Mapping of second messenger and rolipram receptors in mammalian brain. Autoradiographic localizations of major second messengers and a selective cyclic adenosine monophosphate (cyclic-AMP) phosphodiesterase in the brain were visualized in the gerbil and the rat using receptor autoradiography. [3H]Phorbol 12,13-dibutyrate (PDBu), [3H]inositol 1,4,5-trisphosphate (IP3), [3H]forskolin, [3H]cyclic-AMP, and [3H]rolipram were used to label protein kinase C, IP3 receptor, adenylate cyclase, cyclic-AMP-dependent protein kinase (cyclic-AMP-DPK), and Ca2+/calmodulin-independent cyclic-AMP phosphodiesterase (PDE), respectively. Most second messengers and rolipram binding activities were especially found in the limbic system, basal ganglia, and cerebellum. Marked differences were noted in the hippocampus, where cyclic-AMP and rolipram binding activities were very low in gerbils but high in rats. In contrast, regional localization in the binding sites of PDBu, IP3, and forskolin in gerbil brain was relatively similar to that in rat brain. Further, alteration of the cyclic-AMP and rolipram binding sites was studied in the gerbil hippocampus 7 days after 10-min cerebral ischemia. The results suggest that the gerbil differs from the rat with respect to the characteristic neurons or interneurons, especially in the hippocampal formation. This finding may help further elucidate the relationship or difference between gerbils and rats for brain function and behavioral pharmacology. Furthermore, our results suggest that cyclic-AMP and rolipram binding sites are predominantly distributed on the pyramidal cell layer of the hippocampal CA1 sector and that transient cerebral ischemia can cause marked reduction in these binding sites in the hippocampus.


Seasonal and state-dependent changes in brain TRH receptors in hibernating ground squirrels. Quantitative autoradiography was used to localize and quantify thyrotropin-releasing hormone (TRH) receptors in the brain of hibernating (H), winter euthermic (WE), and summer euthermic (SE) animals to further explore the state-dependent physiological and behavioral effects of TRH in ground squirrels. The pattern of [3H]MeTRH binding (Kd 6.7 +/- 0.1 nM) was heterogeneous and highly concentrated in structures primarily associated with the limbic forebrain. Statistically significant seasonal changes (SE vs. WE) were reflected by decreases in TRH receptor binding in the arcuate nucleus, dorsomedial nucleus, and ventral pallidum of WE animals. Increased binding in WE animals was evident in the suprachiasmatic nucleus and choroid plexus of the lateral ventricles. Significant state-dependent changes (WE vs. H) were characterized by decreases in TRH receptor binding in the hypothalamic paraventricular nucleus, medial preoptic area, ventral tegmental area, and choroid plexus of the lateral ventricles of H animals. Increased binding occurred in the anterior cortical nucleus of the amygdala in H animals. The results suggest that naturally occurring changes in central TRH systems may be important in the mediation of physiological and behavioral processes that undergo seasonal and state-dependent adjustments in hibernating mammals.


Negative feedback of corticosterone on the pituitary-adrenal axis is maintained after inhibition of serotonin synthesis with parachlorophenylalanine. The role of the serotonergic transmission on the negative feedback of corticosterone on the pituitary-adrenal (PA) axis was studied in adult male rats. Animals were given p-chlorophenylalanine (PCPA) and 24 h later were administered corticosterone, 2 h before being subjected to 20 min of noise stress. The main results were as follows: First, PCPA administration increased resting levels of both corticotropin (ACTH) and corticosterone but did not alter PA response to noise stress; second, ACTH response to stress was eliminated in rats given PCPA; third, corticosterone reduced ACTH levels in nonstressed rats only and this effect was maintained after PCPA administration. Taken together, the present results suggest a mediator role for serotonin in the noise-stress-induced PA hormone release but no role in the negative feedback of corticosterone on the PA axis.


New drugs for infections in patients with cancer. During the past several years, the treatment and prevention of infections in patients with cancer have improved because of the development of new antimicrobial drugs. A representative antibacterial, antifungal, and antiviral drug is discussed. The fluoroquinolone compounds, such as ciprofloxacin, norfloxacin, and ofloxacin, have broad spectrum bactericidal activity and have proved to be effective in both the treatment and prevention of certain infections in patients with cancer. The triazole drug fluconazole is useful in the treatment of oral, esophageal, and disseminated candidiasis and cryptococcal meningitis. Early studies suggest that a new acyclic nucleoside, ganciclovir, has some effect in the treatment of cytomegalovirus infections, but its precise use in patients with cancer has not been fully established. New drugs currently available to the practitioner will improve management of infections in patients with cancer, but none is totally effective and the search for new compounds must continue.


