The care of people over 75 years old after discharge from hospital: an evaluation of timetabled visiting by Health Visitor Assistants. The objective of this study was to evaluate a programme of timetabled visiting by Health Visitor Assistants (HVAs) to patients over 75 years old who were recently discharged from hospital. An outcome evaluation examined whether patients benefited in measured health status and use of services over the year of the programme. A randomly selected group of patients (the intervention group) who were allocated to the programme of visiting were compared with an equal-sized randomly selected group of patients (the control group) who had no timetabled visiting. A process evaluation examined the actions taken by HVAs during their visits and related the actions taken to patients' measured health status and other characteristics. No overall benefit from the programme of visiting was found in the outcome evaluation. There was wide variation in the numbers of actions recorded for different patients. Numbers of HVAs' actions were related to patient's health status and sex, with more actions being initiated for those in poorer health and women. Neither age nor whether the patient lived alone were found to be related to numbers of HVAs' actions. It was concluded that the lack of demonstrated overall benefit and the wide variation in actions taken on patients' behalf suggest that this type of service cannot be recommended for all discharged patients over 75 years. Poor health at the time of discharge is recommended as a first choice of measure for need of support. Exchange of information between general practice and hospital about previous dependency may aid the identification of patients who need post-discharge support.


Home from hospital: a survey of hospital discharge arrangements in Northamptonshire. The timeliness and adequacy of inpatient discharge communication between hospitals and general practitioners (GPs) in Northamptonshire was examined by a postal questionnaire survey of GPs of patients recently discharged from hospital, with the aim of improving the co-ordination of discharge procedures, and hence improving continuity of care. The questionnaire measured when and how the GP was informed of the discharge, and examined the adequacy of medical, therapeutic and social details in the discharge documents sent out by the hospital. It was found that 67 per cent of discharges had been notified to the GP by the hospital within five days of discharge. With notable exceptions the discharge documents were considered timely. General practitioners were less satisfied with the adequacy of discharge communication in terms of 'social' topics such as transport needs, social services back-up, and whether a patient with a malignancy knew about his or her diagnosis. The GPs of patients under geriatricians were more satisfied with the quality of discharge documents. Comparison with an earlier study suggested that the speed of communication and involvement of GPs in discharge in Northamptonshire is not as satisfactory as that found in Oxford in 1986. It was concluded that within the county there appear to be models of good practice in terms of discharge communication with GPs. These standards should be adopted by other specialties to match or improve on existing good practice.


[Malignant tumor upon pleomorphic adenoma of long development, a case report]. We have studied a 67 years old woman suffering from a 40 years evolution Pleomorphic adenoma. The specimen weighed 250 g, and showed several areas displaying carcinoma. This report confirms the relationship between long-term evolution and malignant transformation.


Molecular pathogenesis of HIV-associated lymphomas. The data presented here indicate that the pathogenesis of AIDS-NHL is variably associated with multiple genetic alterations including monoclonal EBV infection, oncogene activation (c-myc, N-, Ki-ras) and tumor suppressor gene (p53) inactivation. Up to three (3 cases) or four (1 case) different lesions have been observed in the same tumor. The distribution of these lesions among the various histotypes is heterogeneous, although some preferential associations have been found either between lesion and histotype or between lesions. The most notable case involves p53 mutations/loss that is exclusively associated with the SNCC lymphoma subtype. Since alterations of the c-myc gene occur at very high frequency in this same histotype it is possible that both lesions may be required for the pathogenesis of the BL phenotype. The consistent negativity of p53 lesions in other NHLs associated or non associated with HIV infection (18) reinforces this hypothesis. Finally, we note that the frequency of p53 mutations is significantly higher in AIDS-BL than in non HIV-related BL (18), although the significancy of this difference remains to be assessed. This study confirms the relatively low frequency of EBV infection in systemic AIDS-NHL in general, but reinforces the notion that EBV may be required for the pathogenesis of AIDS-LC-IBP, as recently suggested by the high frequency of EBV positivity in primary CNS AIDS-NHL which are mostly represented by LC-IBP (2). Conversely, the low frequency of EBV sequences in the AIDS-SNCC lymphomas appears similar to that observed in sBL. Only in a small minority of cases were ras oncogene mutations found, mostly associated with the BL type.(ABSTRACT TRUNCATED AT 250 WORDS)


EBV-induced human B cell lymphomas in hu-PBL-SCID mice. Epstein-Barr virus (EBV) infection is associated with Burkitt's lymphoma (BL) in normal individuals and immunoblastic B cell lymphomas in immunosuppressed or HIV-infected individuals. SCID mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID) from EBV-seropositive donors also may develop spontaneous B cell lymphomas which histologically and phenotypically resemble post-transplant tumors, and are distinct from BL. These tumors always contain EBV DNA. We have noted three different reproducible outcomes depending upon the EBV-seropositive donor used for generation of hu-PBL-SCID mice: (i) no tumors appear; (ii) tumors appear in a fraction of hu-PBL-SCID mice with a 10-20 wk. latent period; or (iii) tumors appear in all hu-PBL-SCID mice within 6-10 wk. Southern blot analysis of late versus early tumors using a probe specific for the EBV terminal repeat sequences (BamNJ), which allows distinction between circular latent and linear replicating genomes, shows that late tumors do not involve active EBV replication but that early tumors do show replicating genomes. In addition, EBV genomes were monoclonal in late tumors but polyclonal in early tumors. These data suggest two mechanisms for EBV lymphomagenesis, slow outgrowth of rare latently-infected B cells, and more rapid transformation of uninfected bystander B cells by replicating virus. The latter process may be highly amenable to therapy in patients at risk for EBV-related lymphomas. In addition, prospective screening of EBV-seropositive transplant recipients in the hu-PBL-SCID model may predict the risk of post-transplant lymphoma development.


Bronchiolo-alveolar carcinoma: an analysis of survival predictors. Macroscopic and microscopic features of tumours have been analysed in 37 bronchiolo-alveolar carcinomas. Lymphocytes, Langerhans cells, collagen (mature and/or myofibroblastic), were quantitatively or semiquantitatively evaluated. Histology, stage, type of fibrosis, nuclear profile features (area and shape factors), amount and type of mucin secreted, number of mitoses, Langerhans cells, myofibroblasts and LeuM1+ cells were not related to survival. Gross morphology of the tumour and, to a lesser extent, lymphoid infiltrates (in particular UCHL1+ and L26+ peritumoral lymphoid cells) were the only variables significantly related to survival. Estimated survival functions were computed according to Cox's model: well demarcated tumours behaved significantly better than poorly demarcated tumours and even more so than diffuse or multiple mass. Lymphoid infiltrates were significantly more represented in and around well demarcated tumours: however, their survival predicting value was less than that of the gross type.


A pilot study of photodynamic therapy in patients with inoperable non-small cell lung cancer. 26 patients with inoperable non-small cell lung cancer (NSCLC) were treated with photodynamic therapy (PDT) with intravenous Photofrin II 2 mg/kg. 10 out of 11 stage I patients achieved a complete response. The remaining patient, and 11 out of 15 stage III patients had a partial response. No response was seen in 4 patients, 2 of whom had inadequate illumination. Thus, the objective response rate was 85% (22/26). Although lung function did not improve, dyspnoea was ameliorated in 7 (58%) of the partial responders. 4 stage III patients had tumour progression and died of pulmonary haemorrhages 1.5-6 months after PDT. All had received external irradiation, Nd-YAG laser and/or brachytherapy before PDT. 4 patients had grade I-II skin photosensitivity. Although of value in stage I NSCLC, the clinical benefit of PDT in stage III disease was small.


Malignant mesothelioma: clinical characteristics, asbestos mineralogy and chromosomal abnormalities of 41 patients. The clinical characteristics and the results of mineral fibre and cytogenetic analyses were coordinated prospectively for 41 patients with confirmed malignant pleural mesothelioma. A correlation was found between high total fibre concentration, and partial or total loss of chromosomes 1, 4 and 9 and chromosomal rearrangements involving a breakpoint at 1p11-p22. There was also a correlation between crocidolite/amosite as the main fibre type and partial or total loss of chromosomes 1, 3 and 4 and chromosomal rearrangements involving del (3p). Positive prognostic factors were female gender, low total fibre concentration (less than 10(6) fibres per g dried lung tissue), anthophyllite as the main fibre type and normal chromosome 7. In addition, we found 4 patients with malignant mesothelioma who had been exposed mainly to anthophyllite fibres (total lung fibre concentrations of 1.2, 0.4, 0.2 and 0.1 x 10(6) fibres per g dried lung tissue). This would seem to indicate that there may be a carcinogenic role for anthophyllite.


Natural interferon alfa as maintenance therapy for small cell lung cancer. We performed a 3-armed phase III study between 1982 and 1990 to evaluate low dose natural interferon alfa (nIFN-alpha) as a maintenance therapy in small cell lung cancer (SCLC) following induction chemotherapy (CT) and consolidation radiotherapy (RT). All patients received four cycles of CT (cyclophosphamide, vincristine, etoposide), followed by split-course RT (55 Gy in 20 fractions over 7 weeks). 410 patients entered the study. 237 patients who completed induction CT + RT and were classified as responders (complete response + partial response) were randomly assigned to arm 1: low dose nIFN-alpha (91 patients); arm 2: maintenance CT, six cycles of CAP (cyclophosphamide, doxorubicin, cisplatin) (59 patients); or arm 3: control arm (no maintenance treatment) (87 patients). Halfway through the study the CAP arm was discontinued. There was no difference in median survival between the groups (IFN: 11 months, CAP: 11 months, control: 10 months), but a clear difference in long-term survival and in survival in the limited disease group, favouring nIFN-alpha maintenance therapy. Proportional hazards regression analysis also showed a significant effect of IFN treatment on survival. Our results suggest a role for nIFN-alpha in maintaining a clinically disease-free status achieved with other treatment modalities.


Modulation by D,L-buthionine-S,R-sulphoximine of etoposide cytotoxicity on human non-small cell lung, ovarian and breast carcinoma cell lines. Treatment with 25 mumol/l D,L-buthionine-S,R-sulphoximine (BSO) for at least 24 h depleted glutathione (GSH) in human non-small cell lung (SW-1573), ovarian (A2780) and breast carcinoma (MCF-7) cell lines to about 20% of control, and was accompanied by a 2-fold potentiation of the cytotoxicity of etoposide, doxorubicin and cisplatin. Cellular etoposide, but not doxorubicin or cisplatin, concentrations were increased 2-fold due to decreased efflux. This occurred independently of the presence of BSO during 1 h of etoposide exposure, but required prolonged exposure to BSO (at least 24 h). Energy depletion as well as cotreatment, but not pretreatment, of the cells with daunomycin, doxorubicin, vinblastine or vincristine increased cellular etoposide accumulation. Treatment of control cells with verapamil caused similar changes in etoposide cytotoxicity and cellular pharmacokinetics as GSH depletion, but did not further increase etoposide cytotoxicity and accumulation in GSH-depleted cells. Etoposide efflux may have been inhibited, not because of (competitive) inhibition by BSO or disturbance of the energy required for this process, but probably because of plasma membrane alterations.


