compared with that of 900 mg oral aspirin plus 10 mg oral metoclopramide in the acute treatment of migraine. A total of 358 patients treated up to three migraine attacks within 3 months, recording clinical information on a diary card. In attack 1, headache relief after 2 h, defined as a reduction in severity from severe or moderate pain to mild or no pain, was recorded in 56% (74/133) of patients who took sumatriptan and 45% (62/138) of patients who took aspirin plus metoclopramide (p = 0.078). This analysis of the primary efficacy end point was not statistically significant. However, for attacks 2 and 3 (secondary end points), headache relief was achieved in 58 versus 36% of patients (p = 0.001) and 65 versus 34% of patients (p less than 0.001), respectively. Relief from nausea, vomiting, photophobia and phonophobia was similar in both treatment groups. Rescue medication was required by fewer patients treated with sumatriptan than by those who received aspirin plus metoclopramide (attack 1, 34 versus 56%, p less than 0.001; attack 2, 32 versus 51%, p = 0.001, and attack 3, 35 versus 54%, p = 0.001). Sumatriptan also produced a faster improvement and resolution of migraine attacks. Comparing the sumatriptan and aspirin plus metoclopramide treatment groups, complete resolution of the attack occurred within 6 h in 32 versus 19% (attack 1), 35 versus 23% (attack 2) and 32 versus 20% of patients (attack 3).(ABSTRACT TRUNCATED AT 250 WORDS)


Stimulation of neutrophils by insoluble immunoglobulin aggregates from synovial fluid of patients with rheumatoid arthritis. Insoluble immunoglobulin aggregates present in the synovial fluid of patients with rheumatoid arthritis have been examined for their ability to activate reactive oxidant and granule enzyme secretion from bloodstream neutrophils. These insoluble complexes activated luminol chemiluminescence, but did not activate O2-, H2O2 or granule enzyme secretion and did not activate lucigenin chemiluminescence, which also measures reactive oxidant secretion. Hence, the luminol chemiluminescence detected after activation by insoluble immunoglobulin aggregates must be due to intracellularly generated reactive oxidants, i.e. produced within phagolysosomes. Because reactive oxidant and granule enzyme secretion has occurred within rheumatoid joints, other mechanisms of neutrophil activation must exist.


Haemodynamic and renal effects of urodilatin in healthy volunteers. Urodilatin (ANF(95-126)), an analogue of the atrial natriuretic factor (ANF(99-126)), has recently been isolated from human urine. To study haemodynamic and renal effects of synthetic urodilatin, 18 healthy male volunteers (age 26.1 +/- 0.8 years; X +/- SEM) received i.v. bolus injections of urodilatin at doses of 1, 2 or 4 micrograms kg-1 body weight (bw) (n = 6 per dosage group). Urodilatin dose-dependently increased heart rate and cardiac index. A dose-dependent increase in plasma cyclic GMP levels was also observed. Urinary cyclic GMP excretion, urine flow and natriuresis increased 7-fold, 5-fold and 4-fold, respectively. Renal effects were not different between dosage groups. Compared with ANF(99-126), after urodilatin the reduction in mean pulmonary arterial pressure (PAP) was more pronounced (2 micrograms kg-1, n = 6; ANF -1.8 +/- 0.5, URO: -5.5 +/- 1.1 mmHg, P less than 0.05). Furthermore, after urodilatin the reduction of PAP lasted continuously from 2 up to 90 min after injection, while ANF(99-126) produced only a transient decrease of PAP. Similarly the reduction of pulmonary capillary wedge pressure (PCWP) by urodilatin from 9.3 +/- 1.2 to 3.8 +/- 0.9 mmHg (P less than 0.05) was also sustained up to 90 min post administration. These data in healthy volunteers suggest that, due to prolonged reduction of PAP and PCWP with increases of cardiac index and reduction of systemic vascular resistance, urodilatin might exhibit beneficial effects in cardiovascular disease.


