Human papillomavirus type 16 in a homosexual man. Association with perianal carcinoma in situ and condyloma acuminatum. The association of anal carcinoma with human papillomavirus (HPV) type 16 infection is well documented. Anal carcinoma is also frequently associated with a history of anogenital condylomata. More than 90% of anogenital condylomata contain HPV type 6 or 11. It is rare for a condylomatous lesion to contain HPV 16. We report the unusual case of a homosexual man, testing positively for human immunodeficiency virus, with carcinoma in situ evolving within perianal condylomata infected with HPV 16. Microscopic examination of tissue specimens from ulcerated verrucous lesions on the perianal mucosa revealed changes of classic condylomata acuminata with contiguous focal squamous cell carcinoma in situ. Testing for HPV DNA by in situ hybridization identified HPV 16 in both the condylomatous and carcinoma in situ areas. The association of HPV 16-infected condylomata and adjacent carcinoma in situ implies that cutaneous genital condylomata may progress to high-grade lesions. Given that homosexual men are at high risk for perianal carcinomas, HPV typing of perianal condylomata specimens may help identify immunocompromised patients who are at risk for the development of carcinomas.


Reconstruction of surgical defects using the gluteus maximus myocutaneous flap following radical vulvectomy. Radical vulvectomy is often complicated by problems associated with insufficient closure of large skin defects involving postoperative necrosis of the suture line over the mons pubis and the inguinal areas. To resolve these problems, the present study compared radical vulvectomy (RV, 7 cases) with radical vulvectomy followed by reconstructive operation using the gluteus maximus myocutaneous flap (RVR, 5 cases). There was no significant difference in operation time and blood loss between the two groups. Three of the 7 RV patients had wound separations requiring reoperation, while only 1 patient in the RVR group did. The average hospital stay was 86 days in the RV group and 38 days in the RVR. Risks of postoperative infection and wound breakdown were reduced with the flap technique, and ambulation and rehabilitation could begin earlier in this group. Surgical wounds were stable and the quality of life after operation was improved dramatically using the flap technique.


Significance of the serum CA125 level in recurrent ovarian cancer. Thirty-four recurrent cases of 89 patients with ovarian cancer treated in our department between 1985 and 1989 were examined for changes in serum CA125 level. 1) Upon confirmation of recurrence, 17 patients were positive for CA125 and 15 were negative. 2) According to the histopathological type, the rate of CA125 positivity in patients with recurrence was high for serous adenocarcinoma, suggesting that determination of CA125 is useful for detection of recurrence. In contrast, all patients with mucinous adenocarcinoma or endometrioid adenocarcinoma were negative for CA125. 3) The CA125 positivity rate upon confirmation of recurrence was 9% in patients whose CA125 was less than 1,000 U/ml on initial examination, suggesting that close management of such patients is necessary. 4) Elevation of CA125 by 3 steps or more within the normal range (less than 35 U/ml) was useful for predicting recurrence. 5) The cut-off level of CA125 during follow-up should be set at 16 U/ml. 6) It was difficult to evaluate remission with only the serum CA125 level. It is impossible to avoid second look operation at present. These results indicate that pretreatment values and changes of the parameter within the normal range (less than 35 U/ml) have to be considered when using CA125 as a marker of tumor recurrence.


Expression and amplification of cellular oncogenes in human developing placenta and neoplastic trophoblastic tissue. To confirm the expression of cellular oncogenes during normal development, their differential RNA levels in developing human placenta have been studied using radioactive probes such as v-abl, v-erbA, v-fms, v-mos, v-myc, N-ras and v-src. The c-mos and N-ras genes are expressed and amplified at high levels especially in term placenta, while c-abl, and c-erbA are expressed constantly during development. These findings indicate that c-mos and N-ras genes may be closely linked to normal differentiation, although c-abl and c-erbA may participate in overall developmental processes. In contrast, transcripts of c-myc and c-src are enhanced at first trimester and decreased sequentially thereafter, showing that these genes may play a role in early proliferation. Expression patterns of c-fms gene are same as that of c-myc and c-src except reelevation at term. In addition, to characterize the effect of cellular oncogene expression has been also examined in hydatidiform mole and tumor cells such as BeWo and choriocarcinoma. All cellular oncogenes examined in this study were significantly overexpressed. Thus, our results suggest that cellular oncogene activation may be strongly associated with neoplastic change of trophoblast.


Patterns and implications of subclinical vulval human papillomavirus infection: the impact of PCR analysis. This study was performed to elucidate the presence of human papillomavirus (HPV) infection of the vulva by colposcopy, histology and the polymerase chain reaction (PCR). Colposcopy defined 5 patterns of vulval epithelial lesions inconspicuous to the naked eye. Of these 75 subclinical vulval lesions, HPV infection was diagnosed by histology in 20.0% of minor epithelial changes with faint acetowhitening, 52.2% of conspicuous acetowhite lesions, 63.0% of acetowhite areas with satellite lesions, 84.6% of villous lesions, and 85.7% of villous lesions with surrounding acetowhitening. The corresponding HPV DNA positivity rates by PCR were 60.0%, 73.9%, 70.4%, 84.6% and 100% respectively. The oncogenic HPV type 16 was detected by PCR in 37.3% of the samples. These results provide firm evidence for the prevalent existence of subclinical HPV lesions of the vulva. Some of these infections may not produce significant morphologic changes detectable by colposcopy or histology. Subclinical vulval lesions are common and may constitute a reservoir for repeated cervical HPV infections, as well as a source of contamination of cervical samples for HPV DNA detection by sensitive molecular techniques.


The molecular epidemiology of equine herpesvirus 1 (equine abortion virus) in Australasia 1975 to 1989. The restriction endonuclease DNA fingerprints of 57 isolates of equine herpesvirus 1 (EHV1; equine abortion virus) from abortion, perinatal foal mortalities and encephalitis from 15 epidemics that occurred in Australasia between 1975 and 1989 were examined using the enzymes Bam HI, EcoRI and Bgl II. There was a remarkable degree of uniformity in the restriction patterns; mobility differences were observed in only 14 of 52 (27%) of the fragments. Twelve of these 14 fragments were located within the repeat structures that bracket the unique short region of the genome or were located at the left terminus of the 150 kilobase pair genome. Based on the Bam HI fingerprints the commonest virus identified in our study was EHV1.IP (P is for prototype strain). There was a single notable exception in that the Bam HI fingerprints of all 8 isolates from one of 3 Victorian farms that experienced abortion in 1989 resembled a variant EHV1.IB that was identified as a cause of abortion in Central Kentucky in 1970 to 1974. We present evidence that EHV1.IB caused abortion in California in 1964 and has remained unaltered in its Bam HI restriction pattern. No antigenic differences were found among 4 distantly related EHV1 isolates, including the variant IB, using a panel of 5 monoclonal antibodies to glycoprotein C (gC), a glycoprotein recognised to be highly variable. The uniformity of these unrelated EHV1 isolates is further evidence for a recent origin for EHV1 and may help to explain the natural history of this virus in the horse in which it seems to be a cause of serious epidemics of abortion and perinatal mortality, and less commonly of encephalitis.


Comparison of electron microscopy and polyacrylamide gel electrophoresis in the diagnosis of avian reovirus and rotavirus infections. Electron microscopy (EM) and genome electropherotyping by polyacrylamide gel electrophoresis (PAGE) for the detection of avian rotaviruses and reoviruses in intestinal specimens and cell cultures were compared. Fifty-eight field samples of intestine with intestinal contents, referred to as direct specimens, from turkey and chicken flocks located in different regions of California and submitted during 1989 for virus isolation were randomly selected as test samples. Also, 38 field intestinal specimens with suspected viral infection that had been passaged three times in primary chicken embryo kidney (CEK) cell cultures were used in their third passage. The percentage of agreement and the Kappa statistic of positive and negative results between these two tests were calculated. In the comparison, EM was considered the standard test. By statistical analysis, an agreement of 87% was observed in cell-culture samples analyzed by the two virus-detection methods, as contrasted with an agreement of 72% for direct specimens. The analysis of the number of segments and band migration profiles of reference and field virus strains indicated that only reoviruses replicated in CEK cell cultures and mainly rotaviruses were detected by both tests in direct specimens. The Kappa statistic analysis indicated substantial agreement (0.69) between the two tests for CEK samples, with moderate agreement (0.45) for the direct specimens examined.


Tissue-print hybridization using a non-radioactive probe for the detection of infectious bursal disease virus. Tissue-print hybridization was evaluated as a simplified means for detection of infectious bursal disease virus (IBDV) in the bursa of Fabricius from infected chickens. The assay employed a biotin-labeled synthetic oligonucleotide as a probe. The bound probe was detected using a color assay consisting of streptavidin conjugated to alkaline phosphatase. Bursae were imprinted onto nitrocellulose and then hybridized with the biotinylated probe. Bursal prints from IBDV-infected chickens were readily distinguished from control prints by color development and differences in signal intensity.


Digoxigenin-labeled nucleic acid probe for the detection of infectious bursal disease virus in infected cells. A cDNA probe was synthesized from the VP-4 region of a virulent field isolate of infectious bursal disease virus (IBDV). The probe was labeled during synthesis with a non-radioactive steroid hapten, digoxigenin. The probe was used to develop a hybridization assay to detect the presence of IBDV in infected cell-culture and tissue suspensions from the bursa of Fabricius of infected chickens. The test was rapid, reproducible, and sensitive, and it could detect four serologic subtypes of IBDV, including the GLS-5 isolate.


Molecular detection of infectious bursal disease virus by polymerase chain reaction. The polymerase chain reaction (PCR) technique was applied to the detection of infectious bursal disease virus (IBDV). Reverse transcription followed by the PCR was used to amplify a portion of IBDV genome. A set of primers that specify a 150-base-pair segment of IBDV genome was chosen from an Australian strain of IBDV. Standard challenge strain and variant strains A, D, E, G, and GLS-5 of IBDV serotype 1 and OH strain of serotype 2 from infected bursae were subjected to reverse transcription, followed by 30 cycles of PCR. A single band of the PCR product (DNA) of the expected size from each strain of IBDV was visible on polyacrylamide gels stained with ethidium bromide. Using the same primers, no PCR product was detected from genomic nucleic acids of turkey hemorrhagic enteritis virus, infectious bronchitis virus, reovirus, Salmonella enteritidis, Escherichia coli, and uninfected bursae. The PCR could be efficiently performed on serially diluted IBDV RNA and could detect 2 femtograms of IBDV RNA. The identity of the PCR products was confirmed by direct sequencing. The PCR is a specific and sensitive method for the detection of IBDV.