Production of chromogranin A and B derived peptides in human small cell lung carcinoma cell lines. Production of chromogranin (Cg)A and B derived peptides [pancreastatin (PST), GAWK, CCB] was studied using human lung carcinoma derived cell lines. PST-like immunoreactivity (LI) was detected in the culture medium in 3 of 6 small cell lung carcinoma (SCLC) cell lines, while GAWK- and CCB-LIs were detected in 5 of 6 and all the 6 SCLC cell lines, respectively. CCB-LI was produced in large amounts in SCLC cell lines as compared to PST- and GAWK-LIs. In non-SCLC cell lines, on the other hand, PST- and GAWK-LIs were not detected. CCB-LI was detected in 1 of 7 non-SCLC cell lines, but not detected in the remainder. PST, GAWK and CCB-LIs, secreted by these cell lines, consisted of several peaks, and these peaks were different among cell lines. This suggests that processing of CgA and B is different in the cell lines. Production of CgA and B derived peptides seems to be a characteristic feature of SCLC, and among them, CCB LI may be a useful marker for SCLC.


The effective charge number and diffusion coefficient of cationic cytochrome c in aqueous solution. The diffusion coefficient and the effective charge number of cytochrome c as a function of ionic strength, temperature and pH have been measured. The measurements were carried out using a method based on a convective diffusion process across a porous membrane. The effect of ionic strength was studied in an NaCl solution the concentration of which varied from 0.001 to 1.0 M. The temperature range studied was 10-50 degrees C, and the pH values studied were 4.0, 6.5 and 8.25. The diffusion coefficient is fairly constant as a function of ionic strength and pH, and Walden's rule is valid in the temperature range studied. The effective charge number is practically constant (ca. 2) in the concentration range studied, except in 0.001 M solution, where it is the same as the titrated value. The charge number decreases slightly in the temperature range 10-30 degrees C, but seems to drop suddenly to zero at ca. 40 degrees C. Measurements using heavy water (D2O) as a solvent instead of water did not give zero charge at 40 degrees C for cytochrome c.


The mortality of amphibole miners in South Africa, 1946-80. A cohort was established in 1981 of all 7317 white male employees in the amosite and crocidolite mines in South Africa whose names had appeared in the personnel records (initiated between 1945 and 1955) of the major companies. Some of the men had been employed as early as 1925, but only 8% had had more than 10 years of service. Three subcohorts were defined: 3212 men whose only exposure to asbestos was to amosite; 3430 exposed to crocidolite; and 675 to both amphiboles. No deaths or losses to view occurred before 1946, and 5925 men (81%) were known to be alive at the end of 1980. Losses to view numbered 167 (2%), and there had been 1225 deaths (17%), an excess of 331 over the number of deaths expected on the basis of the mortality of all white South African males. The fibre related excesses were of mesothelioma, lung cancer, and other respiratory diseases, but there were other excesses perhaps mainly related to socioeconomic factors including lifestyle. When cause of death was determined according to "best evidence" (after study of clinical, radiological, biopsy, and necropsy reports in conjunction with the death certificate), there were 30 deaths due to mesothelioma (22 pleural, six peritoneal, two other) and 65 due to cancer of trachea, bronchus, and lung. Various analyses of these deaths showed that crocidolite had higher toxicity than amosite for lung cancer and this was most pronounced for mesothelioma; there can now be no question that crocidolite is far more dangerous than amosite at least in so far as mesothelioma is concerned. Nevertheless, crocidolite induced mesothelioma appeared only in men who had been exposed for long periods, at least 12 months, but on average about 15 years.


Constitutive expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor abrogates all requirements for exogenous growth factors. BALB/c3T3 cells are exquisitely growth regulated and require both platelet-derived growth factor and insulin-like growth factor-1 (IGF-1) for optimal proliferation. BALB/c3T3 cells that constitutively express IGF-1 and elevated levels of IGF-1 receptor (IGF-1R) are capable of growth in serum-free medium without the addition of any exogenous growth factors. BALB/c3T3 cells overexpressing only the IGF-1R plasmid required IGF-1 or insulin for serum-free growth. Antisense oligodeoxynucleotides complementary to IGF-1R mRNA inhibited IGF-1-mediated cell growth. Under these conditions, neither the epidermal growth factor receptor nor phospholipase C gamma 1 was autophosphorylated. These findings indicate that constitutive expression of IGF-1 and IGF-1R allows 3T3 cells to grow in serum-free medium without addition of those exogenous growth factors that are required by the parent cell line.


Interleukin 1 alpha mediated inhibition of myogenic terminal differentiation: increased sensitivity of Ha-ras transformed cultures. The commitment of myogenically determined cells to terminal differentiation can be modulated by a variety of agents, including growth factors and activated oncogenes. We have examined the effect of interleukin 1 alpha (IL-1 alpha) on the terminal differentiation of a normal myogenically determined cell line and two myogenically determined, differentiation competent cell lines which contain either one or six copies of the activated c-Ha-ras oncogene. Treatment of all cell lines with IL-1 alpha decreased but did not totally inhibit terminal myogenic differentiation. Over the range of IL-1 alpha concentrations assayed (1-40 ng/ml), the c-Ha-ras transformed cell lines demonstrated a significantly greater sensitivity to the inhibitory effects of IL-1 alpha. The inhibition of differentiation was not the result of enhanced proliferation. Interestingly, transformation with activated c-Ha-ras resulted in a decrease in IL-1 alpha receptor number and affinity. The enhanced IL-1 alpha responsiveness of the ras transformants was not the result of increased proliferation or changes in either ras gene expression or protein kinase C activity. IL-1 alpha treatment decreased the steady-state levels of both MyoD1 and myogenin transcripts in the c-Ha-ras transformed but not the normal myogenic cell line. Further studies are required to determine the mechanism(s) responsible for the increased sensitivity of the c-Ha-ras transformed cultures to the inhibitory effects of IL-1 alpha.


Transposing without ends: the non-LTR retrotransposable elements. Transposable elements have been discovered in animals, plants, fungi, and protozoans which contain open reading frames similar to the gag and pol genes of retroviruses and retrotransposons but which lack long terminal repeats (LTRs). Recent experiments have shown that these non-LTR elements [also called poly(A) type and LINE-like elements] encode functional reverse transcriptase and replicate via an RNA intermediate. Based on phylogenetic analysis of their encoded reverse transcriptase sequences, the non-LTR retrotransposons are the likely progenitors of retroviruses and LTR retrotransposons. Because retroviruses and LTR retrotransposons depend upon their LTRs for key steps in both transcription and integration, the mechanisms utilized by the non-LTR retrotransposons must be fundamentally different. Internal promoter sequences have been found in several non-LTR elements that initiate transcription upstream at the first nucleotide. Current models for retrotransposition of non-LTR elements propose that the 3' ends of staggered nicks at the chromosomal insertion site serve as primers for first- and second-staggered nicks at the chromosomal insertion site serve as primers for first- and second-strand synthesis from the RNA template. These models suggest that the enzymatic machinery of non-LTR elements is likely to be responsible for the integration of SINEs and processed pseudogenes.


Multiple short direct repeats associated with single mtDNA deletions. We have sequenced the breakpoints of deleted mtDNA in muscle from four children with mitochondrial myopathy and multisystem mitochondrial disorders. The deletions were 4884, 6067, 7663 and 7150 base pairs (bp) in size and affected several protein and transfer RNA genes. The sequences needed for transcription and replication of mtDNA were not affected in any case. The deletions were flanked by direct short repeats in all cases. Multiple repeats were found in case 1 and 4. Imperfect repeats were found in case 3 and 4 and this made it possible to distinguish the repeats 5' and 3' to the deletion. In both cases the 3' repeat was retained. The deletion of 7663 bp in case 3 has been reported in two other cases and may represent a second hotspot for mtDNA deletions in addition to the common deletion of 4977 bp found in one third of cases. A comparison of the breakpoint sequence of case 3 with the two other reported cases revealed that when a deletion is formed between the same repeats in different patients either the 5' or 3' repeat can be retained. This study shows that both single and multiple repeats can be associated with single mtDNA deletions and that both 5' and 3' repeated sequences can be retained. These findings are consistent with the slip-replication model for the generation of mtDNA deletions.


Distinguishing between infection, rejection, and the adult respiratory distress syndrome after human lung transplantation. The adult respiratory distress syndrome, bacterial pneumonia, cytomegalovirus pneumonitis, acute rejection, or a combination thereof were the primary causes of radiographic infiltrates or gas exchange abnormalities that occurred early after lung transplantation. The time of occurrence after transplantation, standard measures of clinical assessment as for nontransplant patients (i.e., vital signs, weight, white blood cell count, sputum, and cultures, etc.), bronchoalveolar lavage, and transbronchial lung biopsy were the primary tools used to analyze these situations. Bacterial pneumonia always occurred after postoperative day 2, acute rejection after postoperative day 5, and cytomegalovirus pneumonitis after postoperative day 16. Although cultures of bronchoalveolar lavage fluid were useful to detect pneumonia caused by bacteria, virus, and fungus, the types of cells recovered by bronchoalveolar lavage were not diagnostic of any type of disorder. Transbronchial lung biopsy was necessary to detect acute rejection and cytomegalovirus pneumonitis. Thus the cause of an early radiographic infiltrate or impairment of gas exchange was almost always reliably determined by using standard tools of clinical assessment, knowledge of the usual temporal sequence of the complications, and judicious use of bronchoalveolar lavage and transbronchial lung biopsy.