Trypanosoma cruzi: mechanisms of intracellular calcium homeostasis. Regulation of intracellular Ca2+ homeostasis was characterized in epimastigote forms of Trypanosoma cruzi using the fluorescence probe Fura-2. Despite an increase in extracellular Ca2+, [Ca2+]o, from 0 to 2 mM, cytosolic Ca2+, [Ca2+]i, increased only from 85 +/- 9 to 185 +/- 21 nM, indicating the presence of highly efficient mechanisms for maintaining [Ca2+]i. Exposure to monovalent Na+ (monensin)-, K+ (valinomycin, nigericin)-, and divalent Ca2+ (ionomycin)-specific ionophores, uncouplers of mitochondrial respiration (oligomycin), inhibitors of Na+/K(+)-ATPase (ouabain), and Ca(2+)-sensitive ATPase (orthovanadate) in 0 or 1 mM [Ca2+]o resulted in perturbations of [Ca2+]i, the patterns of which suggested both sequestration and extrusion mechanisms. Following equilibration in 1 mM [Ca2+]o, incubation with orthovanadate markedly increased [Ca2+]i, results which are compatible with an active uptake of [Ca2+]i by endoplasmic reticulum. In contrast, equilibration in 0 or 1 mM [Ca2+]o did not influence the relatively smaller increase in [Ca2+]i following incubation with oligomycin, suggesting a minor role for the mitochondrial compartment. In cells previously equilibrated in 1 mM [Ca2+]o, exposure to monensin or ouabain, conditions known to decrease the [Na+]o/[Na+]i gradient, upon which the Na+/Ca2+ exchange pathways are dependent, markedly increased [Ca2+]i. In a complementary manner, decreasing the extracellular Na+ gradient with Li+ increased [Ca2+]i in a dose-dependent manner. Finally, the calcium channel blockers verapamil and isradipine inhibited the uptake of Ca2+ by greater than 50%, whereas diltiazem, nifedipine, and nicardipine were ineffective. The results suggest that epimastigote forms of T. cruzi maintain [Ca2+]i by uptake, sequestration, and extrusion mechanisms, with properties common to eukaryotic organisms.


Plasmodium falciparum and Plasmodium chabaudi: characterization of glycosylphosphatidylinositol-degrading activities. Merozoites of malaria parasites have a membrane-bound serine protease whose solubilization and subsequent activity depend on a parasite-derived glycosylphosphatidylinositol-phospholipase C (GPI-PLC). The GPI-degrading activities from both Plasmodium falciparum and Plasmodium chabaudi have been characterized and partially purified by phenylboronate chromatography. They are membrane-bound, developmentally regulated, calcium-independent enzymes and as such they resemble GPI-PLC of Trypanosoma brucei. Furthermore, a T. brucei GPI-PLC-specific monoclonal antibody (mAT3) immunoprecipitates the plasmodial GPI-degrading activity. Thin-layer chromatography is suggestive of two activities: a GPI-PLC and a phospholipase A.


EPR spectroscopic characterization of an 'iron only' nitrogenase. S = 3/2 spectrum of component 1 isolated from Rhodobacter capsulatus. The alternative nitrogenase of Rhodobacter capsulatus, isolated from a nifHDK deletion mutant, has been purified to near homogeneity and identified as an 'iron only' nitrogenase. The dithionite-reduced component 1 ('FeFe protein') of this enzyme showed an EPR spectrum consisting of two components: a minor S = 1/2 signal at g = 1.93 and a very characteristic S = 3/2 signal of near-stoichiometric intensity at g = 5.44. This resonance is very close to the highest possible g value (g = 5.46) for the coinciding two intradoublet subspectra of an S = 3/2 system of maximal rhombicity (E/D = 0.33). The deviation from axial symmetry (increasing E/D) correlates with the stability, activity and substrate selectivity of the different (Mo, V, Fe) nitrogenases.