Isolation and propagation of infectious bursal disease virus using the ovine kidney continuous cell line. Twenty-six samples known to contain infectious bursal disease virus (IBDV) were examined by virus-isolation attempts on ovine kidney (OK) cell line, Vero cell line, and chicken embryo fibroblast (CEF) cultures. Virus was isolated from two of 26 samples, three of 26 samples, and three of 25 samples on OK, Vero, and CEF cultures, respectively. However, in contrast to IBDV replication in Vero and CEF cultures, isolated virus was unable to induce serially sustained cytopathic effects (CPE) during successive passages in the OK cell line, unless cell lysates were treated with chloroform between every other passage. The cytopathogenicity of the untreated virus passaged in OK cells was revived and maintained upon passage in Vero cells. An initial single passage of laboratory or field material in OK cells followed by further passages in Vero cells resulted in virus isolation from six of 26 samples, which was better virus recovery than when either cell line was used alone or when CEF cultures were used. Twenty of the 26 test samples were originally positive when examined by nucleic acid hybridization with radiolabeled IBDV cDNA, indicating that some of the samples that were negative upon virus isolation using OK and Vero cells may have contained inactivated virus.


Relationship of common avian pathogen antibody titers in so-called chicken anemia agent (CAA)-antibody-positive chicks to titers in CAA-antibody-negative chicks. Antibody titers for infectious bursal disease virus (IBDV), infectious bronchitis virus, Newcastle disease virus, and reovirus from chicks with chicken anemia agent (CAA) antibodies were compared with antibody titers from their CAA-antibody-negative counterparts. These comparisons were made in 396 chickens that were 1 day, 2 weeks, 8-9 weeks, 10 weeks, 17 weeks, or 29-32 weeks old. Only one serum sample was collected from any given chick or chicken. There were no significant differences between the antibody titers at any age for any antigen, with one exception: at 29-32 weeks, the IBDV titers were higher (t = 2.62, df = 142, P less than 0.01) in chickens with CAA antibody. Although not at all likely, we believe that the observation of high IBDV antibody titers in CAA-antibody-positive chicks could have been a spurious one.


Pathologically and serologically different avian nephritis virus isolates implicated in etiology of baby chick nephropathy. Three virus isolates (WG-3, -4, and -5) from chicks affected by baby chick nephropathy were orally inoculated into 1-day-old specific-pathogen-free chicks of lines PDL-1 and 15I. Additional chicks were orally inoculated with avian nephritis virus (ANV) strain G-4260. Chicks inoculated with isolates WG-3, -4, and -5 died between 2 and 6 days postinoculation (PI), with mortality ranging from 0% to 53.3%. Pathological findings in the dead chicks included nephrosis in chicks inoculated with WG-3, -4, and -5, and nephritis and visceral urate deposition in chicks inoculated with G-4260. The stability of the WG-5 isolate, as well as the size of the particles and the nucleic acid type, were also similar to those of the G-4260 strain. All of the examined chicks inoculated with WG-3, -4, and -5 had interstitial nephritis at 14 days PI. Therefore, the three virus isolates were considered to be ANV. However, there was no serological relationship between the isolates and ANV (G-4260 and M-8 strains).


Detection of type II avian adenoviral antigen in tissue sections using immunohistochemical staining. An immunohistochemical staining technique for the detection of marble spleen disease (MSD) viral antigens and other type II avian adenoviral antigens was developed using a mixture of monoclonal antibodies produced against hemorrhagic enteritis (HE) virus and a commercial streptavidin-biotin peroxidase indicator system. This technique was applied to both frozen and formalin-fixed paraffin-embedded tissue sections. The immunohistochemical staining technique was used on tissues from pheasants with experimental MSD, on tissues from a pheasant with natural MSD, and on tissues from turkeys with natural HE. Staining results were compared with routine hematoxylin-and-eosin (H&E) staining. Additional viral inclusions, not detected with H&E, were found in the liver, lung, bone marrow, and kidney sections using the immunohistochemical technique. The immunohistochemical technique was highly specific and sensitive for the detection of type II adenoviral antigen, and it appears to be useful for studying the pathogenesis of these diseases and for retrospective evaluation of routinely processed diagnostic tissue samples.


Influence of turkey herpesvirus vaccination on the B-haplotype effect on Marek's disease resistance in 15.B-congenic chickens. Eight recently developed 15.B congenic lines of chickens were tested for Marek's disease (MD) resistance by intra-abdominal injection of cell-associated preparations of MD virus of a virulent strain (JM), a very virulent strain (Md5), or Md5 after vaccination with turkey herpesvirus (HVT) strain FC126. Chickens of the 15.N congenic line (B15B21 or B21B21) were very resistant to JM-induced MD, in contrast to chickens homozygous for the B-haplotypes 2, 5, 12, 13, 15, or 19. After Md5 infection, more than 88% of the chickens in all of the congenic lines developed MD. However, when chickens were vaccinated with HVT before being inoculated with Md5, the B5 and B12 homozygotes were more resistant to MD than were the B2, B13, or B19 homozygotes, and B15 and B21 homozygotes had intermediate resistance. B5B5 and B2B5 F2 chicks inoculated with HVT and Md5 had a lower prevalence of MD than B2B2 sibs. These results demonstrate that a protocol involving HVT vaccination of chicks followed by infection with very virulent MD virus will allow the detection of B-haplotypes determining MD resistance, some of which are not detectable in unvaccinated chicks challenged with virulent MD.


Coarse-spray immunization of one-day-old broilers against enteric reovirus infections. Coarse-spray (CS) administration of a commercial S1133 reovirus vaccine was evaluated in 1-day-old specific-pathogen-free broilers for prevention of clinical infection induced by intratracheal challenge with two enteric reovirus isolates. In Expt. 1, chickens were challenged at 4 days of age with either the 2408 or CO8 isolate. In Expt. 2, chickens were challenged at 7 days of age with either isolate. In Expt. 3, chickens were challenged at 3, 5, or 7 days of age with the 2408 isolate. In Expt. 1, vaccinated birds showed significant protection against challenge with either isolate at 4 days of age as measured by morbidity, mortality, gross lesions, and body weight. In Expt. 2, vaccinated birds showed greater protection against challenge at 7 days of age. In Expt. 3, resistance in vaccinated birds increased with time between vaccination and challenge. Vaccinated birds challenged at 3 days of age showed no significant protection, whereas vaccinated birds challenged at 5 or 7 days of age had increased resistance. This vaccine did not induce a drop in weight gain, morbidity, mortality, or microscopic lesions in the tendons.


Transposon mutagenesis used to study the role of complement resistance in the virulence of an avian Escherichia coli isolate. The role of complement resistance in the virulence of an avian Escherichia coli isolate was examined with transposon mutagenesis. A suicide plasmid containing a kanamycin-encoding mini-transposon was used to transform a virulent complement-resistant avian E. coli isolate. A less resistant mutant was identified that contained a transposon insertion in a plasmid and in the chromosome. This loss of complement resistance was associated with a drop in virulence in an embryo assay. No other phenotypic changes were detected in the mutant. These results suggest that complement resistance is associated with the virulence of this organism.


Ethanol modulates epidermal growth factor-stimulated tyrosine kinase and phosphorylation of PLC-gamma 1. A431 cells have an abundance of Epidermal Growth Factor (EGF) receptors which possess intrinsic tyrosine kinase activity. Treatment of membranes isolated from A431 cells with EGF caused a 2-3 fold increase in phosphorylation of a synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly) which is a substrate for tyrosine kinase. Treatment of these membranes with 0.1 to 100 mM ethanol altered basal tyrosine kinase activity in a biphasic manner; increase at 10 mM and decrease at 100 mM ethanol. The treatment of the membranes with the same concentrations of ethanol also altered EGF's ability to stimulate tyrosine kinase activity: increase at 0.1 mM ethanol and decrease at 10 mM. Strikingly, EGF-stimulated tyrosine kinase was more sensitive to ethanol than the basal activity. Experiments with other alcohols showed a relationship between chain length and the inhibitory ability of the alcohol. These data demonstrate a biochemical effect of low concentrations of ethanol on tyrosine kinase. Interestingly, ethanol treatment of A431 cells inhibited EGF-stimulated phosphorylation of PLC-gamma 1 which is a substrate for EGF receptor tyrosine kinase. It is concluded that ethanol at low concentrations has significant modulatory effect on basal and EGF-stimulated tyrosine kinase, as well as PLC-gamma 1 phosphorylation.


Suppressive effect of interleukin-1 on pulmonary cytochrome P450 and superoxide anion production. Interleukin-1 has been shown to prolong the survival of rats exposed to lethal concentrations of oxygen. This oxygen tolerance has been attributed by some workers to an increase of manganese superoxide dismutase. We report here that the administration of interleukin-1 to male adult rats results in (i) significant decrease of pulmonary cytochrome P450 at 24 and 72 hours, (ii) decrease of P450 IIB1 mRNA at 24 and 72 hours and (iii) significant decrease of superoxide anion generation from pulmonary microsomes isolated from treated rats. To the best of our knowledge, this is the first report to demonstrate these effects of interleukin-1 on pulmonary P450 and its oxidase activity (O2- generation). On the basis of these results and several earlier reports in which various P450 depressants have been shown to depress superoxide production from microsomes and to prolong the lives of rodents in hyperoxia, we conclude that oxygen tolerance induced by interleukin-1 administration is likewise mediated, at least in part, by reduced generation of superoxide anion from cytochrome P450 monooxygenase system.


Zinc hydroxide stimulates superoxide production by rat alveolar macrophages. The effect of zinc hydroxide on superoxide (O2-) production by rat alveolar macrophages was determined by chemiluminescence and by cytochrome c reduction. Zinc ions had no effect on the chemiluminescence of unstimulated alveolar macrophages. By contrast, zinc hydroxide (ZnOH2), a neutralized form of zinc ions, increased the chemiluminescence level and O2- release. Increased O2- release was inhibited by pertussis toxin, isoquinoline sulfonamide and pretreatment with EGTA. These findings indicate that zinc hydroxide formation from zinc compounds can stimulate the O2- production by alveolar macrophages by receptor-mediated and Ca(2+)-dependent process.


Activation of TIMP-2/progelatinase A complex by stromelysin. Progelatinase A was purified as a complex with TIMP-2 from the conditioned medium of a human glioblastoma cell line. The TIMP-2/progelatinase complex was resistant to the activation by p-aminophenylmercuric acetic acid (APMA), and showed less than 10% of the activity of the TIMP-2-free active enzyme. When the complex was incubated with stromelysin in the presence of APMA, the 64-kDa progelatinase was effectively converted to the 57-kDa mature enzyme, increasing its gelatinolytic activity about 8-fold. These results suggest that stromelysin is a natural activator of TIMP-2-bound progelatinase A.