Competitive ELISA for serodiagnosis of bluetongue: evaluation of group-specific monoclonal antibodies and expressed VP7 antigen. The performance of 2 competitive enzyme-linked immunosorbent assays (C-ELISA) was compared with the reference C-ELISA I for the detection of antibodies to bluetongue virus (BTV). One of the assays (C-ELISA II) used a group-specific monoclonal antibody (MAb) to BTV, obtained from the American Type Culture Collection (8A3B-6) and tissue culture (TC)-derived BTV antigen (Ag), and the other assay (C-ELISA III) used BTV core protein VP7 (expressed in yeast) and the reference MAb (Pirbright Laboratory, 3-17-A3). Test sera were obtained by sequential blood samples from 22 calves, each inoculated with a different serotype (T) of BTV (South African [SA] T-1-T-16 and T-18-T-20 and USA T-11, T-13, and T-17). Sera were also obtained from 4 calves and 4 sheep inoculated with USA BTV T-10 and from several groups of calves exposed to single or multiple doses of epizootic hemorrhagic disease virus (EHDV) T-1-T-4 grown in TC (BHK-21) or suckling mouse brain (SMB). A total of 618 bovine and ovine field sera collected from BT-free and BT-endemic areas were also tested. The C-ELISA III was more sensitive than the C-ELISA II in the detection of anti-BTV antibody in sera from cattle and sheep early after infection with BTV. Seroconversion was demonstrated by the 3 C-ELISAs in all animals inoculated with BTV by 20 days postinfection (DPI), except in calves that received SA T-3 or USA T-13, which became positive at 40 DPI.(ABSTRACT TRUNCATED AT 250 WORDS)


Evaluation of the sensitivity and specificity of two diagnostic tests for antibodies to pseudorabies virus glycoprotein X. The diagnostic performance of 2 enzyme-linked immunosorbent assays (gX-T, gX-H) for antibodies to pseudorabies virus (PRV) glycoprotein X (gX) were evaluated using 311 serum samples from a nonvaccinated quarantined herd. When the standardized virus neutralization (VN) test, which uses the Shope strain (VN Shope), was used as the comparative diagnostic standard, the gX-T test had a 7% false-negative rate and a 52% false-positive rate, and the gX-H test had a 19% false-negative rate and a 19% false-positive rate. When the VN test with a Bartha recombinant strain (VN Bartha gIIIKa) was used as the diagnostic standard, the gX-T test had a 9% false-negative rate and a 26% false-positive rate, and the gX-H test had a 24% false-negative rate and a 11% false-positive rate. Thus, the gX-T test was more sensitive and the gX-H test was more specific. Additional diagnostic tests on 79 serum samples from a noninfected herd did not produce false positives for the gX-H test, but there was an 8% false-positive rate for the gX-T test. Previous studies from our laboratory have demonstrated that VN Bartha gIIIKa has higher sensitivity than VN Shope, without losing specificity, and thus is a better comparative diagnostic standard. When adding a suspect range to the gX-T test, using the same criteria as the suspect range for the gX-H test, the false-positive rate of the gX-T test was reduced to 5% when evaluated versus VN Bartha gIIIKa in the infected herd and to 1% for the PRV-negative herd.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of the virulence of two isolates of porcine parvovirus in 72-day-old porcine fetuses. Two strains of porcine parvovirus (PPV), designated Kresse and NADL-8, were compared for relative virulence in porcine fetuses. Strain Kresse was injected into the amniotic fluid of all fetuses of 1 uterine horn of each of 2 pregnant gilts at 72 days of gestation. Strain NADL-8 was administered similarly to fetuses of 4 other gilts at the same stage of gestation. All gilts were killed and necropsied 35 days later. Selected tissues of all fetuses were tested for infectious virus and viral antigen. Sera from live fetuses were tested for antibody to PPV. These tests confirmed that most fetuses exposed to PPV by intra-amniotic injection became infected. All of 11 fetuses exposed to strain Kresse by intra-amniotic injection were alive at the time of necropsy, and all appeared clinically normal. In contrast, 8 of 24 fetuses exposed similarly to strain NADL-8 were dead. Many of the fetuses from the uterine horns contralateral to the uterine horns inoculated with virus were infected after 72 days of gestational age by intrauterine spread of the virus. Four such fetuses, 3 infected with the NADL-8 strain and 1 infected with the Kresse strain, were dead at the time of necropsy. These findings were inconsistent with those of a previous report, which indicated that the Kresse strain of PPV was markedly more virulent than the NADL-8 strain of PPV for porcine fetuses. A possible reason for this apparent discrepancy is discussed.


Foot-and-mouth disease virus typing by complement fixation and enzyme-linked immunosorbent assay using monovalent and polyvalent antisera. An indirect "sandwich" enzyme-linked immunosorbent assay (ELISA) using polyvalent and monovalent antisera was compared with the 50% complement fixation (CF50) test for the detection of foot-and-mouth disease (FMD) O, A, and C virus types. ELISA was more sensitive than CF50 tests when polyvalent antisera were used for detecting the 3 types of virus in epithelial samples, whereas ELISA using monovalent antisera was the least sensitive technique. The ELISA performed with polyvalent antisera was 9 times more sensitive for detecting FMD virus than that with monovalent antisera. However, viral isolation in cell culture was the most sensitive detection system. The combined use of ELISA with polyvalent antisera and cell culture inoculations was the most effective procedure for identifying FMD virus in epithelial samples from the field.


Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alcohol. Application to modification at N-terminal serine. The 2-amino alcohol structure -CH(NH2)CH(OH)- exists in proteins and peptides in N-terminal Ser or Thr and in hydroxylysine. Its very rapid oxidation by periodate at pH 7 generates an aldehyde in the peptide and is the first step in a method for site-directed labeling with biotin or a fluorescent reporter. The modifying group is a hydrazide, RCONHNH2, which reacts with the new aldehyde to form a hydrazone-peptide conjugate, RCONHN = CH-peptide. Experiments with two synthetic peptides, Ser-Ile-Gly-Ser-Leu-Ala-Lys and Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly, and with recombinant murine interleukin-1 alpha (an 18-kDa cytokine with N-terminal Ser) demonstrated this method of peptide tagging. The use of a low molar ratio of periodate to peptide minimized the potential for side reactions during the oxidation, and the desired oxidation was rapid and highly specific. The hydrazones formed were stable at pH 6-8 for at least 12 h at 22 degrees C, but were labile at more acidic pH values. Potential uses of this method include the attachment of biotin, reporter groups, metal chelating groups, imaging agents, and cytotoxic drugs to peptides.


Forestomach papillomas in flaky skin and steel-Dickie mutant mice. Gastric papillomas were diagnosed in flaky skin (fsn/fsn) and steel-Dickie (Sl/Sld) mutant mice but not littermate controls. Both mutants suffer from severe anemia of differing causes. Immunohistochemical screening and Southern blot analyses failed to detect any evidence of a papillomavirus in the gastric lesions. Phenotypic expression of the fsn and Sld mutant genes may play an essential role in the spontaneous development of forestomach papillomas in these mouse mutants.


Alpha-MSH peptides inhibit acute inflammation induced in mice by rIL-1 beta, rIL-6, rTNF-alpha and endogenous pyrogen but not that caused by LTB4, PAF and rIL-8. The neuropeptide alpha-melanocyte stimulating hormone [alpha-MSH(1-13)] occurs in the pituitary, brain, skin and other tissues and receptors for this molecule are likewise widespread. In previous research, this tridecapeptide, which shares its amino acid sequence with ACTH(1-13), was shown to have both potent antipyretic activity and a role in the endogenous control of the febrile response. alpha-MSH(1-13) and its COOH-terminal tripeptide were subsequently found to inhibit inflammation induced by general stimuli such as topical application of an irritant. The aim in the present experiments was to determine if these peptides can inhibit acute inflammatory responses induced in mice by injection of individual cytokines, endogenous pyrogen (EP), a natural cytokine mixture, and other mediators of inflammation. Inflammation induced in the mouse ear by rIL-1 beta, rIL-6 or rTNF-alpha was inhibited by alpha-MSH and a D-valine-substituted analog of alpha-MSH(11-13) whereas substantial doses of alpha-MSH(1-13) did not alter inflammation induced by LTB4, PAF and IL-8. Both peptides inhibited edema caused in the mouse paw by local injection of EP. The results indicate that alpha-MSH molecules antagonize the actions of certain cytokine mediators of inflammation, consistent with previous observations of anti-cytokine activity of these peptides. Failure to inhibit edema caused by LTB4, PAF and IL-8 suggests that, in inflammation induced by general stimuli, such as EP, the peptides act prior to the release of these mediators of the inflammatory response. Because of the anticytokine/anti-inflammatory actions of the alpha-MSH molecules they may be useful in understanding the cytokine network and for treatment of inflammatory diseases.


Cerebellar long-term potentiation under suppressed postsynaptic Ca2+ activity. To study the roles of postsynaptic Ca2+ activity in cerebellar synaptic plasticity, we used a patch-recording technique in Purkinje cell dendrites. While the combination of parallel fibre stimulation and 8-bromo cyclic guanosine monophosphate (Br-cGMP) application produced long-term depression (LTD) of the parallel fibre/Purkinje cell transmission, the same stimuli evoked long-term potentiation (LTP) during postsynaptic injection of Ethyleneglycol-bis-(beta-amino-ethylether)-N, N, N', N'-tetraacetate (EGTA). Furthermore, in the presence of alpha-aminobutyric acid (GABA), parallel fibre stimulation plus Br-cGMP produced LTP of extracellular K+ increases following parallel fibre stimulation. These results suggest that postsynaptic Ca2+ activity in Purkinje cells is negatively correlated to the direction of plastic changes and that the Ca2+ changes and cGMP play distinct roles in cerebellar synaptic plasticity.


Prevalence of hepatitis C virus antibodies in chronic liver disease and hepatocellular carcinoma patients in India. The prevalence of antibodies to hepatitis C virus (HCV) was investigated in 129 patients with chronic liver disease (85 with chronic active hepatitis and 44 with cirrhosis) and 53 patients with hepatocellular carcinoma. The commercially available second generation anti-HCV enzyme immunoassay kit was used. Antibodies to hepatitis C virus were detected in 16.2% of the patients with chronic liver disease and in 15.1% with hepatocellular carcinoma. Of the HCV positive patients in all groups 51.7% were positive for hepatitis B virus (HBV) markers indicating present or past infection. Prevalence of HBV markers in all the three groups (CAH, cirrhosis and HCC) was higher as compared with anti-HCV prevalence. These results suggest that HCV infection may not be a major cause of chronic liver disease and hepatocellular carcinoma in India and indicate the presence of other aetiological agents.


TGF beta 1 expression is initiated in adult auditory neurons by sectioning of the auditory nerve. Neuronotrophic factors (e.g. basic fibroblast growth factor, bFGF and nerve growth factor, NGF) have been demonstrated to respectively promote survival and neuritogenesis in cultures of dissociated adult rat spiral ganglia. Transforming growth factor beta (TGF beta 1) has been shown to modulate the response of cultured auditory neurons to bFGF through the induction of high affinity receptors for bFGF in the neurons. In this study, we show that TGF beta is expressed in situ by adult auditory neurons in response to traumatic injury (i.e. transection of the eighth cranial nerve). Based on these in vivo results and on the results from our previous in vitro studies, we propose that TFG beta 1 acts as an early autocrine signal involved in the response to injury by neurons of the peripheral auditor system.