Cytogenetic analysis of an adenoid cystic carcinoma of the Bartholin's gland. A rare, semimalignant tumor of the female genitourinary tract. Cytogenetic analysis has been performed on short-term cultures from a 56-year-old woman suffering from an adenoid cystic carcinoma of Bartholin's gland. Beside a normal female karyotype, the tumor revealed an abnormal cell line with complex chromosome changes involving the chromosomes 1, 4, 6, 11, 22, and 14. The mainly structural and nonbalanced rearrangements led to the loss of the chromosome segments 1p31----qter, 4q22----q28, 6p12----qter, 11p11.2----pter, 14q24----qter, and 22q13----qter. Clonal numerical aberrations were not observed. To our knowledge, such a tumor has to-date not been cytogenetically investigated.


Cytogenetics and molecular genetics of Wilms' tumor of childhood. We describe the way in which application of cytogenetic and molecular genetic techniques to the study of Wilms' tumor (WT) of the kidney and the associated congenital disorders, such as sporadic aniridia and the Beckwith-Wiedemann syndrome, has led to identification of two regions on the short arm of chromosome 11 (11p13 and 11p15) involved in tumor development. In addition, evidence shows that genomic imprinting may be an important factor in transformation. Such investigations have led to cloning of a candidate WT gene (WT1) from 11p13. Linkage studies in familial studies suggest that an additional locus is involved. Analysis of the cytogenetic data available on this tumor suggests that this may be situated on 1p, 16q, or 17p.


Early and multifocal tumors in breast, salivary, harderian and epididymal tissues developed in MMTY-Neu transgenic mice. Transgenic mice carrying various oncogenes driven by mammary gland specific enhancers develop mammary tumors usually arising in a stochastic way. The only exception is a mouse lineage (TG.NF) carrying an activated rat Neu oncogene driven by the murine mammary tumor virus long terminal repeat (MMTV-LTR) that gave rise to rapid and multifocal mammary tumors interpreted as a result of a single-step neoplastic transformation. The effect of the oncogene appeared to be specific for breast tissue, since salivary and Harderian glands as well as epididymis expressed high levels of Neu but only developed hyperplasia (Muller et al., Cell, (1988) 54, p. 105). Here we describe a transgenic mouse lineage for the MMTV-Neu, analysed up to third generation. Multifocal tumors involving mammary glands arose very rapidly in all females independently from pregnancy and in some males. Moreover, multifocal neoplasias occurred also in salivary and Harderian glands and in the epididymis at a very high rate. These data demonstrate that the Neu oncogene can induce tumors in all the tissues where it is expressed at high levels.


Multiple transduction as a means of preserving ganciclovir chemosensitivity in sarcoma cells carrying retrovirally transduced herpes thymidine kinase genes. The potential value of retroviral gene transfer as a means of targeting therapeutic genes to neoplastic cells is threatened by the tendency of occasional cells to lose transduced genes or their expression. To determine whether this threat could be reduced by transducing multiple copies of a therapeutic gene, we compared 8 murine sarcoma sublines carrying from 1 to 6 copies of a retrovirally transduced herpes simplex virus thymidine kinase gene, which sensitizes cells to ganciclovir (GCV). When variability consequent to differences in vector integration site was excluded, increased copy number was associated with an increase in GCV sensitivity and a major reduction in the frequency of GCV-resistant mutants. The results suggest a potential means of preserving the efficacy of future antineoplastic gene therapy strategies.


Development of antibodies against p53 in lung cancer patients appears to be dependent on the type of p53 mutation. Using immunoblotting techniques we studied the sera from small cell lung cancer and non-small cell lung cancer patients for antibodies directed against p53. We have also characterized the majority of these patients' tumors for p53 mutations. In the sera of 13% of the patients (4 of 40 small cell lung cancer and 2 of 6 non-small cell lung cancer) we found antibodies specific for the p53 tumor suppressor gene product. All of the antibody-positive patients tested had p53 missense mutations and expressed detectable p53 antigen in their tumor cell lines. No anti-p53 antibodies were detected in sera from patients whose tumor had p53 stop, splice/stop, splice, or frameshift mutations (n = 10). Thus, while we find that the ability of lung cancer patients to develop anti-p53 antibodies is correlated with the type of p53 mutation, many patients have tumors with missense p53 mutations and did not develop anti-p53 antibodies. The presence of p53 antibodies was not correlated to stage, prior treatment, sex, or survival. None of these lung cancer patient sera had measurable amounts of p53 antigen. By immunoblotting all six anti-p53 antisera we were able to detect a variety of mutant p53 proteins (including those from antibody-negative patients) and detected wild-type p53 protein. The development of anti-p53 antibodies represents an interesting model system for studying immune responses in cancer patients against mutant oncogene products.