Infectious bronchitis virus detection in allantoic fluid using the polymerase chain reaction and a DNA probe. A rapid extraction procedure was developed to purify infectious bronchitis virus (IBV) RNA from the allantoic fluid of inoculated embryonating eggs. Reverse transcription of viral RNA and the polymerase chain reaction (PCR) were used to amplify the viral genome from eight different strains of IBV comprising five different serotypes. A biotinylated DNA probe, prepared to a sequence within the PCR amplification product of the Beaudette strain of IBV, was used in a dot-hybridization assay; it detected the amplification products of all of the IBV strains examined. Reverse transcription and PCR amplification were judged to be specific for IBV. This was because amplification products were not detected by agarose gel electrophoresis or by dot-hybridization when template used in the PCR was extracted from allantoic fluid and the chorioallantoic membrane of uninoculated embryonating eggs or from allantoic fluid of embryonating eggs inoculated with other chicken upper respiratory viruses.


A reversible radioligand specific for the ETB receptor: [125I]Tyr13-Suc-[Glu9,Ala11,15]-endothelin-1(8- 21), [125I]IRL 1620. Suc-[Glu9,Ala11,15]-endothelin(ET)-1(8-21), IRL 1620, is a linear ET-analog specific for the ET-isopeptide-nonselective ETB receptor. The radio-iodinated analog, [125I]IRL 1620, showed a single class of saturable binding to the ETB receptors in porcine lung membranes with a Kd of 18 pM and a Bmax of 930 fmol/mg protein, which are almost comparable to the values obtained with [125I]ET-3 (6 pM and 900 fmol/mg protein). In competitive binding assays with [125I]IRL 1620, unlabeled ET-1, ET-3, IRL 1620 and [monoiodo-Tyr13]-IRL 1620 showed almost identical displacement curves with Ki of 8 to 16 pM. However, [125I]IRL 1620 was dissociated from the binding sites by addition of an excess amount (100 nM) of any of these unlabeled peptides, each with the same t1/2 of 100 min. This was in marked contrast to [125I]ET-3 which was hardly dissociated from the binding sites.


Stable transfection of reticuloendotheliosis virus-transformed lymphoblastoid cell lines. Lymphoblastoid T cell lines were established by infection of chicken splenocytes with reticuloendotheliosis virus (REV). The target cells first were cultured in interleukin-containing conditioned medium or were stimulated by concanavalin A, or both. Most cell lines were T cells expressing CD3 and one of the T cell receptors, and all cell lines were positive for major histocompatibility complex (MHC) class II antigens. Several REV-transformed cell lines were stably transfected using electroporation with a selectable plasmid, pNL1, containing the neor gene. Transfected cell lines were selected using G418 and were maintained for periods up to 137 days. Transfected cell lines were susceptible to MHC class-I restricted lysis by cytotoxic T lymphocytes from REV-infected chickens.


Effect of cell-culture passage on the pathogenicity and immunogenicity of two variant strains of infectious bursal disease virus. Two variant strains of infectious bursal disease virus, IN and E, were adapted and passaged in an established cell line (BGM-70) 30 times and 40 times, respectively. Passage in cell culture resulted in loss of pathogenicity. However, both viruses maintained their antigenicity and immunogenicity, as demonstrated by the immunofluorescence and virus-neutralization tests and by the satisfactory protection induced by vaccinating specific-pathogen-free (SPF) chickens with inactivated preparations of both passaged viruses. No protection was induced when the passaged viruses were given to SPF chickens as live vaccines. It is speculated that the passaged viruses might have lost some ability to replicate in their natural host, resulting in lack of protection.


Immune function in pheasants experimentally infected with marble spleen disease virus. Two experiments were conducted to evaluate the effect of marble spleen disease virus (MSDV) infection on the immune response of pheasants. In the first, 15 ring-necked pheasants were inoculated orally with cell-culture-propagated MSDV and 15 received saline (controls). On days 7, 21, and 35 postinoculation (PI), all birds received sheep erythrocytes intravenously. Hemagglutination titers to sheep erythrocytes were determined for serum samples collected weekly for 6 weeks. The virus-inoculated group had significantly (P less than 0.05) lower hemagglutination titers than the control group. In the second experiment, 30 pheasants were allotted into two groups as above. Whole blood was collected from each bird weekly for 5 weeks. The blood was cultured in microtiter plates with or without optimum concentrations of concanavalin A. Five of 10 MSDV-inoculated pheasants had significantly depressed T lymphocyte transformation on either day 7 or day 14 PI. Overall, the depression of T lymphocyte transformation was transient and mild.


Regulation of 1,25-dihydroxyvitamin D3 receptor gene expression by parathyroid hormone and cAMP-agonists. We studied the effect of parathyroid hormone (PTH) and activation of the cAMP signal pathway on vitamin D receptor (VDR) mRNA levels in the phenotypically osteoblast cell line UMR 106. PTH caused a time- and dose-dependent increase of the VDR mRNA content with a maximum after 2 h. After 24 h the VDR mRNA level in PTH-treated cells returned to control level. In contrast, the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced increase in VDR mRNA did not decline after 24 h. Inhibition of transcription with actinomycin D (10 micrograms/ml) completely abolished the PTH-induced increase of VDR mRNA and inhibition of translation with cycloheximide (1 microgram/ml) resulted in superinduction of VDR mRNA. The role of cAMP in the induction of VDR mRNA was studied with several agents acting via the cAMP pathway. Incubation for 2 and 4 h with forskolin, Bt2cAMP, PTHrP or prostaglandin E2 caused an increase in the level of VDR mRNA comparable to that caused by PTH. The calcium ionophore A23187 did not affect VDR mRNA level. The present study demonstrates that PTH and activation of the cAMP signal pathway cause up-regulation of VDR via induction of VDR gene expression. The effect of cAMP on the VDR gene is suggestive for a cAMP responsive element in the VDR gene.


The endothelin receptor antagonist, BQ-123, inhibits angiotensin II-induced contractions in rabbit aorta. The purpose of this study was to examine the specificity of the cyclic pentapeptide ET(A) receptor antagonist BQ-123. BQ-123 competitively antagonized endothelin-1-induced contractions in rabbit aorta, increases in inositol phosphates in cultured rat vascular smooth muscle A10 cells, and binding of [125I]endothelin-1 to the cloned ETA receptor cDNA expressed in Cos 7 cells. In contrast, BQ-123 was a weak antagonist of [125I]endothelin-3 binding to rat cerebellar membranes and to membranes from Cos 7 cells transfected with the cloned ETB receptor cDNA. BQ-123 shifted concentration-response curves in isolated rabbit aorta elicited by angiotensin II, but did not bind to angiotensin II receptors nor affect angiotensin II-induced increases in inositol phosphates. BQ-123 also did not affect contractions induced by KCl or norepinephrine. These data suggest that endothelin may play a role in angiotensin II-induced contractions of rabbit aorta.


Site-specific mutations of conserved C-terminal residues in aminoglycoside 3'-phosphotransferase II: phenotypic and structural analysis of mutant enzymes. In order to test the biological importance of amino acids in the C-terminal quarter of aminoglycoside 3'-phosphotransferase II enzyme, seven of the highly conserved residues in this region, His-188, Asp-190, Asp-208, Gly-210, Arg-211, Asp-216 and Asp-220, were changed via site-directed mutagenesis. The phenotype of each mutant was compared to wildtype in terms of antibiotic susceptibilities and enzymatic activities. All of the substitutions either abolished or significantly reduced the resistance of the resulting strains to kanamycin, neomycin, butirosin, ribostamycin, paromomycin, gentamicin A, and G-418. Similarly, enzyme activities in crude extracts were substantially reduced for the mutant strains. Affinity of the enzyme for Mg+2-ATP decreased with His-188, Asp-190, Asp-216 and Asp-220 substitutions as revealed by Km measurements. Secondary structure analysis predicted that substitutions at the conserved residues caused severe conformational distortions at the corresponding regions of the protein.


A comparison of human lung, brain, CSF and plasma angiotensin-converting enzyme with regard to neuropeptide metabolism. Human ACE obtained from different tissues and body fluids was assayed with regard to degradative action on tachykinins and various opioid peptides. Substance P (1-9) was easily cleaved, whereas substance P and neurokinin A seemed stable against ACE activity. However, endopeptidase-24.11 easily degraded both of these amidated peptides. When the same peptides were assayed as potential inhibitors of the hydrolysis of hippuryl-His-Leu (specific substrate for ACE activity), substance P and its (1-9) fragment were equally potent, whereas neurokinin A was inactive. The beta-casomorphins, beta-casein derived opioid peptides, with a proline residue at their C-terminus also showed inhibitory action on ACE activity, without being cleaved by the enzyme. These results indicate a modulatory action of these peptides. No differences between ACE originating from different tissues or body fluids could be demonstrated in this regard.


Internalizable insulin-BSA-chlorin E6 conjugate is a more effective photosensitizer than chlorin E6 alone. Experiments with human hepatoma PLC/PRF/5 cells involving the use of two different tests (colony formation and Trypan blue exclusion) have demonstrated a significantly higher photosensitizing activity of chlorin e6 conjugates with bovine serum albumin (BSA) and internalizable ligand insulin as compared to that of chlorin e6 itself. Receptor-mediated internalization of insulin-BSA-chlorin e6 conjugates ensures greater photosensitization of cells than the binding of those conjugates to cell surface receptors. The suitability of such conjugates permitting the delivery of a photosensitizer to most sensitive cell structures is discussed.


Reactions of copper(II)-N-polycarboxylate complexes with hydrogen peroxide in the presence of biological reductants: ESR evidence for the formation of hydroxyl radical. The formation of hydroxyl radicals (.OH) by the reaction of CuII(edta) (edta: ethylenediaminetetraacetic acid) with hydrogen peroxide (H2O2) in the presence of biological reductants, such as L-ascorbic acid and L-cysteine, has been demonstrated for the first time by ESR spectroscopy using water-soluble spin-traps, 5,5-dimethyl-1-pyrroline N-oxide (DMPO, 1), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN, 2) and 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS, 3). Ethylenediaminetetraacetic acid (edta) is one of the polyamine-N-polycarboxylate chelating agents and it is commonly used by chemists and biochemists. Edta can chelate several metal ions. It is known that the CuII(edta) complex is usually less active than free copper ions in radical reactions, whereas complexes of edta with Fe(II) or Fe(III) still react with hydrogen peroxide (H2O2) or superoxide ion (O2-) (1). In our previous papers (2-4), we also have shown that copper(II) complexes with polyamine-N-polycarboxylates, such as edta and dtpa (diethylenetriaminepentaacetic acid), do not react with H2O2, whereas CuII(en)2 (en: ethylenediamine) can easily do so to give hydroxyl radical (.OH) as a reactive intermediate. Further, we assumed that the change of redox potential of Cu(II) ions as a result of ligation with different ligands causes the difference in reactivity of Cu(II) complexes towards H2O2. To verify this assumption, the reactions of CuII(edta), which was chosen as a Cu(II)-polyamine-N-polycarboxylate complex, with H2O2 were investigated in the presence of some biological reductants, using an ESR-spin trapping method.(ABSTRACT TRUNCATED AT 250 WORDS)


Ca pump messenger RNA expression in pregnant rat uterus. Rat uterus expresses transcripts for the plasma membrane Ca pump PMCA1 and for the sarcoplasmic reticulum Ca pump SERCA2. Uterus contains two mRNA isoforms for PMCA1, one encoding a cyclic nucleotide insensitive Ca pump (PMCA1a) and the other encoding a cyclic nucleotide sensitive pump (PMCA1b). The relative ratio of mRNA to 28S RNA for PMCA1a or PMCA1b and the amount of poly A+ RNA per microgram total RNA obtained from uteri of rats pregnant for 15 days and for parturient rats was similar but the ratio of SERCA2 mRNA to 28S RNA was significantly higher in the parturient rats.