Decreased interleukin-2 receptor beta chain expression by peripheral blood lymphocytes in chronic liver disease. To investigate the decrease in natural killer (NK) activity in chronic liver disease, interleukin-2 receptor beta chain (IL-2R beta) expression was assessed by peripheral blood lymphocytes (PBL) using flow cytometry and an IL-2R beta chain-specific mouse monoclonal antibody. The percentage of IL-2R beta chain-positive PBL was significantly decreased in patients with chronic viral hepatitis, liver cirrhosis and hepatocellular carcinoma in comparison with normal controls (P less than 0.01). Among chronic viral hepatitis patients, it was significantly less in those with chronic active hepatitis than in those with chronic persistent hepatitis (P less than 0.05). Two-colour flow cytometry revealed that the IL-2R beta chain was mainly expressed by CD8+ or CD16+ cells in both the controls and the liver disease patients. CD8dull+ cells (NK cells) constituted more than 60% of the CD8+ cells expressing the IL-2R beta chain. Expression of the IL-2R beta chain with CD8 or CD16 was also significantly decreased in chronic liver disease patients compared with controls. In chronic viral hepatitis, there was a significant correlation between NK activity and the percentage of IL-2R beta+ PBL (P less than 0.001, r = 0.916), as well as between NK activity and the percentage of PBL co-expressing both the IL-2R beta chain and CD16 (P less than 0.001, r = 0.850). These findings suggest that decreased expression of the IL-2R beta chain by PBL may result in diminished NK activity in chronic liver disease.


Glycinergic transmission influences the development of dendrite shape. We are interested in the trophic influence of inhibitory synaptic transmission during neuronal maturation. The morphology of dendrites in the gerbil lateral superior olive (LSO) was examined following pharmacological blockade of glycine receptors during postnatal development. The normally occurring change in dendrite morphology during the third postnatal week was prevented. Dendrites had a significantly greater number of branch points, and their arbors were more spread out along the frequency axis in the region of LSO where glycine receptor density is greatest. An identical result has recently been obtained when the glycinergic projection to the LSO was functionally denervated during development. We conclude that glycinergic transmission modulates the maturation of dendrite form during development.


Presynaptic long-term changes in excitability of the corticostriatal pathway. We employed measurements of striatal terminal excitability to monitor the presynaptic effects of tetanic stimulation of corticostriatal fibers. Cortical tetanic stimulation (CTS) initiated a long-lasting decrease in terminal excitability. With higher current CTS, a transient increase in excitability preceded the decrease. However, a long-term increase was induced (1) by a second tetanus applied during the brief elevation in excitability initiated by a previous CTS or (2) when dopamine and GABA transmission were disrupted. A long-term increase also occurred following tetanic stimulation of the striatal terminal field (STS). The direction of the long-lasting change in excitability may depend on the level of polarization of the membrane. These presynaptic mechanisms could be important for the long-term selective modification of striatal synaptic transmission.


CRH-mediated pituitary-adrenal responses are inhibited by nifedipine in humans. Sustained CRH-stimulated ACTH release in vitro depends on Ca2+ influx and is inhibited 30-40%, but not delayed, by dihydropyridine Ca2+ channel blockers. In five normal humans, we found that nifedipine pretreatment reduced integrated ACTH responses to the CRH-mediated stimulus of fenfluramine by 28% and cortisol responses by 34%, results comparable with those from in vitro reports. Nifedipine did not alter the timing of peak hormonal responses. We conclude that (1) in humans, nifedipine inhibits ACTH release by fenfluramine by blocking Ca2+ influx via L-type channels in corticotrophs; (2) the magnitude of fenfluramine-stimulated CRH release is probably unaltered by nifedipine and (3) because the timing is unaltered, nifedipine does not affect the rate of CRH delivery to the corticotroph.


Promoter methylation and progressive transgene inactivation in Arabidopsis. Agrobacterium-transformed Arabidopsis plants were generated and the stability of their T-DNA-encoded resistance to kanamycin was examined. Of seven families, each homozygous for a single insertion event, two showed progressive inactivation of resistance over four generations of inbreeding. Loss of resistance was associated with methylation of an Sst II site in the nos promoter of the kanamycin resistance gene. Treatment of plant roots from inactive lines with the demethylating agent 5-azacytidine restored the ability of such lines to form callus on kanamycin-containing media. These observations are consistent with the view that methylation is a factor in the progressive inactivation of transgenes in Arabidopsis.


Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacterium Anabaena sp. strain PCC 7120. Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacterium Anabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of the Anabaena ADPGlc PPase gene and its expression in Escherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48,347 Da which is in agreement with the molecular mass determined by SDS-PAGE for the Anabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and the E. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in the Anabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in an E. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the native Anabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be the Anabaena enzyme.


Structural heterogeneity in the R173 family of rye-specific repetitive DNA sequences. The rye-specific R173 family of repeated DNA sequences consists of ca. 15,000 individual copies per diploid rye (Secale cereale) genome and is distributed over all 7 rye chromosomes in a dispersed manner. Individual R173 elements vary in size between 3 and 6 kb, are generally not arranged as tandem repeats and are flanked by both multi-copy and single-copy sequences. DNA sequence analysis of three R173 elements (R173-1, R173-2 and R173-3) demonstrated a high degree of homology in conserved domains. The structure of R173-1 was quite different from the other two elements: long direct repeats, which represent a rye-specific repetitive sequence, were found at the ends and a 600 bp long domain was replaced by an unrelated sequence of approximately equal size. R173-2 and R173-3 were extremely similar to each other with the exception of a terminal truncation of R173-2. No open reading frames for proteins greater than 20 kDa were present and a database search failed to detect significant homologies to published protein sequences. Despite the transposon like genomic organisation of the R173 family, individual elements lacked sequence features frequently associated with transposons and retrotransposons. In contrast, two of the regions flanking R173 elements showed strong DNA homologies to a 850 bp long region of a proposed wheat retrotransposon and to a 300 bp long region downstream of the wheat Glu-D1 gene.


Growth factors and cutaneous wound repair. The healing of an adult skin lesion is a well studied but complex affair of some considerable clinical interest. Endogenous growth factors, including the EGF, FGF, PDGF and TGF beta families, are released at the wound site and presumed to be a necessary part of the natural wound healing machinery. Moreover, members of each of these families have been shown to enhance healing if added exogenously to a wound site. In this review we shall briefly discuss what is known about the mechanics and cell biology of adult wound healing, describe the normal cellular source of growth factors during the healing process and, with reference to their known capacities in tissue culture, speculate as to how particular growth factors might be able to enhance healing.


Diverse receptors for fibroblast growth factors. The development and maintenance of multicellular organisms requires a complex interplay between cells in different tissues. Many of the factors mediating cell-cell communication are polypeptides, which were originally identified because of their ability to stimulate cell growth. In addition to growth signalling several of these factors have been observed to modulate cell survival, chemotaxis and differentiation both in vitro and in vivo. Fibroblast growth factors are a good example of polypeptide mitogens eliciting a wide variety of responses depending on the target cell type. Our knowledge of the cell surface receptors mediating the effects of FGFs has recently expanded remarkably. Perhaps not surprisingly, the complexity of the FGF family and FGF induced responses is reflected as diversity and redundancy of the FGF receptors.


Transfer of the ADA gene into human ADA-deficient T lymphocytes reconstitutes specific immune functions. Peripheral blood lymphocytes obtained from a patient affected by adenosine deaminase (ADA) deficiency and severe combined immunodeficiency were infected with a retroviral vector containing two copies of a human ADA minigene, and injected into bg/nu/xid (BNX) immunodeficient mice. Six to 10 weeks after injection, human T cells were cloned from the spleens of recipient animals and analyzed for proliferative potential, T-cell surface markers, expression of ADA activity, integration of retroviral sequences, T-cell receptor (TCR) beta gene rearrangement, and specificity of antigen recognition. Efficient gene transfer and expression restored proliferative potential in vitro and long-term survival in vivo. All clonable human T lymphocytes obtained from the spleen of recipient animals had high levels of vector-derived ADA enzyme activity and showed predominantly the CD4+ phenotype. Retroviral integrations and TCR-beta gene rearrangements demonstrated the presence of a variety of different clones in the spleens of recipient mice. Furthermore, the combined analyses of vector integration and TCR rearrangement provided evidence that a circulating progenitor cell was transduced by the retroviral vector, giving rise to different and functional TCRs. Evaluation of antigen-specificity demonstrated both alloreactive and foreign antigen specific immune responses. These results suggest that restoration of enzyme activity in human ADA-deficient peripheral blood T cells by retroviral-mediated ADA gene transfer allows in vivo survival and reconstitution of specific immune functions. Therefore, retroviral vector-mediated gene transfer into circulating mononuclear cells could be successful not only in maintaining the metabolic homeostasis, but also for the development of a functional immune repertoire. This is a fundamental prerequisite for the usage of genetically engineered peripheral blood lymphocytes for somatic cell gene therapy of ADA deficiency.


Restoration of superoxide generation to a chronic granulomatous disease-derived B-cell line by retrovirus mediated gene transfer. Failure of a superoxide generating system, the NADPH oxidase, present in neutrophils and other phagocytes gives rise to chronic granulomatous disease (CGD), a group of single-gene inherited disorders all characterized by an extreme susceptibility to pyogenic infection, with potentially fatal consequences. About 30% of CGD cases are caused by an autosomally inherited deficiency of a 47-Kd cytoplasmic component of the oxidase (p47-phox). Epstein-Barr virus (EBV) immortalized B-lymphocyte lines established from these CGD patients also express this NADPH oxidase defect and consequently are rendered incapable of generating superoxide on stimulation. We have used a p47-phox-deficient EBV-transformed B-cell line as a recipient for retroviral transfer of a functional p47-phox cDNA. The presence and activity of the retrovirally encoded p47-phox in the transduced cells is demonstrated and we show that this restores their capacity to generate superoxide.


Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation. Thrombomodulin (TM) is a surface glycoprotein that forms a 1:1 complex with thrombin, thereby interacting to form the basis of a major physiologically relevant natural anticoagulant mechanism. Although initially described as a vascular endothelial cell receptor, TM has been reported to be present in several other cells, including megakaryocytes, platelets, monocytes, and several cultured cells. Other investigators have reported that neutrophils (PMN) may play a role in the hemostatic mechanism by supporting transformation of prothrombin to thrombin. To determine whether PMN might contribute further to the regulation of the coagulation system, we have evaluated these cells for the expression of TM. Large numbers of human leukocytes were isolated by standard techniques, and the PMN fraction was extracted and shown to be free of platelets and monocytes. Membrane preparations were affinity purified on an anti-TM-Affigel-10 matrix and the eluted material was examined by Western blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and silver staining. The purified material was identical in apparent molecular weight to TM from human placenta and human umbilical vein endothelial cells (HUVEC). Using a sensitive and specific immunoassay, we estimated that there are a minimum of 5,220 +/- 1,658 molecules of TM per PMN, as compared with more than 50,000 in HUVEC. Northern analysis of RNA from PMN indicates that specific messenger RNA for TM, as identified by a single 3.8-kb band, is identical to that from HUVEC, and thereby confirms that PMN can also synthesize the receptor. Localization of TM in PMN was attempted by immunofluorescence, and the receptor was visualized only in permeabilized PMN, but was not seen on the surface of nonpermeabilized cells. Flow cytometry was also used, and could detect TM in 10% to 15% of nonpermeabilized PMN, whereas the antigen was present in greater than 80% of permeabilized cells. Biologic function of the PMN-derived TM, as tested by thrombin-dependent activation of protein C, was absent. Our results suggest that TM is synthesized by PMN, but under nonstimulated conditions, the protein is largely excluded from the membrane surface, and lacks the ability to promote activation of protein C by thrombin. TM from PMN may provide a further link between inflammation and thrombosis and may also be a significant source of plasma TM.