Differential expression of two sodium channel subtypes in human brain. Two partial human brain sodium channel cDNA sequences (designated HBSC I and II) have been cloned and mapped to chromosome 2q23-2q24 by chromosome microdissection-PCR (CMPCR). The distribution of HBSC I and II mRNA in human brain was studied by means of a novel approach based on the ligase detection reaction. These studies demonstrate that HBSC I and II mRNA is heterogeneously distributed in brain, and that the relative ratio of the two forms can vary as much as 7-fold between different regions.


Multiple genes for Xenopus activin receptor expressed during early embryogenesis. Four distinct cDNAs for activin receptor designated as XSTK2, 3, 8 and 9 have been cloned from a Xenopus laevis cDNA library. The protein structures deduced from the cDNAs have shown that they all have a putative extracellular ligand-binding domain, a single transmembrane domain and cytoplasmic Ser/Thr kinase domain, except that XSTK2 is extremely similar to the XSTK3 gene but lacks a carboxyl-terminal part of the kinase motif. Northern blot analysis showed that all transcripts are maternally inherited. The levels of transcript for XSTK2, 3 and 8 appeared to fluctuate during early development while those for XSTK9 maintain constant.


Regional hypothermia protects against tourniquet neuropathy. Ischaemic nerve injury has been suggested as the mechanism for post-tourniquet limb paralysis. As hypothermia has been shown to prolong ischaemia time in many tissues, we tested the hypothesis that cold protection would reduce tourniquet induced neural injury. In 16 male Wistar rats a tourniquet was applied to the right hind limb for 3 h. In eight rats both hind limbs were maintained at room temperature (27 degrees C) while the limb temperature was decreased to 4 degrees C by application of cold polyglycol gel packs in the remaining eight animals. Motor nerve conduction velocity (MNCV) and the amplitude of muscle motor units (AMMU) in response to nerve stimulation were measured at 0 h, 1 h and 1 week post-tourniquet release. Application of the tourniquet for 3 h abolished nerve conduction in all animals. Hypothermia resulted in a significant improvement in AMMU (5.8 +/- vs. 0.6 +/- 0.4 mv, p less than 0.05) at 1 h and in MNCV (26.6 +/- 1.6 vs. 8.1 +/- 4.4 ms-1, p less than 0.05) and AMMU (18.5 +/- 2.0 vs. 9.7 +/- 4.5 mv, p less than 0.05) at 1 week. Hypothermia without ischaemia also reduced MNCV (44.2 +/- 9.4 vs. 74.3 +/- 8.9 ms-1, p less than 0.05) and AMMU (28.1 +/- 2.8 vs. 36.3 +/- 8.4 mv, N.S.). These data indicate that hypothermia reduces normal MNCV and protects nerve function during tourniquet ischaemia. These results support the ischaemic hypothesis for post-tourniquet nerve injury. Cold protection may have clinical applications for surgical procedures performed with tourniquet ischaemia.


[A case of latent Addison's disease with hypogonadotropic hypogonadism and dwarfism]. A case of latent Addison's disease accompanied by hypogonadotropic hypogonadism and dwarfism is described. A 20-year-old man was admitted to our department complaining of short stature and immature development of the external genitalia. Pigmentation was most evident on the fingers and face. Endocrinologically, serum ACTH level was very high, and serum cortisol level was in the lower limit of the normal range. Serum aldosterone and adrenal androgen levels were below their normal ranges. Based on these clinical and laboratory findings, the patient was diagnosed as having latent Addison's disease. Serum LH did not respond to a bolus injection of LH-RH. However, after 3 days administration of LH-RH, the response of serum LH to a bolus injection of LH-RH was enhanced. Serum testosterone level was not increased after the administration of hCG. These findings suggested a hypothalamic cause for the hypogonadism. It was indicated that short stature was apparently caused by GH neurosecretory dysfunction, since nocturnal GH secretion was below that in normal males and the response of GH to the administration of arginine was normal. In regard to the thyroid function, the peak of serum TSH after a bolus injection of TRH was delayed compared with normal subjects, and although serum T4 level was high, the basal metabolic rate was very low. This suggests that there is tissue resistance to the elevated thyroid hormone.