Effect of GM1-ganglioside on gastric mucosal epidermal growth factor and platelet-derived growth factor receptor expression. The effect of intragastric administration of GM1-ganglioside on the expression of gastric mucosal epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptors was investigated. Gastric mucosal cell membranes were isolated from the stomach of groups of rats, one receiving twice daily for 3 consecutive days a dose of 0.25 mg/100g GM1-ganglioside, and the other only vehicle. Binding assays revealed the presence of both types of receptors, activation of which led to the elevation of tyrosine kinase activity as evidenced by a marked increase in membrane protein tyrosine phosphorylation patterns. The specific receptor binding in the control group was 2.47 fmol/mg protein for EGF and 1.46 fmol/mg protein for PDGF, whereas the respective binding values in the GM1-ganglioside treated group increased by 45% and 38%. The results suggest that GM1-ganglioside is capable of enhancement of gastric mucosal EGF and PDGF receptor activities.


Transplacental immunity to varicella-zoster virus in extremely low birthweight infants. Sixteen infants in our intensive care nursery (ICN) were inadvertently exposed to an adult who presented the next day with chickenpox. Since a majority of the patients were extremely premature, we were concerned that they might not have received transplacental varicella-zoster virus (VZV) antibody, and consequently would be at risk for contracting chickenpox. Serum samples were obtained from each infant and examined for the presence or absence of VZV antibody. Fourteen of the 16 infants had antibody levels equal to or in excess of 1:16 by indirect fluorescent antibody, including six of eight infants with birthweights equal to or less than 1000 gm, and eight of ten infants with gestational ages equal to or less than 28 weeks. Passive transfer of immunity to VZV appears to be common in infants as young as 24 to 28 weeks' gestation.


Epstein-Barr virus serology and isoelectrofocusing pattern of serum immunoglobulins in cyclosporin or placebo-treated type I diabetics. Epstein-Barr virus (EBV) serology was serially studied for a period of 18 months in 49 recently diagnosed type I diabetics randomly assigned to receive cyclosporin (CsA) (n = 25) or placebo (n = 24). Additionally, isoelectrofocusing of serum (IEF) was serially performed in 59 CsA- and 38 placebo-treated diabetics, over a 36 month period (687 sera) in order to detect restriction of heterogeneity of circulating immunoglobulins. Patients were continuously treated with CsA at a dose of 7.5-10 mg/day during the first 6 months and a dose not exceeding 5 mg/kg/day, thereafter. Before treatment anti-EBV antibody levels were in the normal range for the whole group. In the placebo group, Epstein-Barr nuclear antigen (EBNA) and viral capsid antigens (VCA) IgG were moderately raised during the first 6 months in comparison with baseline level (0.33 +/- 0.13 and 0.30 +/- 0.18 two-fold dilutions respectively). In contrast, in the CsA group, EBNA and VCA IgG decreased slightly (0.26 +/- 0.16 and 0.26 +/- 0.17 dilutions). Changes between the two groups at 3 and 6 months were significantly different (P less than 0.05), but the difference disappeared subsequently. No significant changes in anti-early antigen (EA) IgG and in EA and VCA IgA were observed. No IEF abnormalities appeared in the CsA group. One CsA-treated patient who was initially EBV seronegative developed normal serological signs of recent EBV infection at 12 months without restriction of immunoglobulin heterogeneity or clinical symptoms of infectious mononucleosis. This study indicates that CsA, at moderate dosage, slightly reduces the titer of pre-existing anti-EBV antibodies but does not alter the response to recent EBV infection. These results do not provide any evidence of clinically latent EBV-induced benign or malignant lymphocyte proliferation.


Antibodies against polypeptides of purified Epstein-Barr virus in sera from patients with connective tissue diseases. Antibodies to Epstein-Barr virus (EBV) were investigated in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) by immunoblotting using purified virus. Compared with sera from Epstein-Barr virus seropositive healthy individuals who served as control, sera from patients less frequently recognized several polypeptides. In particular, a 100 kDa envelope polypeptide was recognized by 92% of healthy subjects and only 11% of patients (P less than 0.001). On the other hand, 62% of the patient sera had antibodies to the 42 kDa envelope polypeptide, whereas these antibodies were only present in 4% of the sera from the control subjects (P less than 0.001). These results could reflect either perturbations of the immune response associated with connective tissue disorders or a possible pathogenic role of EBV.


Monitoring antigenemia is useful in guiding treatment of severe cytomegalovirus disease after organ transplantation. We investigated the value of monitoring CMV antigenemia during and after antiviral therapy for CMV disease. During the study period, 10 out of 214 renal transplant recipients were treated for CMV disease, receiving a total of 14 courses of treatment. Antigenemia decreased within 7 days after onset of treatment in eight of nine courses associated with a rapid clinical recovery. In three courses with a slow or absent response, antigenemia levels initially increased. Monitoring antigenemia was helpful in differentiating persisting CMV disease from other opportunistic infections and rejection. Relapses of CMV disease were preceded by rises in antigenemia. Viral isolation became negative within 3 days after initiation of ganciclovir, irrespective of the clinical response. Antigenemia is a marker of the effect of ganciclovir on CMV replication in vivo, and its monitoring may be valuable in the management of patients with severe CMV disease.


Protein-tyrosine phosphorylation: an essential component of Fc epsilon RI signaling. Mast cell and basophil activation can be achieved by antigen-mediated aggregation of cell surface Fc epsilon RI molecules. At least two signaling pathways are triggered by this activation. Both involve tyrosine phosphorylation. This aspect of Fc epsilon RI signaling is examined here in detail and its position in a complex network of post-binding events assessed.


Cerebrospinal fluid norepinephrine, 3-methoxy-4-hydroxyphenylglycol and neuropeptide Y levels in Parkinson's disease, multiple system atrophy and dementia of the Alzheimer type. Neuropeptide Y, one of the most abundant polypeptides within the nervous system, is co-stored with catecholamines, especially norepinephrine (NE), thus suggesting its possible involvement in pathologies characterized by a noradrenergic impairment. In Parkinson's disease (PD), as well as in multiple system atrophy (MSA), a central noradrenergic deficit has been demonstrated, and in the dementia of Alzheimer type (DAT) an impaired noradrenergic transmission has been postulated. In this study we determined CSF NE and MHPG levels in 29 PD, 15 MSA, 22 DAT patients and in 36 controls, while CSF NPY-immunoreactivity (NPY-ir) levels were measured in 10 PD, 7 MSA, 10 DAT patients and 20 controls. PD, MSA, and DAT patients showed a significant reduction in CSF NPY-ir and NE levels compared with controls, while CSF MHPG levels resulted in a reduction in only the MSA group. Furthermore, an inverse correlation between either NE or MHPG levels and the duration of the orthostatic hypotension was found in MSA patients while for DAT patients the MHPG levels were directly correlated to the severity of cognitive impairment, and inversely to the duration of illness.


Survey of neuropeptide gene expression in tumor cell lines. The presence of 3 different neuropeptide mRNAs with a strict cell-specific expression in vivo was investigated in 13 tumor cell lines from neuroendocrine and in 23 tumor cell lines from non-neuroendocrine origin. Northern blots showed no expression of mRNA for vasopressin (VP) in the 36 tested cell lines. Very low oxytocin (OT) mRNA hybridization signals were detected in the rat pituitary tumor cell line GH4C2 and the rat pancreas tumor cell line RIN5. Both the rat pituitary tumor cell line AtT-20 and the human myeloid leukemia cell line K562, contained proopiomelanocortin (POMC) mRNA. The low incidence of VP, OT and POMC gene expression in the tested tumor cell lines was not influenced by treatments inducing differentiation. In contrast, the cholecystokinin (CCK) gene which is widely present in nervous and endocrine systems was abundantly expressed in the human primitive neuroepithelioma cell line SK-N-MC and its clonal derivative SK-N-MC-IX-C. The results indicate that the expression of neuropeptide genes is very rare in tumor cell lines. The lack of expression in undifferentiated cells agrees with the appearance of expression after day 13 of the embryogenesis when maturation of neurons begins.


Expression of the vasopressin and gastrin-releasing peptide genes in small cell lung carcinoma cell lines. Various polypeptide hormones including vasopressin (VP) and gastrin-releasing peptide (GRP) are produced by small cell lung carcinomas (SCLC). VP as well as GRP have mitogenic effects on several cell types and are proposed to be autocrine growth factors. In this study the presence of VP mRNA, oxytocin (OT) mRNA and GRP mRNA was investigated in cell lines derived from SCLCs. Out of 26 cell lines 3 contained low amounts of VP mRNA (GLC-8, SCLC-21H and NCI-H345) and 7 contained abundant GRP mRNA (GLC-16, GLC-1-M13, SCLC-22H, NCI-H249, NCI-H345, NCI-H449 and NCI-H450). The GRP mRNA-containing cell lines belong to the classic SCLC type, whereas VP mRNA was found in two classic and one variant cell line. None of the SCLC cell lines contained detectable levels of OT mRNA. Of the three VP-expressing SCLC cell lines, GLC-8 had the highest level of VP mRNA. Both the length of the transcript and the hybridization with different probes containing exons A and C of the VP gene suggest that the detected transcript is a normal VP messenger. SCLC GLC-8 contained low levels of VP immunoreactivity and VP receptors. In GLC-8 an autocrine role of VP may be suspected.


In vivo modulation of oligodendrocyte function by an anti-receptor antibody. The receptor for reovirus serotype 3 (Reo3R) is biochemically, pharmacologically, and antigenically related to the adrenergic receptors. Previous studies have demonstrated that anti-Reo3R antibodies and Reo3R-binding peptides alter oligodendrocyte differentiation in culture. In the present studies, antibodies and peptides that bind the Reo3R were found to alter myelin morphology in vivo. Microinjection of purified anti-Reo3R antibody into guinea pig optic nerves produced expansion of the adaxonal oligodendrocyte cytoplasm, separation of myelin lamellae, widening of Schmidt-Lanterman clefts, myelin vesiculation, and demyelination. A divalent Reo3R-binding peptide reproduced some of these changes. Anti-Reo3R antibodies and Reo3R-binding peptides alter oligodendrocyte function in vivo resulting in myelin changes. These effects appear to be mediated directly by Reo3R perturbation, at least in part, rather than through activation of additional effector mechanisms.