Human B-cell interleukin-10: B-cell lines derived from patients with acquired immunodeficiency syndrome and Burkitt's lymphoma constitutively secrete large quantities of interleukin-10. A recent addition to the lymphokine network is human IL-10 (hIL-10). This novel lymphokine has striking homology to BCRF1 protein, the product of a previously uncharacterized open-reading frame in the Epstein-Barr virus (EBV) genome. To date, IL-10 expression has been described in several T clones induced with anti-CD3 and phorbol myristate acetate (PMA), in monocytes stimulated with lipopolysaccharide (LPS), and in murine B-cell lymphomas. We sought to determine whether human B cells express hIL-10 and, if so, its relationship to EBV and to other B-cell lymphokines. We studied 21 EBV-positive B-cell lines derived from patients with acquired immunodeficiency syndrome (AIDS) and Burkitt's lymphoma (n = 6), American Burkitt's (n = 3), African Burkitt's (n = 5), and normal lymphoblastoid cell lines (n = 7), in comparison with seven EBV-negative cell lines. All cell lines were activated with the tumor promoters PMA and teleocidin and were studied by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunoadsorbent assay (ELISA). We demonstrated that EBV-positive cell lines derived from patients with American Burkitt's lymphoma, and especially those from patients with AIDS, constitutively express large quantities of hIL-10 by Northern blot analysis and ELISA (range, 3,101 to 25,915 pg/mL), and that both teleocidin and PMA induce hIL-10 in these cell lines. In contrast, six of seven EBV-negative cell lines did not express hIL-10 even by RT-PCR, and hIL-10 was not triggered by PMA or teleocidin. To assure that the 350 bp amplified by PCR was hIL-10 and not BCRF1, we used PCR primers, which do not amplify a fragment from plasmid templates containing BCRF1. Cloning and sequencing of the 350 bp product also demonstrated that B-cell IL-10 is identical to hIL-10 from the T-cell clone B21. Correlation of hIL-10 with other B-cell lymphokines secreted by these B-cell lines demonstrated that hIL-10 secretor cell lines also constitutively secrete or can be induced to secrete IL-6, although to a much lesser amount. Since both lymphokines influence B-cell growth and differentiation, we suggest that hIL-10 may contribute to the polyclonal B-cell activation and hyperglobulinemia seen in AIDS patients. Finally, several reports support the hypothesis that EBV is an important cofactor in the development of human immunodeficiency virus type 1 (HIV-1)-related B-cell lymphomas. Detection of large quantities of hIL-10 in B-cell lines derived from AIDS patients, the close association between EBV and hIL-10 shown in this report, and the ability of BCRF1 to capture hIL-10 activities, make hIL-10/BCRF1 an attractive candidate as a factor causing B-cell growth and immortalization in patients with AIDS and B-cell lymphomas.


The type B receptor for tumor necrosis factor-alpha mediates DNA fragmentation in HL-60 and U937 cells and differentiation in HL-60 cells. Tumor necrosis factor-alpha (TNF) binds to two specific cell surface receptors, types A and B, which are both present on HL-60 and U937 cells, and induces monocytoid differentiation in HL-60 cells and early DNA fragmentation in HL-60 and U937 cells. To further define the receptors' roles, we studied how monoclonal antibodies (MoAbs) against each receptor affected TNF-induced cellular responses. HTR-9, an MoAb against the type B (low affinity, 55 Kd) receptor, reproduced all of these effects in a dose-dependent manner. UTR-1, an MoAb against the type A (high affinity, 75 Kd) receptor, had no effect in saturating doses, but supersaturating doses enhanced DNA fragmentation threefold. TNF and interferon gamma (IFN-gamma) synergistically induced morphologic differentiation and monocytic antigen expression, while the antitype B receptor MoAb was synergistic for morphologic response, but not antigen expression. Our results indicate that (1) the type B receptor mediates some responses to TNF in HL-60 and U937 cells, (2) the type A receptor does not stimulate these responses, (3) the TNF molecule is not necessary for some of these actions, and (4) TNF-induced morphologic changes and surface antigen expression in HL-60 cells may be regulated by separate postreceptor pathways.


Cytomegalovirus antigen detection in peripheral blood leukocytes after allogeneic marrow transplantation. Detection of cytomegalovirus (CMV) antigenemia was compared with shell vial centrifugation cultures for rapid detection of CMV infection. In a prospective study, 59 CMV seropositive patients were monitored weekly during the first 100 days after allogeneic marrow transplantation for virus excretion from urine, throat, and blood and for antigenemia by direct staining of peripheral leukocytes using an antibody pool directed against pp 65. Antigenemia was present in 21 of 22 patients with culture-proven CMV infection and in 3 of 37 without culture-proven CMV infection (sensitivity 95%, specificity 91%). The median time of onset of antigenemia and shell vial cultures was day 47 and 55 after transplant, respectively (P = .0006). Among patients who developed CMV disease without preceding cultures, antigenemia was detected in all patients with CMV pneumonia (N = 6) and in two of three patients with gastrointestinal disease by a median of 10 and 7 days, respectively, before the onset of disease (P = .0002). Levels of antigenemia were significantly higher in patients with disease or viremia than in patients with excretion from urine or throat (P less than .05). Whether the antigenemia assay is more sensitive than rapid culture methods to focus antiviral prophylaxis in marrow transplant patients must be determined in controlled studies.


Non-invasive breast carcinoma.

Non-invasive breast carcinoma. Non-invasive breast cancer is comprised of two distinct entities: lobular carcinoma in-situ (LCIS) and ductal carcinoma in-situ (DCIS). The natural history of each clinical entity is described and a biologic interpretation of the available data is offered. Lobular carcinoma in-situ is considered only a risk marker rather than a precursor for the subsequent development of invasive cancer, so that once the diagnosis is established, further operative intervention is unnecessary and serial follow-up alone is recommended. The treatment of ductal carcinoma in-situ must take into account that breast-preserving therapy is now considered optimal treatment of invasive cancer of the breast, the disease we are trying to prevent. The pitfalls of recommending treatment based on retrospective data is emphasized and the need to support clinical trials designed to determine the optimal therapeutic management of intraductal carcinoma is affirmed.


Value of cytometric analysis for distinction of intraductal carcinoma of the breast. The DNA nuclear content of 83 'pure' intraductal cancers (DCIS), 30 DCIS with an invasive component, 7 intraductal papillomas, 26 atypical hyperplasias, and 5 'hobnail' lesions was determined with a Cell Analysis Systems Image Analyzer (CAS 200). No consistent quantitative alteration of DNA or nuclear grade assessed by routine microscopy was observed for any ductal change diagnostic of DCIS. Fifty-eight percent of all DCIS were considered to be aneuploid, whereas all papillomas and atypical hyperplasias were diploid with normoproliferative S-phase and good nuclear grade. Use of ploidy, S phase, or nuclear grade for the distinction of DCIS and papillomas and atypical hyperplasia is limited, since their estimates would be comparable in 42%, 46%, and 40% of instances respectively. Poor nuclear grade was significantly more frequent in solid than cribriform types of DCIS. There were no significant differences in any parameters in DCIS with and without comedo necrosis. This represents further evidence against recognition of a unique comedo form of DCIS. DNA histograms of nuclei comprising the 'hobnail' lesions were aneuploid. Despite this their biologic nature is uncertain. No consistent differences in DNA content were noted between DCIS with and without an invasive component. This obfuscates insight into the role of DCIS in the development of invasive cancer of the breast.


Immunohistochemical markers in the identification of metastatic breast cancer. A panel of nine monoclonal and polyclonal antibodies were tested regarding specificity for metastatic breast cancer. A hundred metastatic tumors were stained, 50 of breast origin and 50 of other origins. Antibodies used were anti-alpha-lactalbumin, anti-lactoferrin, anti-casein, E29 (Dako-EMA), anti-secretory component, anti-gross cystic disease fluid protein (GCDFP15), BRST1, BRST2, and MC5. Analyses of the results were performed using chi-square and logistic regression. Positivity for MC5, BRST1, BRST2, lactoferrin, EMA, and GCDFP15 was significantly higher in tumors of breast origin than in others (p less than 0.05). Analyses of the whole panel indicated that GCDEP15 and MC5 were the best markers for identification of breast cancer metastases. When both were positive (58% of breast origin cases), the predicted probability of breast origin was 98%, compared to only 5% when both were negative. Comparison of anti-GCDFP15 with BRST2, a monoclonal antibody against the same protein, showed a slightly better sensitivity of the former, and a similar degree of specificity for breast tissue. In conclusion, a panel of antibodies can be used to securely differentiate metastatic breast cancer from other cancers in a large number of metastatic tumors of unknown origin.


Cloning and characterization of a pectate lyase gene from the soft-rotting bacterium Pseudomonas viridiflava. Pseudomonas viridiflava is a soft-rotting pathogen of harvested vegetables that produces an extracellular pectate lyase (PL) responsible for maceration of plant tissue. A pel gene encoding PL was cloned from the genome of strain SJ074 and efficiently expressed in Escherichia coli. After a series of deletion subclonings and analysis by transposon mutagenesis, the pel gene was located in a 1.2-kb PstI-BglII genomic fragment. This fragment appears to contain a promoter at the PstI end required for pel gene expression. The PL produced by pectolytic E. coli clones is identical to those produced by strain SJ074 and by other strains of P. viridiflava in terms of molecular weight (42 kDa) and pI (9.7). A mutant of strain SJ074, designated MEI, which had Tn5 specifically inserted in the pel locus was constructed by site-directed mutagenesis. The MEI mutant produced 70- to 100-fold less PL than the wild type and failed to cause tissue maceration in plants. PL production and soft-rot pathogenicity in MEI and in a Pel- mutant previously isolated from strain SF312 were restored to the wild-type level by complementation in trans with the cloned pel gene. By using the 1.2-kb fragment as a probe, pel homologs were detected in four bacteria that are pathologically unrelated to P. viridiflava. These include three pathovars of P. syringae (pv. lachrymans, pv. phaseolicola, and pv. tabaci) and Xanthomonas campestris pv. malvacearum. No DNA fragments showing homology to pel of P. viridiflava were detected in genomic digests prepared from two strains of soft-rot erwinias.(ABSTRACT TRUNCATED AT 250 WORDS)


Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fiber protein MP70. The crystalline lens is an attractive system to study the biology of intercellular communication; however, the identity of the structural components of gap junctions in the lens has been controversial. We have cloned a novel member of the connexin family of gap junction proteins, Cx50, and have shown that it is likely to correspond to the previously described lens fiber protein MP70. The N-terminal amino acid sequence of MP70 closely matches the sequence predicted by the clone. Cx50 mRNA is detected only in the lens, among the 12 organs tested, and this distribution is indistinguishable from that of MP70 protein. A monoclonal antibody directed against MP70 and an anti-Cx50 antibody produced against a synthetic peptide identify the same proteins on western blots and produce identical patterns of immunofluorescence on frozen sections of rodent lens. We also show that expression of Cx50 in paired Xenopus oocytes induces high levels of voltage-dependent conductance. This indicates that Cx50 is a functional member of the connexin family with unique physiological properties. With the cloning of Cx50, all known participants in gap junction formation between various cell types in the lens are available for study and reconstitution in experimental systems.