Collagenases from human and rat skin fibroblasts purified on a zinc chelating column reveal marked differences in latency as a result of serum culture conditions. 1. Fibroblasts from both human and rat skin were grown in the presence or absence of serum and the collagenase activity in the medium was partially purified on zinc-Sepharose. 2. During chromatography, using a discontinuous elution gradient, the rat collagenase elutes at different pH and ionic strength than the human collagenase. Both latent and active collagenases of both species are retarded by the affinity matrix. 3. Latency of collagenase in media obtained from fibroblast cultures appears to be influenced by the presence of a serum component in the culture medium. 4. The results demonstrate that collagenases secreted by fibroblast cultures established from the same tissue but obtained from different species are biochemically diverse and that, within one species, the amount of active enzyme depends on the presence of a serum factor.


Alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium. Selective activation by manganese and effect of other divalent cations. 1. Alkaline p-nitrophenylphosphate phosphatase (pNPPase) activity of Halobacterium halobium is selectively stabilized and stimulated by Mn2+ ions. 2. Mn2+ binding to native pNPPase is characterized by a dissociation constant of 0.35 mM at pH 8.5, 37 degrees C, with a Hill coefficient of 0.988. 3. Mn2+ behaves as a mixed type nonessential activator, increasing the Vmax value (beta = 6.09, pH 8.5) and decreasing the Km value for pNPP (alpha = 0.56, pH 8.5). The Ki value for inorganic phosphate (a competitive inhibitor) was also decreased in the presence of Mn2+. 4. Activation of native pNPPase by preincubation with Mn2+ is a slow temperature-dependent process, which can be described by an exponential relationship vs time. However, a weak but immediate activation was also detected. 5. Zn2+, Cu2+ and Ni2+ were found to inhibit both native and Mn(2+)-stimulated pNPPase, whereas Co2+ and Cd2+ inhibited the Mn(2+)-stimulated pNPPase but had no effect on the native enzyme form.


The effects of freeze-drying on the potency and stability of live varicella virus vaccine. Investigations into methods for improving the potency and stability of live varicella-zoster virus (Oka strain) vaccines have included the use of different lyophilization procedures which yielded products with different moisture levels. Three procedures were used: an 8-hour controlled-vacuum (0.47 mBars) procedure, a 14-hour controlled-vacuum (0.14 mBars) procedure, and a 48-hour high-vacuum (less than 0.07 mBars) procedure. Samples were stored for 24 months at -24 degrees C, -15 degrees C (in a frost-free freezer), and 4 degrees C. Potency was determined by a plaque assay in MRC-5 cells; moisture content was measured by the Karl Fisher method. Moisture content was 6 to 8 percent for the product made using the 8-hour procedure, 2 to 7 percent for the 14-hour procedure, and 0.5 to 1.5 percent for the 48-hour procedure. In addition to higher moisture, the 8-hour procedure resulted in a higher initial potency, indicating a lower loss during lyophilization, and better stability than did the 14- and 48-hour procedures. Although the initial potency from the 14-hour procedure was not statistically different from that for the 48-hour procedure, the product made with the 14-hour procedure did have better stability characteristics than that made with the 48-hour procedure.


Regulation of blood coagulation by the protein C system. Protein C is a plasma, vitamin K-dependent zymogen of a serine protease that can inhibit blood coagulation. Protein C is regulated by a series of reactions known as the protein C pathway. The importance of this pathway is seen in the occurrence of thrombosis in individuals with deficiencies in elements of the pathway like protein C and protein S. Work on several steps in this pathway has revealed that mechanisms involved in activation of protein C and the expression of its anticoagulant activity have features that allow for the expression of the anticoagulant activity away from sites in which procoagulant reactions occur, but not systemically. Thrombin, the principal procoagulant enzyme at the site of an injury, is converted to an anticoagulant enzyme at distant sites through its interaction with the endothelial cell protein thrombomodulin. Structural and functional studies have revealed the importance of several domain structures in the modulation of thrombin activity. Structural features of both activated protein C and its substrates (coagulation factors V and VIII) are such that they require the localization of enzyme and substrate on the surface of phosphatidyl serine containing membranes for optimum activity.