Purification and characterization of two different thymidine-5'-triphosphosphate-hydrolysing enzymes in human serum. Two different enzymes capable of hydrolysing dTTP to the corresponding diphosphate were purified from human serum in order to investigate their enzymatic properties. A specific dTTPase was purified to apparent homogeneity with a purification factor of ca. 10,000 and showed a molecular mass of 46,000 Da, consisting of two identical subunits. This enzyme revealed an isoelectric point of 5.8 and a Km value of 38 microM. The other enzyme showed substrate specificity for dTTP and dCTP and was purified with a factor of ca. 5,000. It seems to be a multifunctional enzyme of one subunit (96,000 Da) with two different catalytic sites for dTTP and dCTP. The isoelectric point was 5.2, the Km values were 20 microM for dTTP and 17 microM for dCTP, respectively. Both enzymes were sensitive to inorganic phosphate, but the dTTPase to a minor extent. In contrast to the dCTPase-dTTPase, the dTTPase was strongly inhibited by ZnSO4. Physico-chemical and biochemical data suggest the purification of two different enzymes.


Cervical metastatic glioblastoma multiforme. Glioblastoma multiforme is an anaplastic neoplasm of glial origin. In spite of the aggressive histologic features and poor prognosis, metastasis outside the cranial vault is distinctly unusual. A patient with glioblastoma multiforme metastatic to the neck is presented. We also review the topic of metastatic intracranial tumors.


Undifferentiated small-cell neoplasm of the petrous apex. A case report. We present a rare case of undifferentiated small-cell neoplasm involving the temporal bone petrous apex. The symptoms, physical examination, importance of roentgenographic findings, and pathologic findings are reviewed. While not absolutely conclusive, the collective evidence in this case supports a diagnosis of small-cell carcinoma of the lung with metastasis to the petrous apex. A discussion of temporal bone malignancies, their frequencies, and characteristics is included. To our knowledge, a review of the literature over the past 25 years reveals no other published cases of an undifferentiated small-cell carcinoma in the temporal bone.


Structural dependence of diurnal fluctuations of radon progeny in residential buildings. Movement of radon progeny inside houses is a complex process that depends both on atmospheric conditions and on building structure. The indoor working level (WL) monitored in four houses of differing structures shows regular diurnal fluctuations related to solar warming of the atmosphere. In the two houses with full basements, radon is removed by indoor/outdoor pressure-driven airflow, and basement WL varies inversely with outdoor temperature. In the two houses with half basements open to crawl spaces, radon is drawn into the basement faster than it is removed, so that basement WL varies directly with outside temperature. Average WL's in basements are about twice as high as first floor WL's and as much as 18 times as high as outdoor WL's. Each house shows an individual pattern of radon progeny movement throughout the building.


Degree of neutrophil chemotaxis is dependent upon the chemoattractant and barrier. Stimulated neutrophil migration across lung endothelial and epithelial barriers is important in lung inflammatory processes. To better understand the interaction between chemoattractants, neutrophils, and endothelium and epithelium, we compared the ability of leukotriene B4 (LTB4), formylmethionylleucylphenylalanine (FMLP), and platelet-activating factor (PAF) to induce human neutrophil migration across 3-microns-pore filters alone and human umbilical vein endothelial (HUVE) cells and two different epithelial cell types, Madin-Darby canine kidney (MDCK) cells and human lung A549 cells, cultured in monolayers on these filters. LTB4, FMLP, and PAF induced neutrophil migration through naked filters, endothelial cells, and epithelial cells in a dose-related fashion. At optimal chemoattractant doses, LTB4, FMLP, and PAF induced relatively equivalent neutrophil migration through filters and endothelial and epithelial monolayers. However, the doses at which optimal neutrophil migration was observed to occur as well as the degree of neutrophil migration through the three barriers varied depending upon the chemoattractant. Based on dose-response experiments, the relative rank order of potency for the three chemoattractants was: LTB4 = FMLP greater than PAF for filter alone barrier; LTB4 greater than FMLP greater than PAF for HUVE cell barrier; and FMLP greater than LTB4 greater than PAF for MDCK and A549 epithelial cell barriers. Our data suggest that neutrophil chemotactic and subsequent lung inflammatory responses are interrelatedly influenced by both the quantity and type of chemoattractant present and the barrier through which the neutrophil must migrate.


Purification of type I and type II tumor necrosis factor receptors from human lung tissue. Two receptors for tumor necrosis factor-alpha (TNF) were purified from detergent-solubilized human lung tissues by adsorption to TNF-Sepharose, followed by elution with low pH. By SDS-PAGE analysis, the two proteins had molecular weights of 75 and 55 kD. Using a soluble receptor assay, a binding affinity of approximately 1.2 nM was calculated for the isolated lung receptors. Each protein, isolated by electroelution from polyacrylamide gels, specifically bound TNF. Antibodies raised against the mixture of type I and II receptors bound specifically to both purified receptors by immunoblot analysis. Both the 75- and 55-kD receptors could be precipitated from 125I-surface-labeled or 35S-methionine-labeled U937 cells using TNF-Sepharose or anti-receptor antibodies. In addition, the anti-TNF receptor antibodies partially blocked binding of TNF to U937 cells and specifically immunoprecipitated 125I-TNF cross-linked to its receptors on U937 cells. These results demonstrate that both type I and II TNF receptors can be isolated from human lung tissue by ligand affinity chromatography, and that U937 cells express both TNF receptor types.


A technique to harvest viable tracheobronchial epithelial cells from living human donors. The ability to obtain airway epithelial cells from the lower respiratory tract in living human donors will facilitate study of the biologic properties of these cells. We report our experience harvesting tracheobronchial epithelial cells from living human donors by brushing the mucosal surface of the trachea and mainstem bronchi. Cells were obtained on 21 occasions from 18 healthy adult subjects under direct vision with a brush-tipped catheter during fiberoptic bronchoscopy. The average number of cells harvested per subject was 14 +/- 2 x 10(6), and cell viability determined by trypan blue exclusion averaged 36 +/- 4%. Of note, cell viability was significantly enhanced when lidocaine was confined to the nares. Lidocaine was also observed to diminish cell viability in vitro in a dose-dependent fashion. Morphologic and staining properties were used to classify harvested cells into the three major cell types present in the mucosa (i.e., ciliated, secretory, and basal cells). All three subtypes were obtained. The percentage of ciliated, secretory, and basal-like cells was 24 +/- 2%, 11 +/- 1%, 29 +/- 1%, respectively, while the remaining 36% were difficult to type. In one subject in whom brushing was performed on three occasions over a 7-wk period, the percentage of each of the three subtypes was similar across procedures. Harvested cells could be successfully placed in primary culture with a plating efficiency of 50 to 60% and could be subcultured for up to seven passages. Acutely dissociated cells could be used to study the beta-adrenergic receptor adenylyl cyclase system since they produced cAMP in response to isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)


Attenuation of ozone-induced airway permeability in rats by pretreatment with cyclophosphamide, FPL 55712, and indomethacin. Exposure of rats to ozone (O3) produces an increase in airway permeability and a concomitant influx of polymorphonuclear leukocytes in the lung. These observations raise the possibility that the inflammatory cells play a role in the cellular injury and increased airway permeability after O3 exposure. This study was therefore designed to determine if the inflammatory cells or their products are essential for the O3 effect. In a series of experiments, rats were rendered leukopenic with cyclophosphamide, treated with leukotriene B4 (LTB4), or with the inhibitors of lipoxygenase or cyclooxygenase products of arachidonic acid, followed by exposure to O3. A 2-h exposure to 0.8 ppm O3 caused a significant increase in the flux of proteins and albumin in bronchoalveolar lavage (BAL) and elevated the transport of 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA) from trachea to blood. The treatment with cyclophosphamide caused a significant reduction in the circulating and pulmonary leukocytes and prevented an increase in tracheal mucosal permeability to 99mTc-DTPA and the protein and albumin flux in BAL. While the intratracheal instillation of LTB4 did not affect the permeability, tracheal permeability and albumin levels in BAL in rats treated with LTD4 antagonist FPL 55712 and exposed to O3 were lower than in the untreated O3-exposed rats. Pretreatment with indomethacin also prevented the O3 effects, as reflected by the decreased protein and albumin flux in BAL and 99mTc-DTPA transport from trachea to blood. These data show a reduction in the effect of O3 by agents that affect leukocytes or their products. The results support a mechanism of increased permeability that is dependent upon inflammatory cells and their products.


[Peripheral neuropathy and correlation with risk factors in material from autopsies and amputations]. A morphological study of a series of 100 human peroneal nerves proceeding from autopsies and amputations of patients hospitalized for different diseases was performed. A relation between the morphologic findings and the risk factors of neuropathy to which the patients may have been exposed is established. Following the analysis of their results the authors found that peripheral neuropathy is more frequent than suspected in clinical practice. The peripheral nerve is relatively resistant to regional ischemia. The risk factors considered in the casuist included diabetes as that which most influences in determining morphological alterations compatible with neuropathy.


Electrophoretic transfer of DNA from gels to filters by using a homemade apparatus. Electroblotting is a rapid and high-efficiency method of transferring DNA from gels to the variety of membranes available, in very short time frames when compared with capillary-blotting techniques and with much less preparation required. A simple apparatus made of two food savers, one dialysis membrane, one sponge, and two removable electrodes was used to transfer DNA molecules up to 21 kb, from agarose gels to nitrocellulose filters, in less than 30 min. A recombinant pAT153 plasmid carrying the human cytomegalovirus BamH I-P fragment (7.2 kb) was rapidly identified by hybridization, using our simplified transfer procedure.


A single-blind study of the efficacy and safety of intravenous granisetron compared with alizapride plus dexamethasone in the prophylaxis and control of emesis in patients receiving 5-day cytostatic therapy. The Granisetron Study Group. 200 cancer patients who were due to receive fractionated chemotherapy (cisplatin greater than or equal to 15, ifosfamide greater than or equal to 1.2 or etoposide greater than or equal to 120, all mg/m2 per day) for 5 days, entered a multicentre study. Patients were randomised single-blind to receive either prophylactic intravenous granisetron (40 micrograms/kg) or alizapride (4 mg/kg followed by 4 mg/kg at 4 and 8 h post-treatment) plus dexamethasone 8 mg. Granistron was superior to the combination in preventing nausea and vomiting (54% vs. 43% complete responders). The differences were in the cisplatin-treated group. The time to first episode of moderate to severe nausea was significantly longer in the granisetron group (P = 0.03). Dosing with granisetron was more simple, with over 85% of patients requiring only a single prophylactic dose. Fewer patients receiving granisetron experienced adverse events (48% vs. 62%, P = 0.047). The frequency of constipation was, as expected, significantly higher in the granisetron group. Extrapyramidal effects, which were not noted by any granisetron patient, occurred in 5.3% of comparator patients.