Immunological characterization of avian MAP kinases: evidence for nuclear localization. The subcellular distribution and regulation of MAP kinase isoforms in chicken hepatoma DU249 cells was investigated with antibodies directed against peptides patterned after sequences in the mitogen-activated protein (MAP) kinases, sea star p44mpk, and rat p44erk1. MonoQ chromatography of cytosol from these cells afforded the resolution of at least four peaks of myelin basic protein (MBP) phosphotransferase activity, but only one of these (peak II) was stimulated in extracts from phorbol ester-treated cells. A 40- to 41-kDa (p41) doublet on Western blots detected with three different MAP kinase antibodies was coincident with peak II, and it probably corresponded to the avian homolog of p42mapk/erk2. Immunofluorescent studies with DU249 cells and chicken embryo fibroblasts revealed that most of the cross-reactive protein with at least two different MAP kinase antibodies was distributed in the nucleus. Subcellular fractionation studies confirmed a predominantly nuclear localization for p41 MAP kinase. Nocodazole arrest of DU249 cells was exploited for the detection of an M-phase-activated MBP kinase that was resolved from p41 MAP kinase by phenyl-Superose chromatography. Western blotting analysis with antibodies for the cdc2-encoded protein kinase and p13suc1-agarose binding studies allowed positive identification of this MBP kinase as p34cdc2.


Cloning and heterologous expression of the phenazine biosynthetic locus from Pseudomonas aureofaciens 30-84. Pseudomonas aureofaciens strain 30-84 suppresses take-all disease of wheat caused by Gaeumannomyces graminis var. tritici. Three antibiotics, phenazine-1-carboxylic acid, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine, were responsible for disease suppression. Tn5-induced mutants deficient in production of one or more of the antibiotics (Phz-) were significantly less suppressive than the parental strain. Cosmids pLSP259 and pLSP282 from a genomic library of strain 30-84 restored phenazine production and fungal inhibition to 10 different Phz- mutants. Sequences required for production of the phenazines were localized to a segment of approximately 2.8 kilobases that was present in both cosmids. Expression of this locus in Escherichia coli required the introduction of a functional promoter, was orientation-specific, and resulted in the production of all three phenazine antibiotics. These results strongly suggest that the cloned sequences encode a major portion of the phenazine biosynthetic pathway.


Autonomic dysfunction in peripheral nerve disorders. The autonomic nervous system is affected by most conditions that cause peripheral neuropathy. Autonomic dysfunction may be clinically significant in some conditions such as diabetes, amyloidosis, Guillain-Barré syndrome and porphyria, but in many the autonomic disturbances are of only minor clinical importance. Clinical tests of both sympathetic and parasympathetic function are necessary to establish the diagnosis.


Excitatory amino acid receptors and disease. Recent advances in the molecular biology of excitatory amino acid receptors are reviewed. Evidence that drugs blocking the excitatory action of glutamate at the N-methyl-D-aspartate (NMDA) and non-NMDA receptors may be of clinical use in epilepsy, Parkinson's disease, cerebral ischaemia and trauma, acquired immune deficiency syndrome (AIDS) encephalopathy and neuropathic pain is summarized.


New anticonvulsants.

New anticonvulsants. The development of antiepileptic drugs has been rather slow, and this may be related partly to the regulatory requirement to show unequivocal benefit of a new agent over existing drugs. There is a need to reconsider trial protocols to achieve this objective. Five new drugs, vigabatrin (GVG), lamotrigine (LTG), gabapentin (GPT), felbamate and oxcarbazepine (OCBZ) appear to be the most widely tested and promising agents. Of the others, loreclezole and stiripentol (STP) are showing the highest potential for therapeutic application. Clobazam appears to be more effective than implied from earlier reports.


Congenital, hereditary and developmental spinal cord disease. A number of papers have been published during 1991 in the area of congenital, hereditary and developmental spinal cord disorders, which discuss a wide range of rare spinal anomalies; there have, however, been no significant advances. The diagnosis of these disorders has been enhanced by the use of magnetic resonance imaging (MRI).


Neurophysiological evaluation of disorders and procedures affecting the spinal cord and the cauda equina. Neurophysiological tests that asses afferent or efferent pathways of the spinal cord and of nerve roots are used for clinical diagnosis, intraoperative monitoring, and judgement of prognosis. Most tests can readily be performed in awake subjects, whereas many difficulties arise in anesthetized patients in whom neurophysiological testing is the only way of monitoring functional integrity of neural tissue. The research, therefore, that has been carried out in this field during the past year is of special interest.


Morphology and functional responses of isolated inner adrenocortical cells of rats infused with interleukin-beta. The effects of the prolonged infusion with interleukin-1 beta (IL-1 beta) (20 pM.kg-1.min-1) on the function and morphology of the isolated inner cells of the rat adrenal cortex were investigated. After 3 and 5 days of IL-1 beta infusion, the level of circulating ACTH was below the control level, while the plasma concentration of corticosterone was strikingly elevated. After 5 days of infusion, isolated inner adrenocortical cells showed an enhanced basal and ACTH-stimulated corticosterone secretion, and showed a conspicuous hypertrophy. The acute exposure to IL-1 beta 10(-6) M did not affect the secretory activity of dispersed cell from either control or IL-1 beta-infused rats. These findings indicate that the prolonged exposure to high levels of circulating IL-1 beta, like those occurring during chronic inflammatory diseases, is able to enhance the growth and steroidogenic (glucocorticoid) capacity of the rat inner adrenocortical zones. Moreover, they suggest that the mechanism underlying this adrenocorticotrophic effect of IL-1 beta does not involve either a stimulation of the hypophyseal ACTH release or a direct stimulatory effect of monokine on adrenocortical cells. It is suggested that IL-1 beta may activate an intra-adrenal paracrine regulatory mechanism.


Hypophyseal pathology in AIDS.

Hypophyseal pathology in AIDS. One hundred and eleven pituitary glands of patients (93 males, 18 females; mean age 32 years, 5 months) who died of fully developed AIDS or ARC were examined under light microscopy with the aid of immunohistochemistry. On post mortem (p.m.) examination a wide series of multiorgan alterations was noticed. Microscopically various lesions in both adeno- and neurohypophysis were seen. These ranged from vessel damage to secondaries to systemic infections, neoplasms and functional derangements. Necrotic lumps due to recent infarction could appear in both parts of the gland, while old fibrous scars sustained a previously overcome necrosis. Different pathogens (mainly fungi) could be seen either within the gland or arising from its meningeal surroundings. Examples of tumour pathology were provided by microadenomas, gliosis/gliomas; the frequency of adenomas (11.7%) was similar to that typical of senility. The functional impairment was mainly connected with ACTH cell hyperplasia, which seems in keeping with corticoadrenal or ACTH-receptor damage.


[Gonadotropin releasing hormone and analogs. Physiology and pharmacology]. The natural gonadotropin-releasing hormone (GnRH) is secreted by hypothalamic neurons and acts at the level of anterior pituitary gonadotrophs releasing the peptides luteinizing hormone and follicle-stimulating hormone. GnRH is a decapeptide sensitive to peptidases, resulting in a short half-life. The synthesis of highly potent analogues of GnRH provides peptides with a longer half-life and a higher affinity to the GnRH-receptor. The presently available GnRH analogues for clinical use act initially as agonists by upregulating GnRH receptors in the pituitary. A few days after continuous application of GnRH analogues down-regulation of receptors and desensitization of the pituitary occur. In addition to receptor mechanisms, a number of postreceptor actions at the level of second messengers (protein kinase C, leukotriene and inositol phosphates) are responsible for the inhibition of adequate gonadotropin secretion. This paradoxical entire fertility action of the analogues is the basic principle for its clinical use, resulting in a reversible suppression of pituitary and thereby ovarian function.


Long term follow up of women with borderline cervical smear test results: effects of age and viral infection on progression to high grade dyskaryosis. To follow up and assess the significance of borderline change in cervical smears. Retrospective study of women undergoing routine cervical cytological screening in 1981. Avon Cervical Screening Programme, covering 250,000 women in Bristol and Weston super Mare. 437 women showing borderline cervical changes in 1981 and 437 age matched controls with normal results in 1981. Cytological progression to high grade dyskaryosis (cervical intraepithelial neoplasia grade III or invasive carcinoma). During follow up ranging from 13 to 106 months 98 of the 437 women (22.4%) with borderline cytological changes on routine cervical cytology screening had a subsequent smear test showing high grade dyskaryosis compared with three of the 437 women (0.9%) in the control group. The risk of progression was greater in women aged 20 to 39 than in those aged 40 and over. Human papillomavirus infection had initially been diagnosed cytologically in 101 of the 437 (23%) women with borderline results. Significantly fewer of these women developed high grade dyskaryosis (13/98 (13%) v 88/339 (26%), p less than 0.05). Women with borderline smear test results are at increased risk of developing high grade dyskaryosis, particularly if the borderline changes occur without cytological features of human papillomavirus infection. Progression occurs within three years in 50% of cases, although a linearly increasing risk was sustained over the nine years of follow up and was greatest in women aged 20 to 39. Careful follow up of these women is indicated.