Signal transduction from cell surface to nucleus in development and disease. Recent studies indicate that extracellular signals affect cell proliferation and differentiation by modulating transcription factor activity via protein phosphorylation cascades. This review discusses the basic outline of the eukaryotic signal transduction systems used to transmit information from the cell surface to the transcriptional machinery in the nucleus. Several examples that illustrate how these pathways control cell proliferation, differentiation and development are discussed.


Glutathione mobilization during cerebral ischemia and reperfusion in the rat. 1. Cerebral ischemia applied for 15 min and followed by a 30 min reperfusion did not change the glutathione (GSH) levels and beta-adrenoceptor density (Bmax) in brain cortex. 2. A significant increase in erythrocyte-lysate GSH concentration (vs control) and a significant decrease of Bmax values in erythrocyte membranes (vs control) was found at the same time. 3. Pretreatment with the alpha-adrenoceptor antagonist phentolamine (5 mg/kg i.p.) prevented the erythrocyte GSH increase but not the decrease of Bmax value. Pretreatment with the beta-antagonist propranolol (2 mg/kg i.p.) did not influence the increase in erythrocyte GSH but circumvented the decrease of Bmax.


Cholera toxin augments the release of endothelium-derived relaxing factor evoked by bradykinin and the calcium ionophore A23187. 1. Experiments were designed to examine the effect of cholera toxin and forskolin on the release of relaxing factor(s) from superfused cultured endothelial cells under basal conditions and upon stimulation with bradykinin, adenosine diphosphate or the calcium ionophore A23187. 2. Exposure of cultured porcine aortic endothelial cells to cholera toxin (30 micrograms/ml, for 3 hr) and forskolin (10(-6) M, for 45 min) significantly increased the intracellular content in cyclic AMP. Cholera toxin but not forskolin stimulated the accumulation of cyclic GMP. 3. Exposure to cholera toxin did not modify the basal release of endothelium-derived relaxing factor nor that induced by adenosine diphosphate, but significantly increased that evoked by bradykinin and the calcium ionophore A23187. Forskolin did not significantly affect the basal or the stimulated release of endothelium-derived relaxing factor. 4. These results suggest that cholera toxin potentiates the release of endothelium-derived relaxing factor (presumably nitric oxide) from endothelial cells by a mechanism other than augmented production of cyclic AMP.


Long-term treatment with different calcium- and calmodulin-antagonists induces changes in rat brain alpha-adrenoceptors. 1. The binding characteristics (Bmax and Kd) of the alpha-adrenoceptor radioligand [3H] WB4101 in crude membrane fraction (fraction P2) from cerebral cortex were studied after 13-day oral treatment of male Wistar rats with the Ca(2+)-antagonists nifedipine (20 mg/kg), verapamil (50 mg/kg), flunarizine (10 mg/kg) and with the calmodulin-antagonist trifluoperazine (TFP) (3 mg/kg). 2. A significant reduction of the binding sites (Bmax) for [3H] WB4101 was established after the three Ca(2+)-antagonists as well as after TFP treatment. 3. Different changes in the affinity constant (Kd) of brain adrenoceptors were observed depending on the type of the Ca2+ or CaM-antagonist used: nifedipine did not change the Kd value, verapamil and TFP decreased whereas flunarizine increased the Kd value. 4. Relationships between Ca ions and alpha-adrenoceptor functions are suggested.