Radiation therapy, an important mode of treatment for head and neck chemodectomas. Between 1970 and 1990, 22 patients with 44 chemodectomas in the head and neck region were seen at the Netherlands Cancer Institute in Amsterdam. All patients were treated with radiation therapy (17 patients with radiation therapy only and 5 in combination with surgery). One patient was treated two times with an interval of 12 years at each side of the neck. Standard dose was 50 Gy in 25 fractions over 5 weeks. A radiation portal arrangement with oblique fields with paired wedges was used most frequently. The follow-up period ranged from 1 year to 20 years. Two recurrences at 2 and 9 years after treatment were observed. The actuarial local control rate was 88% at 10 years follow-up. Comparison of the results of surgery and radiotherapy demonstrates that radiation therapy is an effective treatment modality without mutilation or severe late morbidity for chemodectomas in the head and neck region.


Immunocytochemistry and in situ hybridisation of epidermal growth factor receptor and relation to prognostic factors in breast cancer. The breast tumour distribution of epidermal growth factor receptor (EGFR) was studied in 193 patients with primary breast cancer by immunocytochemistry on frozen sections. EGFR was correlated (P = 0.0009) with growth fraction assessed by Ki-67, and negatively correlated with oestrogen receptor (ER, P = 0.0001) and progesterone receptor (PR, P = 0.0001) status. In 47 patients, in-situ hybridisation for EGFR mRNA showed good agreement with the immunocytochemically assessed EGFR protein. There were, however, several tumours in which EGFR mRNA could be detected in the absence of EGFR protein and there were differences between the ER and PR status of those tumours in which translation of EGFR mRNA was not seen. The cause of these differences is unclear, but these findings may represent a clue as to the differential control of breast cancer cell receptors.


Increased myelosuppression during cytostatic treatment and pleural effusion in patients with small cell lung cancer. 30 patients with small cell lung cancer (SCLC) and malignant pleural effusion were compared with 30 matched patients with SCLC but without pleural effusion. In the 30 with pleural effusion, white blood cell and platelet counts fell significantly after initial chemotherapy, necessitating dose reduction. Of the patients with pleural effusion, 16 developed severe (WHO grade IV) leukopenia, 7 had severe thrombocytopenia, and 2 patients died of infection. Accordingly, exhaustive aspiration of radiologically verified pleural effusion before starting chemotherapy in patients with SCLC is recommended.


Immunohistochemical and biochemical characterisation of the expression of a human embryonal carcinoma cell proteoglycan antigen in human germ cell tumours and other tissues. In the embryonal carcinoma (EC) cell line GCT 27, monoclonal antibody GCTM-2 recognises an epitope on a 200 kD pericellular matrix keratan sulphate proteoglycan. Immunohistochemical analyses demonstrated staining of tissue sections from 21 out of 22 human non-seminomatous germ cell tumours, and from 22 out of 28 sections of seminomas. In normal human fetal tissues gut epithelium and muscle stained strongly, and certain other epithelia stained moderately. In adult tissues, the distribution of the epitope was similar, but staining intensity was weaker. Neoplastic tissues showed reactivity with embryonal rhabdomyosarcoma and colorectal carcinoma, but no other non-germ cell tumours. Immunofluorescence microscopy showed that GCTM-2 also stained cell lines from human colorectal carcinoma, embryonal rhabdomyosarcoma and choriocarcinoma. In contrast to EC cells the epitope in these other cell types required permeabilization of the cells to be visualised, and the protein bands in immunoblots lacked extensive modification with keratan sulphate and were smaller. Thus, GCTM-2 reacts with an epitope which has a previously unrecognised tissue distribution; its expression as a pericellular matrix proteoglycan is predominantly a characteristic of human EC cells.


Fatty acid composition of normal and malignant cells and cytotoxicity of stearic, oleic and sterculic acids in vitro. The aim of this study was to investigate the hypothesis that saturated fatty acids are differentially cytotoxic to cancer cells. Three studies were undertaken to: (1) measure the toxicities of stearic and oleic acids to normal and malignant cells in vitro, (2) assess if there is any relationship between toxicity and relative fatty acid composition and (3) determine whether the relative fatty acid composition of a cancer cell line could be modified by sterculic acid, an inhibitor of delta-9-desaturase. Stearic (18:0) and oleic (18:1) acids inhibited the colony-forming abilities of five human cancer cell lines and two non-neoplastic cell lines in a dose-dependent fashion. The concentration of oleic acid required to reduce colony formation ability by 50% was 2.5-6.0-fold greater than that of stearic acid. Addition of sterculic acid to a cancer cell line resulted in steady-state levels of stearic acid and increasing percentage of oleic acid.


A comparison of granisetron as a single agent with conventional combination antiemetic therapies in the treatment of cytostatic-induced emesis. The Granisetron Study Group. The safety and efficacy of intravenous granisetron were compared with combinations of conventional antiemetics in two single-blind, parallel-group studies which have been reported previously. In this review updated data from both studies is presented. In both studies granisetron (40 micrograms/kg) was given as a single 5-min infusion before chemotherapy with two additional doses allowed to control subsequent nausea and vomiting. All patients were naive to chemotherapy. Patients due to receive cisplatin (greater than 49 mg/m2) were randomly assigned to receive either granisetron alone or metoclopramide (3 mg/kg) plus dexamethasone (12 mg) given prophylactically followed by an 8-h infusion of metoclopramide (4 mg/kg). In the 24 h after the start of chemotherapy 70% of granisetron-treated patients and 67% of comparator group were complete responders. In patients due to receive moderately emetogenic chemotherapy, granisetron was compared with chlorpromazine (up to 200 mg/24 h) plus dexamethasone (12 mg). Twenty-four hour efficacy was significantly higher in the granisetron group with complete response in 68% of patients compared to 47% in the comparator group (P less than 0.001). A subset of 40 patients in this study were crossed over to receive the alternative antiemetic on their next cycle of chemotherapy. A significant majority of patients (32/34; 94%) preferred granisetron (P less than 0.001). Around 80% of the granisetron-treated patients in both groups required only a single prophylactic dose of granisetron. Following the first additional dose of granisetron, around 87% of patients reported symptoms to be improved or resolved. Adverse experience reporting was higher in the comparator groups with somnolence and extrapyramidal reactions representing the most common events. Headache was the most commonly reported adverse experience in granisetron-treated patients. Granisetron has proved safe and effective in controlling chemotherapy-induced emesis and is more convenient to administer than conventional antiemetics.


Dose granisetron remain effective over multiple cycles? The Granisetron Study Group. Intravenous (i.v.) granisetron was made available to patients who had received the drug during their first course of chemotherapy and requested granisetron prophylaxis at subsequent cycles. Initially at each further cycle patients received 40 micrograms/kg in each further cycle; this was later simplified to 3 mg. 574 patients were treated with 40 micrograms/kg over 1966 cycles of emetogenic chemotherapy in study 1. 335 patients (81 of whom transferred from study 1) received 3 mg over 785 cycles in study 2. With either regimen about 60% of all patients gained complete protection of symptoms over the 24-h postchemotherapy for up to 8 cycles. Complete response was maintained at around 70% in the subgroup of patients treated with moderately emetogenic regimens. Efficacy decreased over 5 cycles of cisplatin (greater than or equal to 50 mg/m2) from approximately 59% at the first additional cycle to around 37% at the fifth. This could, in part, be explained by a reversal in the proportions of males (low risk) to females (high risk) during the study. Withdrawal was largely due to completion of chemotherapy courses; approximately 15% of patients discontinued treatment for reasons possibly related to poor emetic control and 10% for unspecified reasons. Granisetron was well tolerated and no new toxicity developed following repeated exposure. In conclusion, granisetron maintained its efficacy over repeated cycles in most patients, although some fall-off occurred with high-dose cisplatin. 40 micrograms/kg and 3 mg were equally effective.


Are all 5-HT3 receptor antagonists the same? A number of 5-HT3 receptor antagonists are currently in clinical development as antiemetics. In this paper we focus on two of these antagonists, granisetron and ondansetron, and compare their antimetic activity against cisplatin (10 mg/kg i.v.)- or whole body X-irradiation (200 rads)-induced emesis in the conscious ferret. The results presented here have been discussed in the light of the recently published literature. Our data suggest that in comparison to ondansetron, granisetron is a more potent, longer acting and pharmacologically "cleaner" compound with a more conventional dose-response profile. The possible impact of these features upon the performance of these compounds in the clinic is discussed particularly with respect to dosing regimens and clinical efficacy. Differences appear to be emerging between granisetron and ondansetron in both these respects, although a direct head-to-head clinical comparison has yet to be carried out. This would involve studies monitoring a sufficiently high number of patients receiving severely emetogenic regimes to allow real clinical differences to be detected with the appropriate statistical power.


Fractionated chemotherapy--granisetron or conventional antiemetics? The Granisetron Study Group. Two randomised single-blind comparative studies were carried out in patients receiving 5-day fractionated chemotherapy. The first which has been reported previously [1] compared granisetron (40 micrograms/kg) (n = 103) with alizapride (12 mg/kg) plus dexamethasone (8 mg) (n = 94) while the second compared granisetron (40 micrograms/kg) (n = 143) with metoclopramide (7 mg/kg) plus dexamethasone (12 mg) (n = 141). Granisetron, unlike alizapride or metoclopramide is a specific 5-HT3 antagonist. The percentage of complete responders (patients with no vomiting and no worse than mild nausea) over the 5-day treatment period was higher for granisetron than for alizapride/dexamethasone (54% vs. 42.7%) (P = 0.121) or for metoclopramide/dexamethasone (46.8% vs. 43.9%). The percentage of complete responders in the first 24 h was significantly higher for granisetron (90.3%) than for alizapride/dexamethasone (65.9%) (P less than 0.001) or for metoclopramide/dexamethasone (87.4% vs. 67.9% P less than 0.0001). Granisetron was also superior to both comparators in terms of the time to the first episode of moderate/severe nausea and to less than a complete response. Significantly fewer granisetron patients were withdrawn than in the alizapride/dexamethasone group (P = 0.017) or the metoclopramide/dexamethasone group (P less than 0.0001). In both studies more comparator patients were withdrawn due to lack of efficacy and adverse events. Significantly fewer granisetron patients experienced adverse events than in either the alizapride/dexamethasone group (47.6% vs. 61.7%, P = 0.047) or the metoclopramide/dexamethasone group (60.8% vs. 77.3% P = 0.003). Granisetron patients experienced a significantly higher occurrence of constipation in both studies (10.7% vs. 3.2% and 12.6% vs. 2.8%). Headache and fever were also more frequent in the granisetron group, while extrapyramidal effects, reported by 5.3% of the alizapride/dexamethsone group and 20.6% of the metoclopramide/dexamethasone group, were not reported in any granisetron patients.