Opiate receptor blockade in juvenile macaques: effect on affiliative interactions with their mothers and group companions. The aim of this study was to test the hypothesis that, in non-human primates, the offspring-mother attachment and other social bonds within the group have in common neural mediating mechanisms involving brain opioids. The subjects were 10 juvenile macaques living in a stable social group with their mothers and other group companions. A within-subjects design, balanced for the order of drug administration (naloxone 1 mg/kg i.m. and saline), was used. In the naloxone condition, the juveniles increased their relative role in maintaining proximity with their mothers, made more grooming solicitations, and received more grooming. We found no evidence for differential effects of naloxone on the affiliative bonds the subjects had with their mothers and other group companions. The subjects' increased demands for social comfort were evenly addressed to, and responded to by, both their mothers and other group companions. This finding supports the hypothesis that, at a neural level, the endogenous opioid peptides form a common substrate for different types of social attachments in primates.


Role of spleen or spleen products in the deficiency in morphine-induced analgesia in diabetic mice. We examined the possibility that the spleen or factor(s) derived from spleen mononuclear cells are involved in the deficient mu-opioid receptor-mediated analgesia encountered in diabetic mice. Splenectomized diabetic mice had a significantly higher sensitivity to morphine analgesia than untreated or sham-operated diabetic mice. Naive recipient mice injected with mononuclear spleen cells from diabetic mice exhibited a lower sensitivity to morphine analgesia than vehicle-treated naive mice. These results suggest that some factor(s) derived from spleen mononuclear cells may play an important, direct or indirect role in the selective reduction in mu-agonist-mediated analgesia in diabetic mice.


The effect of the kappa-opioid receptor agonist CI-977 in a rat model of focal cerebral ischaemia. The effect of a novel, highly potent and selective kappa-opioid receptor agonist CI-977 upon ischaemic brain damage and brain swelling has been examined in a rat model of focal cerebral ischaemia. Focal ischaemia was produced by the permanent occlusion of the left middle cerebral artery (MCA) during a brief period of halothane anaesthesia. The animals were sacrificed 24 h after MCA occlusion and the amount of ischaemic brain damage and swelling was assessed in coronal sections at 8 predetermined stereotactic planes. Treatment with CI-977 (0.03, 0.3 or 3 mg/kg), initiated 30 min prior to MCA occlusion (and at multiple times thereafter) produced dose-dependent reductions in the volumes of infarction and of brain swelling, with the most marked reductions being noted with CI-977 (0.3 mg/kg) in both infarction (reduced by 38% from controls; P less than 0.02) and swelling (reduced by 31%; P less than 0.002). There was an excellent correlation between the volume of brain swelling and ischaemic damage which was similar with saline-treated and CI-977-treated animals (overall correlation coefficient r = 0.896). These results indicate that CI-977 is effective in reducing infarction in a model of focal cerebral ischaemia, and that the reduction in brain swelling occurs in parallel with the reduction in ischaemic damage.


Melanocortins stimulate proliferation and induce morphological changes in cultured rat astrocytes by distinct transducing mechanisms. Melanocyte stimulating hormone (MSH), adrenocorticotropic hormone (ACTH), and several peptides derived from pro-opiomelanocortin, are present in the dorsolateral hypothalamus and arcuate nucleus of several vertebrate species. These peptides affect central nervous system (CNS) functions including behavior, memory, and foetal brain development. In this study we investigated the effects of ACTH1-24, ACTH1-17, ACTH4-10, alpha-MSH, beta-MSH, and a potent analog (Nle4,D-Phe7)-alpha-MSH (melanocortins) on immunocytochemically defined astroglial cells prepared from primary cultures of 1-2-day-old rat brains. A cyclic adenosine 3',5'-monophosphate (cAMP) response to the melanocortins was only detected in astrocytes and not in other cell types in the culture. The extent of the cAMP response was greatest on day 21, the latest time tested. On the other hand, (methyl3H)-thymidine incorporation in astrocytes was significantly stimulated (1.5-2-fold) by melanocortins only in 7 and not in 14 and 21 day cultures. This mitogenic activity of melanocortins was not mimicked by other agents such as forskolin or isoproterenol which efficiently stimulate cAMP production in astrocytes. ACTH1-17 as a melanocortin representative induced significant morphological changes in 7 and 14 day cultures which included rounding of the cell body and process extension. This response, however, resembled that induced by forskolin and hence appears to be cAMP mediated. These findings suggest that astrocytes in the CNS may serve as a target for melanocortins. These peptides appear to affect differentiation and proliferation of these cells during certain developmental periods. While the morphological effects of melanocortins seem to be cAMP mediated, induction of proliferation of the astrocytes by melanocortins appears to involve an alternative signal transduction pathway.


Adrenalectomy and stress modulate GABAA receptor function in LS and SS mice. The effects of manipulation of adrenal steroids by adrenalectomy (ADX) or stress on GABAA receptor function were characterized in long-sleep (LS) and short-sleep (SS) mice. 36Chloride flux was not altered in either line of mouse after ADX; however, exposure to a behavioral stressor resulted in a highly significant inhibition of ion channel activity measured in cortical membranes from both LS and SS mice. Adrenalectomy also had no effect on [3H]FNZ binding; whereas exposure to stress differentially altered benzodiazepine binding in LS and SS mice. In LS cortex both Bmax and Kd values increased, whereas in SS cerebellum, Bmax and Kd values were decreased after stress. In SS mice ADX did not affect GABA-enhancement of [3H]FNZ binding. In LS mice, however, ADX resulted in a potentiation of GABA-enhanced [3H]FNZ binding in cortex and an inhibition of enhancement in cerebellum. Corticosterone (CCS) replacement in ADX-LS mice returned enhancement values to those of sham-operated mice, indicating a role for basal levels of CCS in maintaining normal receptor coupling function in this line of mouse. These results suggest that GABAA receptor sensitivity is more labile under stressful conditions. Differential receptor responses to adrenal manipulation between LS and SS mice may be due to genetic variation in GABAA receptor subunit combinations in these lines of mice.


AMPA/kainic acid glutamate receptor antagonism in the zona incerta dorsal to the subthalamic nucleus inhibits amphetamine-induced stereotypy bur not locomotor activity. The effect of 6,7-dinitroquinoxaline-2,3-dione (DNQX), an alpha-amino-3- hydroxy-5-methyl-4-isoxazole-propionate (AMPA)/kainic acid glutamate receptor antagonist, injected into the zona incerta (ZI) was investigated to determine whether the behavioral responses to systemic amphetamine involve AMPA/kainic acid receptors in this brain region. Rats were injected bilaterally in the ZI with either vehicle or DNQX (1 microgram/0.5 microliter) and immediately given a systemic injection of D-amphetamine (0.5, 1.0 or 10.0 mg/kg, s.c.). Locomotor activity was recorded for 1 h. DNQX did not significantly affect hypermotility stimulated with 0.5 and 1.0 mg/kg amphetamine, but markedly increased the level of locomotor activity elicited by the higher dose, 10 mg/kg. To test the hypothesis that the enhanced locomotor response to high dose amphetamine was due to an inhibition of stereotyped behavior, the effect of DNQX in the ZI on amphetamine and apomorphine-induced stereotypy was investigated. DNQX significantly inhibited stereotypy induced by amphetamine (10 mg/kg) and apomorphine (1 mg/kg), with the onset of inhibition of amphetamine-induced stereotypy corresponding to the onset of enhanced locomotor activity. Ibotenic acid lesions of the ZI produced similar results, having an insignificant effect on locomotor activity stimulated by low dose amphetamine (1 mg/kg) and an attenuation of apomorphine-induced stereotypy which was of a magnitude comparable to that produced by DNQX. Thus, the AMPA/kainic acid subtypes of glutamate receptors in the ZI may be involved in the regulation of motor function mediated via striatal output but not mesolimbically generated locomotor activity.


Intrathecal MK-801 and local nerve anesthesia synergistically reduce nociceptive behaviors in rats with experimental peripheral mononeuropathy. The hyperalgesia and spontaneous pain that occur following peripheral nerve injury may be related to abnormal peripheral input or altered central activity, or both. The present experiments investigated these possibilities by examining the effects of MK-801 (a non-competitive N-methyl-D-aspartate, NMDA, receptor antagonist) and bupivacaine (a local anesthetic agent) on thermal hyperalgesia and spontaneous nociceptive behaviors in rats with painful peripheral mononeuropathy. Peripheral mononeuropathy was produced by loosely ligating the rat's common sciatic nerve, a procedure which causes chronic constrictive injury (CCI) of the ligated nerve. The resulting hyperalgesia to radiant heat and spontaneous nociceptive behaviors was assessed by using a foot-withdrawal test and a spontaneous pain behavior rating method, respectively. CCI rats receiving 4 daily intraperitoneal (i.p.) MK-801 injections (0.03, 0.1, 0.3 mg/kg) beginning 15 min prior to nerve ligation exhibited less hyperalgesia (i.e., longer foot-withdrawal latencies) on days 3, 5, 7, 10, and 15 after nerve ligation as compared to those receiving saline injections. Thermal hyperalgesia also was reduced when a single MK-801 injection was given intrathecally (i.t.) onto the spinal cord lumbar segments on Day 3 after nerve ligation. This effect of postinjury MK-801 treatment was dose-dependent (2.5-20 nmol) and lasted for at least 48 h after injection. Moreover, i.t. injection of MK-801 (10 nmol) reliably lowered spontaneous pain behavior rating scores in CCI rats compared to those in the saline group. The spinal site of MK-801 action is situated within the caudal (probably lumbar) spinal cord, since i.t. injection of MK-801 (10 nmol) onto the spinal cord thoracic segments did not affect thermal hyperalgesia.(ABSTRACT TRUNCATED AT 250 WORDS)


Nasal mucosa sensitization with toluene diisocyanate (TDI) increases preprotachykinin A (PPTA) and preproCGRP mRNAs in guinea pig trigeminal ganglion neurons. Toluene diisocyanate (TDI) induces respiratory allergy in mammals. Using immunohistochemistry and in situ hybridization histochemistry, the present study examined effects of nasal mucosa sensitization by TDI on the immunoreactivity for substance P (SP) and calcitonin gene-related peptide (CGRP) and on the expression of their mRNAs in guinea pig trigeminal ganglion and their terminals. Single intranasal application of TDI (acute experiment) did not induce nasal allergy-like behaviours and failed to cause changes of SP and CGRP immunoreactivity and in the expression of preprotachykinin A (PPTA) mRNA and preproCGRP mRNA coding for SP and CGRP respectively in the trigeminal ganglion neurons. However, repeated application of TDI (chronic experiment) caused a dramatic increase of SP and CGRP immunoreactivity in peripheral neurites of sensory nerves in the nasal mucosa but a slight increase in the spinal trigeminal nucleus, a decrease of the same immunoreactivities in the cell bodies of the trigeminal ganglion neurons, and an increase of the expression of PPTA and preproCGRP mRNA in the same neurons. These findings suggest that chronic exposure of the nasal mucosa to TDI apparently causes enhancement of both the biosynthesis of SP and CGRP and their axonal transport in the trigeminal system.