Turnover of R1 (type I) and R2 (type II) retrotransposable elements in the ribosomal DNA of Drosophila melanogaster. R1 and R2 are distantly related non-long terminal repeat retrotransposable elements each of which inserts into a specific site in the 28S rRNA genes of most insects. We have analyzed aspects of R1 and R2 abundance and sequence variation in 27 geographical isolates of Drosophila melanogaster. The fraction of 28S rRNA genes containing these elements varied greatly between strains, 17-67% for R1 elements and 2-28% for R2 elements. The total percentage of the rDNA repeats inserted ranged from 32 to 77%. The fraction of the rDNA repeats that contained both of these elements suggested that R1 and R2 exhibit neither an inhibition of nor preference for insertion into a 28S gene already containing the other type of element. Based on the conservation of restriction sites in the elements of all strains, and sequence analysis of individual elements from three strains, nucleotide divergence is very low for R1 and R2 elements within or between strains (less than 0.6%). This sequence uniformity is the expected result of the forces of concerted evolution (unequal crossovers and gene conversion) which act on the rRNA genes themselves. Evidence for the role of retrotransposition in the turnover of R1 and R2 was obtained by using naturally occurring 5' length polymorphisms of the elements as markers for independent transposition events. The pattern of these different length 5' truncations of R1 and R2 was found to be diverse and unique to most strains analyzed. Because recombination can only, with time, amplify or eliminate those length variants already present, the diversity found in each strain suggests that retrotransposition has played a critical role in maintaining these elements in the rDNA repeats of D. melanogaster.


Delineation of cis-acting sequences required for expression of Drosophila mojavensis Adh-1. The control of expression of the Adh-1 gene of Drosophila mojavensis has been analyzed by transforming ADH null Drosophila melanogaster hosts with P element constructs which contain D. mojavensis Adh-1 having deletions of different extent in the 5' and 3' ends. Adh-1 expression in the D. melanogaster hosts is qualitatively similar to expression in D. mojavensis, although expression is quantitatively lower in transformants. Deletions of the 5' end indicate that information required for normal temporal and tissue expression in larvae is contained within 70 bp of the transcription start site. However, deletion constructs to -70 are deficient in ovarian nurse cell expression, whereas the additional upstream sequences present in constructs containing deletions to -257 do support expression in the ovary. Comparison of the nucleotide sequence in the -257 to -70 region of Adh-1 of four species: D. mojavensis and Drosophila arizona, which express Adh-1 in the ovary, and Drosophila mulleri and Drosophila navojoa, which do not, has led to the identification of regions of sequence similarity that correlate with ovary expression. One of these bears a striking similarity to a conserved sequence located upstream of the three heat shock genes that have constitutive ovarian expression and may be an ovarian control element. We have identified an aberrant aspect of Adh-1 expression. In transformants which carry an Adh-1 gene without a functional upstream Adh-2 gene Adh-1 expression continues into the adult stage instead of ceasing at the onset of metamorphosis. In transformants with a functional Adh-2 gene, Adh-1 expression ceases in the third larval instar stage and aberrant expression in the adult stage does not occur.


Insertional mutagenesis of the maize P gene by intragenic transposition of Ac. The P-rr allele of the maize P gene regulates the synthesis of pigments derived from flavan-4-ol in the pericarp, cob glumes and other floral organs. We characterized 21 P alleles derived by intragenic transposition of Ac from three known positions. Ac transpositions can occur in either direction in the P gene, and with no apparent minimum distance: in one case Ac transposed just 6 bp from its original insertion site. However, the distribution of transposed Ac elements was markedly nonrandom: of 19 transposed Ac elements derived from a single Ac donor, 15 were inserted in a 1.1-kb region at the 5' end of P, while none had inserted in an adjacent 3.2-kb intronic region. All of the Ac insertions affect both pericarp and cob glume pigmentation, providing further evidence that the P-rr allele contains a single gene required for both pericarp and cob glume pigmentation. The distribution of the inserted Ac elements and the phenotype conditioned by each allele suggests a structure of P-rr which is similar to that previously determined molecularly. Possible explanations for the nonrandom distribution of transposed Ac elements are discussed.