Oral granisetron--simple and effective: a preliminary report. The Granisetron Study Group. This randomised, double-blind, study was carried out in 930 patients in order to examine the efficacy and safety of oral granisetron in the dose range 0.25, 0.5, 1.0 and 2.0 mg twice daily. Oral granisetron was administered for either 7 or 14 days according to the chemotherapy regimen used. A total of 930 patients were enrolled into this study in order to be able to detect a difference of 15% between groups (80% power). The preliminary results showed that at 7 days efficacy was significantly greater for 1.0 mg b.d. (58.5%) than for 0.25 mg b.d. (43.7%) and indicated that, of the doses examined, the 1.0 mg b.d. dose was optimal in patients receiving moderately emetogenic chemotherapy. In agreement with this there were more withdrawals from the 0.25 and 0.5 mg groups due to lack of efficacy. The adverse events most frequently reported (in greater than 5% of patients) were constipation, headache, abdominal pain, fever, leucopenia and asthenia. The latter three are recognised side effects of the primary disease and of chemotherapy. The incidence of headache was similar to that seen in previous granisetron studies. Abdominal pain may have been related to constipation. The incidence of constipation (25.9%) was higher than that reported in previous granisetron studies but was not dose related. Thus oral granisetron at 1.0 mg twice daily is an effective antiemetic, offering a convenient dosing regimen without significant adverse events.


Are granisetron and ondansetron equivalent in the clinic? There are no published direct trials of granisetron vs. ondansetron. Difficulties exist in comparing reported trials because of differences in methodology, especially in response criteria. In this review, a comparison is made between ondansetron and granisetron by recalculating the complete response criterion for granisetron, standardising it against that in the ondansetron programme (i.e. no vomiting). Weighted means have been calculated for three areas of study. Against cisplatin-induced emesis the (weighted) mean percentage of complete responders were calculated at 64% (range 49-77%) for granisetron and 49% (range 40-55%) for ondansetron. Against moderately emetogenic stimuli, the response rates were 76% (range 68-80%) and 73% (range 60-87%) respectively. For fractionated chemotherapy the response rates were 57% and 27% for granisetron and ondansetron respectively. Although not shown by formal statistical analysis, these results suggest that a clinical advantage for granisetron may exist.


Intravenous granisetron--establishing the optimal dose. The Granisetron Study Group. Three double-blind, dose-ranging studies, involving 996 chemotherapy-naive patients, were conducted to determine the optimal prophylactic dose of intravenous (i.v.) granisetron for prevention of cytotoxic-induced emesis. The antiemetic efficacy of prophylactic i.v. granisetron doses ranging from 2-40 micrograms/kg (study 1) and 40-160 micrograms/kg (study 2) were examined in patients receiving high-dose cisplatin regimens. In study 3, i.v. doses of 40 and 160 micrograms/kg were compared in patients receiving other emetogenic cytotoxic therapies. In study 1, 67.9% (36/53) of patients were complete responders at 24 h following the 40 micrograms/kg dose compared with 61.5% (32/52) and 30.8% (16/52) in the 10 and 2 micrograms/kg groups, respectively (40 vs. 2 micrograms/kg; P less than 0.001). There were no significant differences between doses of 40 and 160 micrograms/kg in any efficacy parameter in Studies 2 and 3. Granisetron was well tolerated across the dose range examined and no dose-related toxicity was observed. In conclusion, a single 40 micrograms/kg prophylactic dose provides optimal control of cytotoxic-induced nausea and vomiting. A simple 3 mg single-dose i.v. regimen (equivalent to 40 micrograms/kg in a 75 kg person) is recommended for prevention of acute emesis associated with all cytotoxic regimens.


The Scottish trial of adjuvant tamoxifen in node-negative breast cancer. Scottish Cancer Trials Breast Group. This paper presents results, 5 years after closure, of a randomized trial comparing adjuvant with postrelapse tamoxifen in 747 women with histological node-negative breast cancer. Two hundred thirty-six disease-free patients on adjuvant therapy were secondarily randomly assigned at 5 years to stop or to continue tamoxifen until relapse. Adjuvant tamoxifen, taken for a median duration of 60 months, has significantly prolonged disease-free survival overall (P = .0001), in the 214 premenopausal group (P = .018), in the 533 postmenopausal group (P = .0026), and for the 246 patients with estrogen receptor levels greater than 19 fmol/mg of protein (P = .0032); distant relapse has also been delayed overall (P = .029). Total survival comparison by Kaplan-Meier life-table analysis shows a nonsignificant trend in the same direction (P = .07). Adjuvant tamoxifen has also reduced the incidence of contralateral breast cancer and of death from myocardial infarction. No increase in the incidence of endometrial cancer has been found.


Continuous culture of neuronal cells from adult human olfactory epithelium. Cells from the olfactory epithelium of adult human cadavers have been propagated in primary culture and subsequently cloned. These cells exhibit neuronal properties including: neuron-specific enolase, olfactory marker protein, neurofilaments, and growth-associated protein 43. Simultaneously, the cells exhibit nonneuronal properties such as glial fibrillary acidic protein and keratin, the latter suggesting properties of neuroblasts or stem cells. These clonal cultures contain 5-10% of cells sufficiently differentiated to show odorant-dependent cyclic adenosine 3',5'-monophosphate (cAMP) or calcium-release responses when challenged with submicromolar concentrations of odorants. The potential of culturing neuronal cells from patients with neuropsychiatric disorders, such as Alzheimer's disease or schizophrenia, could enable the study of the pathophysiology of these neurons in the culture dish and allow new approaches to the study of mental illness.


Actions of neomycin on neuronal L-, N-, and non-L/non-N-type voltage-sensitive calcium channel responses. The effects of neomycin on neuronal voltage-sensitive calcium channel (VSCC) responses were investigated by evaluating its effects on calcium-dependent neuronal responses that are sensitive and insensitive to the N-type voltage-sensitive calcium channel antagonist omega-conotoxin GVIA and the L-type VSCC antagonist nitrendipine. Chick synaptosomal 45Ca2+ influx and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices were omega-conotoxin GVIA sensitive and nitrendipine insensitive, suggesting that these responses are mediated predominantly by N-type VSCC. The K(+)-evoked increase of intracellular calcium in cortical neurons and the K(+)-evoked release of [3H]norepinephrine from rat brain cortical slices was partially sensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses are mediated by N-, L- and non-L/non-N-type VSCC. Rat synaptosomal 45Ca2+ influx and the K(+)-evoked release of [3H]D-aspartate from rat hippocampal slices were completely insensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses were mediated predominantly by non-L/non-N-type VSCC. Neomycin caused a concentration-dependent and virtually complete inhibition of all response parameters, with IC50 values ranging from 90 to 400 microM. The results suggest that neomycin is a nonselective inhibitor of neuronal responses mediated by L-, N-, and non-L/non-N-type VSCC.


Pleomorphic adenoma of the lacrimal gland. To reduce the risk of recurrence and malignant transformation, pleomorphic adenomas of the lacrimal gland should be removed intact, without prior biopsy. Seventy one of the 78 patients in this series were referred without previous surgery, and, on clinical or radiological evidence, 63 (89%) tumours were correctly diagnosed and totally excised, with preservation of a margin of the surrounding normal tissue. Sixty three patients had tumour within the body of the gland and 55 (84%) had radiological signs or satisfied clinical criteria for pleomorphic adenoma that we suggested previously; that is, over 1 year of symptoms and absence of pain. Eight (16%) orbital lobe tumours were misdiagnosed preoperatively and biopsied; in all these patients symptoms had been present for less than 1 year, and four patients had pain. Eight patients with tumours arising in the palpebral lobe had a short history of an upper lid mass, and their tumours were excised without biopsy. With the surgical techniques described in this paper, there has been no recurrence of tumour in patients with follow-up as long as 21 years. A modification of surgical technique, with preservation of the palpebral lobe of the gland, has reduced the incidence of postoperative dry eye and distortion of the upper eyelid.


Primary malignant neoplasms of the lacrimal gland. The clinical characteristics and outcome of 50 primary malignant neoplasms of the lacrimal gland are reviewed: 38 (76%) adenoid cystic carcinomas, six (12%) carcinomas arising in pleomorphic adenoma, and six (12%) adenocarcinomas or other types of carcinoma. Most patients presented with a short history and pain, though pain tended to occur less often and later with adenocarcinoma than with adenoid cystic carcinoma. Pain was unrelated to the duration of symptoms, invasion of bone, loss of trigeminal nerve function, or the frequency and time of tumour recurrence. The estimated disease-free survival for patients with adenoid cystic carcinoma was significantly (p less than 0.01) reduced where half or more of the biopsy specimen showed basaloid differentiation. Eleven patients underwent extended cranio-orbital resection, and the others received a combination of total dacryoadenectomy adenectomy and/or radiotherapy. Survival after adenoid cystic carcinomas appears to be significantly (p less than 0.05) greater when tumour resection is combined with radiotherapy than after radiotherapy alone. At present, however, the rate of disease-free survival after treatment of adenoid cystic carcinoma appears unaltered by cranio-orbital resection, though these latter patients form a relatively greater proportion of those surviving for more than 10 years. Further long-term follow-up is needed to see if this technique does influence survival.


Non-traumatic acquisition of herpes simplex virus infection through the eye. Primary ocular herpes is usually seen as a follicular conjunctivitis and blepharitis, with or without involvement of the cornea. It is unknown, however, to what extent asymptomatic and/or subclinical primary disease occurs, and whether primary ocular herpes follows direct droplet spread to the eye. Previous models of murine ocular herpes have used trauma (scarification) to introduce virus into the cornea, producing disease which results in significant corneal scarring. To mimic a likely route of infection in humans, a droplet containing virus was placed on the mouse eye and clinical disease recorded. At least 1 month after inoculation, serum was assayed for neutralising antibodies and the cornea, iris, and trigeminal ganglion were investigated for evidence of herpes simplex virus type 1, by cocultivation and the polymerase chain reaction. Some animals showed a severe ulcerative blepharitis with little to no involvement of the cornea, while disease was undetectable in others. The development of disease depended on the dose and strain of virus and age of the animal, with older mice appearing more resistant. Virus was isolated from the trigeminal ganglion of younger animals inoculated with higher doses of virus, after 21 days in culture, suggesting that latency had been established. Neutralising antibodies were present in most mice irrespective of the presence of recognisable clinical disease. Using primers for the thymidine kinase and glycoprotein C regions of the viral genome, herpes simplex virus type 1 DNA was found in the cornea, iris, and trigeminal ganglion of most animals and showed a good correlation with the presence of neutralising antibodies. It would thus appear that herpes simplex virus type 1 is able to accede into the cornea, iris, and trigeminal ganglion following nontraumatic application of virus onto the mouse eye. This model mimics primary ocular disease in humans and may be useful for studies on recurrent disease and the spread of ocular herpes.