Co-localization of basic fibroblast growth factor-like immunoreactivity and its receptor mRNA in the rat spinal cord and the dorsal root ganglion. In the present study, we examined the localizations of basic fibroblast growth factor-like immunoreactivity (bFGF-LI) and its receptor mRNA in the spinal cord and the dorsal root ganglion of the rat. Anti-bFGF peptide antibody and cRNA probe were employed to visualize the localizations of bFGF-LI and FGF receptor (FGF-R) mRNA, respectively. In the spinal cord, we observed that a number of neurons including the motor neurons and interneurons were positive for both substances. In the dorsal root ganglion (DRG), the large neurons preferentially showed co-localization of bFGF-LI and FGF-R mRNA, while the small neurons were not always positive for both. Given the fact that FGF-R is a membrane-spanning protein, these findings suggest the following two possibilities: (1) bFGF acts on the neurons of the spinal cord and the DRG in an autocrine and/or paracrine manner; (2) FGF-R mRNA-positive neurons take up bFGF from innervating neurons and/or surrounding glias in a receptor-mediated fashion.


Calcium currents in rat motor nerve terminals. Ca2+ currents in response to an action potential were recorded extracellularly under non-voltage clamped conditions from rat motor nerve terminals. The Ca2+ current was blocked by Cd2+, Co2+, and Ni2+. A residual component that could not be blocked by inorganic cations was inhibited completely by tetrodotoxin (TTX). The Ca2+ current was also moderately sensitive to the N- and L-type Ca2+ channel-blocker omega-conotoxin but was insensitive to the L-type channel-specific dihydropyridines. When a fraction of the terminal K+ currents was blocked by 10 mM tetraethylammonium (TEA), the Ca2+ current duration decreased only slightly as stimulation frequency increased from 0.5 to 20 Hz. When K+ currents were blocked by TEA plus 3,4-diaminopyridine (250 microM) though, the Ca2+ current duration decreased from greater than 70 ms to 8-10 ms as stimulation frequency increased from 0.5 to 20 Hz. Recovery of the duration following 20-Hz stimulation occurred faster during subsequent stimulation at 0.5 Hz than at 2 Hz. ATP and ACh inhibit Ca2+ currents at stimulation frequencies ranging from 0.5 to 20 Hz; however, when the purinergic and cholinergic autoreceptors are blocked by theophylline (100 microM) and pirenzepine (3 microM), respectively, the frequency-induced decrease in current duration persisted. Thus, motor nerve terminal Ca2+ current duration is determined by stimulus repetition frequency; this appears to involve intracellular Ca2+ accumulation, although effects secondary to variability in the time course of changes in terminal membrane potentials cannot be ruled out.


Coactivation of dopamine D1 and D2 receptors increases the affinity of cholecystokinin-8 receptors in membranes from post-mortem human caudate-putamen. The effects of dopamine in vitro were investigated on the binding sites for cholecystokinin-8 (sulphated, CCK-8) and neurotensin in membrane preparations of the caudate-putamen and nucleus accumbens of post-mortem human brains. Dopamine reduced the IC50 value of competition curves with CCK-8 for [125I]CCK-8 binding in membranes from the caudate-putamen, but not the nucleus accumbens, with a maximal decrease of -25 +/- 9% at 300 nM of dopamine. This decrease could be antagonized by 100 nM of SCH 23390 or 100 nM of raclopride. Kinetic analysis of [125I]CCK-8 binding showed a decrease in the first order dissociation rate constant and in the kinetic Kd (-22 +/- 6% and -24 +/- 6%, respectively) at 300 nM of dopamine, without any significant effect on the apparent or actual association rate constant. Competition curves with neurotensin versus [125I]neurotensin were not affected by dopamine (10-1000 nM) in membranes from the caudate-putamen or the nucleus accumbens. These results suggest that dopamine, by synergistic stimulation of both D1 and D2 receptors, selectively increases the affinity of CCK-8 receptors in the human caudate-putamen, by a selective inhibition of ligand dissociation. This increase may reflect a positive feed-back mechanism, further enhancing the modulatory effects of CCK-8 on dopamine neurotransmission.


Post-injury treatment with GM1 ganglioside reduces nociceptive behaviors and spinal cord metabolic activity in rats with experimental peripheral mononeuropathy. In a rat model of painful peripheral mononeuropathy, this study examined the effects of post-injury treatment with a monosialoganglioside, GM1, on abnormal nociceptive behaviors and spinal cord neural activity resulting from loose ligation of the rat common sciatic nerve (chronic constrictive injury, CCI). Thermal hyperalgesia and spontaneous pain behaviors of CCI rats were assessed by measuring foot-withdrawal latencies to radiant heat and by rating spontaneous hind paw guarding positions, respectively. Neural activity within different regions of the spinal cord was inferred in both CCI and sham-operated rats by employing the [14C]-2-deoxyglucose (2-DG) autoradiographic technique to measure spinal cord glucose metabolism. Intraperitoneal (i.p.) GM1 treatment (10 mg/kg) initiated 1 h or 24 h after injury and once daily for the first 9 post-injury days reduced thermal hyperalgesia of the hind paw ipsilateral to nerve ligation and lowered spontaneous pain behavior rating scores in CCI rats. Sciatic nerve ligation reliably increased basal 2-DG metabolic activity of CCI rats in all four sampled regions (laminae I-IV, V-VI, VII, VIII-IX) of spinal cord lumbar segments (L2-L5) both ipsilateral and contralateral to nerve ligation 10 days after injury. Consistent with the drug's effects on spontaneous pain behaviors, 10 daily GM1 treatments (10 mg/kg, i.p.) initiated 1 h after nerve ligation reduced spinal cord 2-DG metabolic activity in laminae V-VI and VII ipsilateral to nerve ligation and in all four sampled regions contralateral to nerve ligation. This attenuation of the increased spinal cord glucose utilization that occurs in the absence of overt peripheral stimulation may reflect an influence of GM1 on increased neural activity contributing to spontaneous pain. Since gangliosides are thought to protect neurons from excitotoxic effects of excitatory amino acids, these results suggest that ganglioside treatment may result in attenuation of excitatory neurotoxicity that may occur following peripheral nerve injury. Thus, ganglioside treatment could provide a new approach to the clinical management of neuropathic pain syndromes following peripheral nerve injury.


Distribution of the endogenous digitalis-like substance (EDLS)-containing neurons labeled by digoxin antibody in hypothalamus and three circumventricular organs of dog and macaque. Endogenous digitalis-like substance (EDLS) is a newly discovered humoral agent which causes sodium-diuresis. EDLS is well known to have inhibitory activity to Na+,K(+)-ATPase and cross-immunoreactivity to digoxin antibody; however, its precise chemical structure has not yet been determined. We had previously developed a histochemical technique for EDLS, i.e., digoxin-immunohistochemistry, and demonstrated that EDLS was produced in the hypothalamic neurons. In the present study, the distribution of EDLS-containing neurons in the hypothalamus of dog and macaque was investigated using this technique, because anti-EDLS antibody cannot be obtained yet. In both species, EDLS neuronal somata were mainly localized in the paraventricular nucleus and the supraoptic nucleus and its accessory nuclei. A number of somata were also scattered in the other hypothalamic areas. The processes of these neurons ran from the area where the somata were located, through the lateral and basal area of the hypothalamus, to the infundibulum. These nerve fibers with varicosities were associated with the primary capillaries of hypophysial portal veins. A few immunopositive nerve fibers were also seen in the pituitary posterior lobe of both species. Intensive immunoreactivities were observed in the subfornical organ and organum vasculosum laminae terminalis. There were no differences between dog and macaque.


Long-lasting decreases of type II calmodulin kinase expression in kindled rat brains. The kindling model of epilepsy is associated with long-lasting changes in type II calmodulin kinase (CaM kinase) activity and immunoreactivity. In order to determine the mechanism of these alterations, we measured gene expression of CaM kinase using in situ hybridization in septally kindled rat brains and paired controls using a 35S-labeled riboprobe for the beta subunit of the enzyme. We found CaM kinase mRNA concentrated in the hippocampus and other limbic structures. Kindling decreased hippocampal CaM kinase mRNA by 30% in CA1, 34% in CA2, 35% in CA3 41% in CA4, and 29% in the dentate gyrus. Hybridization was also decreased by 21% in the cerebral cortex but not in the lateral septum. These changes are similar in distribution and direction to those previously measured by immunohistochemistry. These data suggest that altered CaM kinase activity and immunoreactivity associated with kindling reflect long-lasting alterations in gene expression of this important synaptic protein, and provide further evidence for its possible importance in the kindling phenomenon.


Intrathecal GM1 ganglioside and local nerve anesthesia reduce nociceptive behaviors in rats with experimental peripheral mononeuropathy. Our previous experiments demonstrated that systemic treatment with GM1 ganglioside reduces nociceptive behaviors and spinal cord metabolic activity in a rat model of painful peripheral mononeuropathy produced by experimental sciatic nerve ligation (chronic constrictive injury, CCI). In the present study, we examined the effects of intrathecal (i.t.) GM1 treatment on thermal hyperalgesia and spontaneous pain behaviors resulting from nerve ligation in order to determine the locus of GM1 action. In addition, a local anesthetic agent, bupivacaine, given alone or combined with i.t. GM1, was applied to the injured sciatic nerve to determine if peripheral nerve anesthesia would influence post-injury nociceptive behaviors. Thermal hyperalgesia to radiant heat decreased in a dose-dependent manner when GM1 (10-80 nmol, i.t.) was administered once daily onto the lumbar segments of the spinal cord beginning 1 h after experimental nerve injury and continued for the first 9 days after nerve ligation. Moreover, this GM1 (80 nmol) treatment regimen reliably lowered spontaneous pain behavior rating scores in CCI rats suggesting the possible attenuation of spontaneous pain. The central site of i.t. GM1 action is located at the caudal (probably lumbar) spinal cord, since i.t. injection of 20 nmol GM1 onto the cervical spinal cord did not produce any protective effect. A single perinerve injection of a local anesthetic agent, bupivacaine (0.5%, 0.6 ml), on the 3rd day after nerve ligation reduced thermal hyperalgesia for at least 24 h following injection, a duration longer than that of the local anesthetic action of bupivacaine. Neither a single bupivacaine injection nor four daily i.t.(ABSTRACT TRUNCATED AT 250 WORDS)


The spatial relationship between Müller cell processes and the photoreceptor output synapse. Glutamate is the neurotransmitter released by photoreceptors in the retina. The postsynaptic action of glutamate is terminated partly by uptake into glial (Müller) cells. The anatomical distribution of Müller cell processes around the synaptic terminals of photoreceptors was investigated electron microscopically in the tiger salamander retina. Müller cells wrap around the synaptic terminals of both rods and cones and come within 1-3 microns of the sites of glutamate release, close enough to contribute to terminating the synaptic action of glutamate.