Fitness effects of Ty transposition in Saccharomyces cerevisiae. It has been suggested that the primary evolutionary role of transposable elements is negative and parasitic. Alternatively, the target specificity and gene regulatory capabilities of many transposable elements raise the possibility that transposable element-induced mutations are more likely to be adaptively favorable than other types of mutations. Populations of Saccharomyces cerevisiae containing large amounts of variation for Ty1 genomic insertions were constructed, and the effects of Ty1 copy number on two components of fitness, yield and growth rate were determined. Although mean stationary phase density decreased with increased Ty1 copy number, the variance and range increased. The distributions of stationary phase densities indicate that many Ty1 insertions have negative effects on fitness, but also that some may have positive effects. To test directly for adaptively favorable Ty1 insertions, populations containing large amounts of variability for Ty1 copy number were grown in continuous culture. After 98-112 generations the frequency of clones containing zero Ty1 elements had decreased to approximately 0.0, and specific Ty1-containing clone families had predominated. Considering that most of the genetic variation in the populations was due to Ty1 transposition, and that Ty1 insertions had, on average, a negative effect on fitness, we conclude that Ty1 transposition events were directly responsible for the production of adaptive mutations in the clones that predominated in the populations.


P element transposition contributes substantial new variation for a quantitative trait in Drosophila melanogaster. The P-M system of transposition in Drosophila melanogaster is a powerful mutator for many visible and lethal loci. Experiments using crosses between unrelated P and M stocks to assess the importance of transposition-mediated mutations affecting quantitative loci and response to selection have yielded unrepeatable or ambiguous results. In a different approach, we have used a P stock produced by microinjection of the ry506 M stock. Selection responses were compared between transposition lines that were initiated by crossing M strain females with males from the "co-isogenic" P strain, and ry506 M control lines. Unlike previous attempts to quantify the effects of P element transposition, there is no possibility of P transposition in the controls. During 10 generations of selection for the quantitative trait abdominal bristle number, none of the four control lines showed any response to selection, indicative of isogenicity for those loci affecting abdominal bristle number. In contrast, three of the four transposition lines showed substantial response, with regression of cumulative response on cumulative selection differential ranging from 15% to 25%. Transposition of P elements has produced new additive genetic variance at a rate which is more than 30 times greater than the rate expected from spontaneous mutation.


The evolution of insertion sequences within enteric bacteria. To identify mechanisms that influence the evolution of bacterial transposons, DNA sequence variation was evaluated among homologs of insertion sequences IS1, IS3 and IS30 from natural strains of Escherichia coli and related enteric bacteria. The nucleotide sequences within each class of IS were highly conserved among E. coli strains, over 99.7% similar to a consensus sequence. When compared to the range of nucleotide divergence among chromosomal genes, these data indicate high turnover and rapid movement of the transposons among clonal lineages of E. coli. In addition, length polymorphism among IS appears to be far less frequent than in eukaryotic transposons, indicating that nonfunctional elements comprise a smaller fraction of bacterial transposon populations than found in eukaryotes. IS present in other species of enteric bacteria are substantially divergent from E. coli elements, indicating that IS are mobilized among bacterial species at a reduced rate. However, homologs of IS1 and IS3 from diverse species provide evidence that recombination events and horizontal transfer of IS among species have both played major roles in the evolution of these elements. IS3 elements from E. coli and Shigella show multiple, nested, intragenic recombinations with a distantly related transposon, and IS1 homologs from diverse taxa reveal a mosaic structure indicative of multiple recombination and horizontal transfer events.


The Drosophila sloppy paired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors. The sloppy paired locus is involved in the establishment of the metameric body plan of the Drosophila embryo. We have cloned the sloppy paired locus by P-element-mediated enhancer detection. The locus is composed of two genes, slp1 and slp2, that are structurally and functionally related. They belong to a novel class of putative transcription factors containing a fork head domain that has also been found in mammalian hepatocyte transcription factors. The spatial expression patterns of the two transcripts are very similar, suggesting common regulation of the two genes. We recovered additional sloppy paired alleles by remobilization of an enhancer detector transposon. Genetic analysis suggests that both genes contribute to the segmentation phenotype that has characteristics of both, pair-rule and segment polarity genes, and that they interact functionally. The two genes appear to share an enhancer element situated upstream of slp1 that acts on both the proximal slp1 promoter and the distal slp2 promoter.


Selective disruption of genes expressed in totipotent embryonal stem cells. Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.