Spectroscopic characterization of triplet forming states in photosystem II. Fluorescence and electron paramagnetic resonance (EPR) measurements have been applied to characterize chlorophyll triplet formation in the reaction center of photosystem II (PSII). A highly triplet forming state was generated in PSII membranes by chemical double reduction of the primary electron acceptor QA. In triplet forming PSII centers, the steady-state yield of chlorophyll fluorescence decreased to about 70% of the maximal fluorescence yield observed in closed PSII centers in which QA is singly reduced. The results are well interpreted in the framework of a model where the charge state of QA electrostatically controls the yield of primary charge separation [Schatz, G. H., Brock, H., & Holzwarth, A. R. (1988) Biophys. J. 54, 397-405]. Thus, high triplet yield and decreased, although still quite high, fluorescence indicate a charge-neutralized state of PSII in which QA is singly or doubly reduced and protonated or absent. The EPR signal of the triplet primary chlorophyll donor, 3P680, is suppressed by illumination at 77 K concomitant with the formation of a cationic radical (g = 2.0025-2.0027, and 0.92 mT wide) that is stable in the dark. This is attributed to the oxidation of an accessory chlorophyll (Chl) in the vicinity of P680. Electrostatic repulsion between Chl+ and P680+ is likely to prevent primary charge separation, and in turn triplet formation, providing a further example of electrostatic control of primary charge separation. The triplet P680 EPR signal is also suppressed in the presence of oxygen. This effect, which is almost completely reversible by removing the oxygen, is attributed to the interaction of triplet P680 with triplet O2.


Redox titrations of carbon monoxide dehydrogenase from Clostridium thermoaceticum. Redox titrations of carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum were performed using the reductant CO and the oxidant thionin. Titrations were followed at 420 nm, a wavelength sensitive to redox changes of the iron-sulfur clusters in the enzyme. When CODH was oxidized by just enough thionin to maximize A420, two molecules of CO per mole of CODH dimer (4 equiv/mol) reduced the enzyme fully. Likewise, 4 equiv/mol of thionin oxidized the fully-reduced enzyme to the point where A420 maximized. The four n = 1 redox sites which titrated in this region were designated group I sites. They include at least two iron-sulfur clusters, [Fe/S]A and [Fe/S]B, and two other sites, A' and B'. The [Fe4S4]2+/1+ cluster in CODH is included in this group. [Fe/S]B and B' have reduction potentials (at pH 8) below -480 mV vs NHE; [Fe/S]A and A' have reduction potentials above that value. The reduction potential of either [Fe/S]B or B' is near to the CO/CO2 couple at pH 8 (-622 mV). When CODH was oxidized by more than enough thionin to maximize A420, some of the excess thionin oxidized the so-called group II redox sites. These sites have reduction potentials more positive than group I and do not exhibit changes at 420 nm when titrated. Titration of group II sites required 1-2 equiv/mol. EPR of reduced group II sites exhibited the gav = 1.82 signal. When these sites were oxidized, the only signal present had g values at 2.075, 2.036, and 1.983.(ABSTRACT TRUNCATED AT 250 WORDS)


Vesicle fusion in protein transport through the Golgi in vitro does not involve long-lived prefusion intermediates. A reassessment of the kinetics of transport as measured by glycosylation. The well-characterized cell-free assay measuring protein transport between compartments of the Golgi [Balch, W. E., Dunphy, W. G., Braell, W. A., & Rothman, J. E. (1984) Cell 39, 405-416] utilizes glycosylation of a glycoprotein to mark movement of that protein from one Golgi compartment to the next. Glycosylation had been thought to occur immediately after vesicles carrying the glycoprotein fuse with their transport target. Therefore, the kinetics of glycosylation were taken to reflect the kinetics of vesicle fusion. We previously isolated and raised monoclonal antibodies against a protein (the prefusion operating protein, POP) which is required in this assay at a step after vesicles have apparently been formed and interacted with the target membranes, but long before glycosylation takes place. This was therefore presumed to be a reaction involving targeted but unfused vesicles. Here we report that POP is identical to uridine monophosphokinase, as revealed by molecular cloning. We show that POP is not active in transport per se but instead enhances the glycosylation used to mark transport. This indicated that, contrary to previous assumptions, glycosylation might lag significantly behind vesicle fusion. We directly show this to be true. This alters the interpretation of several earlier studies. In particular, the previously reported existence of a late, prefusion intermediate, the "NEM-resistant intermediate", can be seen to be due to effects on glycosylation and not indicative of true fusion events.


Essential groups in synthetic agonist peptides for activation of the platelet thrombin receptor. Thrombin appears to activate platelets by a novel mechanism that involves the cleavage of its receptor, and it has been proposed that the newly generated N-terminal region of the receptor then acts as a tethered ligand [Vu, T. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. Peptides with sequences corresponding to those of the tethered ligand are capable of activating the receptor. In the present study, groups within this tethered ligand peptide that are important for activation of the receptor have been identified by synthesizing a series of peptides. A 14-residue peptide based on the tethered ligand stimulated the aggregation of gel-filtered platelets with an EC50 of 7 microM, and a concentration of 10 microM was the minimum concentration necessary to yield a full aggregation response in platelet-rich plasma. Truncation of the peptide from the C-terminus to nine residues did not markedly affect the response to the peptide. Shorter peptides of five, six, and eight amino acids retained their agonist activity, but the minimal concentration necessary to achieve a full aggregation response in platelet-rich plasma was 2-5-fold higher. Side chains within the tethered ligand peptide that are important for receptor activation were identified by synthesizing a series of peptides in which residues were sequentially replaced by alanine. The results indicated that the side chains of phenylalanine, leucine, and arginine in positions 2, 4, and 5, respectively, are essential for full activity. Most notably, substitution of phenylalanine in the second position resulted in complete loss of agonist activity at concentrations up to 800 microM.(ABSTRACT TRUNCATED AT 250 WORDS)


Probing structural factors stabilizing antisense oligonucleotide duplexes: NMR studies of a DNA.DNA duplex containing a formacetal linkage. The duplex formed by annealing the formacetal backbone modified dodecamer d-(CGCGTTOCH2OTTGCGC) to its complementary strand, d(GCGCAAAACGCG) (duplex I), has been studied by NMR techniques and analyzed with reference to its unmodified counterpart (duplex II). Comparison of parameters such as 2D cross-peak intensities, coupling constants, and spectral patterns indicates that structural perturbations caused by the incorporation of the formacetal linkage are minimal and localized to the central T4.A4 block. Duplex I adopts a B-type helical conformation with regular Watson-Crick base pairing and normal minor groove width. The methylene group is accommodated along the phosphate backbone in a conformation similar to that of the PO2 group found in the B-form DNA family. The central T6-T7 base pairs of duplex I melt simultaneously with the duplex, indicating a cooperative transition to single strands. Although the formacetal linkage affects global melting, as evidenced by a 3 degree C reduction in Tm for duplex I with respect to duplex II, the present study indicates that this is not the result of localized premelting at the formacetal site of duplex I but rather reflects the subtle interplay of several structural and energy factors which need to be further explored.


Binding of oligopeptides to d-AGATCTAGATCT and d-AAGCTTAAGCTT: can tryptophan intercalate in DNA hairpins? The interactions of three tryptophan-containing peptides, KWK, KGWK tert-butyl ester, and KGWGK, with two self-complementary dodecamers of the same base composition but different sequence were studied by UV, CD, and fluorescence spectroscopy. The oligonucleotides, d-AGATCTAGATCT and d-AAGCTTAAGCTT, contain tandem repeats of the recognition site for the restriction enzyme BglII in the former and HindIII in the latter. Thermal transition data in dilute solutions and in 0.01 M NaCl indicate these dodecamers to be present in hairpin forms. Binding of peptides to these hairpins was followed by tryptophan fluorescence quenching titrations at 10 mM Na+; the data suggest intercalation of the indole ring. The association constants for the peptide-oligonucleotide (PN) complexes are an order of magnitude higher (10(5) M) than those reported with polynucleotides [10(4) M; Rajeswari et al. (1987) Biochemistry 26, 6825]. The pentapeptide, KGWGK, discriminates between BglII and HindIII sequences with higher affinity for the HindIII dodecamer. The CD maximum of KGWGK, at 220 nm, is drastically diminished upon interaction with oligonucleotides. The ellipticity at 220 nm is halved at 10 times less P/N ratio with the HindIII dodecamer than the BglII dodecamer, suggesting stronger binding to the HindIII dodecamer. The results are discussed in terms of two different modes of binding of oligopeptides to the DNA hairpins.


Bifunctional intercalator [N-MeCys3,N-MeCys7]TANDEM binds to the dinucleotide TpA. The binding of [N-MeCys3,N-MeCys7]TANDEM has been examined by DNase I footprinting and diethyl pyrocarbonate modification of several synthetic DNA fragments containing AT-rich regions. DNase I footprinting reveals that at low concentrations the ligand binds preferentially to the center of (AT)n regions. A fragment containing the tetranucleotide AATT was unaffected by the ligand. Diethyl pyrocarbonate modification of several fragments containing blocks of (AT)n revealed a pattern in which alternate adenines were rendered more reactive in the presence of the ligand. These reactive adenines were staggered across the two DNA strands in the 3'-direction, consistent with ligand binding to the dinucleotide TpA. In sequences of the type (TAA)n.(TTA)n, binding of [N-MeCys3,N-MeCys7]TANDEM resulted in strong modification of the second adenine in the sequence TAA, i.e., the base on the 3'-side of the ligand binding site. Data for binding to (AT)n are best explained by suggesting that the adenines sandwiched between the quinoxaline chromophores are rendered most reactive to diethyl pyrocarbonate.


Pulsed EPR studies of the type 2 copper binding site in the mercury derivative of laccase. The nuclear modulation effect in pulsed EPR spectroscopy was used to study the type 2 copper binding site in the mercury derivative of laccase (MDL) in which the type 1 copper is substituted by Hg(II). By comparing the three-pulse electron spin-echo modulations and Fourier transform spectra of MDL and several model compounds, we conclude that the imidazole groups of two histidyl amino acid residues are equatorially coordinated to Cu(II) in the type 2 site. Computer simulations of these data suggest that the remote nonbonding nitrogens of the two imidazoles possess nuclear quadrupole parameters e2qQ = 1.47 MHz and eta = 0.83. A(iso) values of these two nitrogens are not identical, being 1.5 and 2.0 MHz. We have also used samples of the enzyme exchanged with D2O to examine the coordination of the water to the type 2 copper site. The deuterium modulation that is resolved by taking the ratio of the time domain ESEEM data from native and D2O-exchanged enzyme indicates that there is an equatorial water ligand, and further data show that this water is displaced by azide.