In vivo growth of C6 glioma cells transfected with connexin43 cDNA. In order to examine the possible role of intercellular communication via gap junctions in the control of tumor growth, we have transfected C6 glioma cells with connexin43 cDNA. We obtained several clones with variable expression of connexin43. The growth rate of these clones in culture was inversely related to the degree of expression of the transfected cDNA. To examine the growth of these transfected cells in vivo, cells were grown in spinner culture flasks to form spheroids 250-300 microns in diameter. Spheroids of nontransfected C6 cells produced large gliomas. Immunohistochemical and in situ hybridization analyses revealed relatively high levels of connexin43 protein and mRNA in the host tissue, while little of this protein was detected in the glioma. In contrast, spheroids of connexin43-transfected cells grew more slowly and exhibited elevated levels of connexin43 protein and mRNA. These findings suggest that the expression of connexin43 may be associated with the control of brain tumor growth in vivo.


DNA topoisomerase II immunostaining in human leukemia and rhabdomyosarcoma cell lines and their responses to topoisomerase II inhibitors. DNA topoisomerase II is an enzyme that affects nuclear structure and function and is the target of a number of anticancer drugs in clinical use, including teniposide (VM-26). We have used our polyclonal antisera that recognize both the M(r) 170,000 and 180,000 forms of topoisomerase II to examine the nuclear distribution of topoisomerase II in cytospin preparations of drug-sensitive (CEM) and VM-26-resistant (CEM/VM-1 and CEM/VM-1-5) human leukemic lymphoblasts. We have also examined the nuclear distribution of topoisomerase II in monolayer cultures of a human rhabdomyosarcoma (Rh30) cell line. In the absence of drug, we observed a focal "patchy" staining of nuclear topoisomerase II in all cell lines, that was especially notable in the lymphoblastic cells. Treatment of CEM and Rh30 cells with VM-26 under conditions that increase the number of covalent topoisomerase II-DNA complexes increased both the intensity and the homogeneity of nuclear topoisomerase II staining in a subpopulation of cells; focal staining was less evident after treatment with drug. These responses were roughly proportional to the concentration of VM-26 used and required only brief (approximately 25-min) incubation with drug. We also found that treatment of CEM cells with 4'-(9-acridinylamino)methanesulfon-m-anisidide similarly increased the intensity and homogeneity of nuclear topoisomerase II immunostaining. In contrast, 4'-(9-acridinylamino)methanesulfon-o-anisidide and 1-beta-D-arabinofuranosylcytosine, agents that do not inhibit topoisomerase II, did not produce this effect. Finally, the VM-26-mediated alteration in topoisomerase II staining intensity and distribution was attenuated in proportion to the degree of VM-26 resistance in the CEM/VM-1 and CEM/VM-1-5 sublines. These results appear to be related to the ability of the drug to stabilize DNA-topoisomerase covalent ("cleavable") complexes in intact cells. Our findings indicate that anti-topoisomerase II drugs, such as VM-26, have profound effects on the ability to detect topoisomerase II in the nucleus and provide a novel way of examining drug-stabilized DNA topoisomerase II complexes in intact single tumor cells.


Induction of transforming growth factor beta 1 in human breast cancer in vivo following tamoxifen treatment. We have investigated the ability of tamoxifen to regulate members of the transforming growth factor beta (TGF-beta) family in human breast cancers in vivo. Using immunohistochemical techniques, we find that 3 months of tamoxifen treatment causes a consistent induction of extracellular TGF-beta 1 in breast cancer biopsies, compared with matched pretreatment samples from the same patient. The induced TGF-beta is localized between and around stromal fibroblasts and appears to be derived from these cells. Lower levels of TGF-beta 1,-beta 2, and -beta 3 seen in epithelial cells were not altered by tamoxifen treatment. The increased stromal staining of TGF-beta 1 occurred in estrogen receptor-negative as well as estrogen receptor-positive tumors. These results provide in vivo evidence for a novel, estrogen receptor-independent mechanism of action for tamoxifen, involving the stromal induction of a potent growth inhibitor for epithelial cells.


Role of the WT1 gene in Wilms' tumour.

Role of the WT1 gene in Wilms' tumour. Wilms' tumour is a paediatric kidney cancer which, in a substantial number of cases, has been associated with a genetic predisposition. Susceptibility to Wilms' tumour can be manifested by the presence of bilateral tumours, and in rare cases by a family history of this tumour or by associated congenital malformations. Like retinoblastoma, Wilms' tumour has been postulated to result from the inactivation of a tumour suppressor gene, although genetic studies implicate more than a single genetic locus. The recent isolation of the WT1 gene, which maps to chromosome 11, band p13, has provided the first molecular clue to Wilms' tumorigenesis. WT1 is specifically inactivated in a number of Wilms' tumours, and mutations have been found in the germline of susceptible individuals. This gene appears to encode a transcription factor with complex alternative splices, whose expression is strictly regulated in the developing kidney. Functional studies will be required to elucidate the role of WT1 in normal kidney development and in tumorigenesis.


Interactions of HPV E6 and E7 oncoproteins with tumour suppressor gene products. The HPVs associated with anogenital cancers encode two oncoproteins, E6 and E7. Both E6 and E7 can form specific complexes with tumour suppressor gene products. The E7 protein binds to the retinoblastoma tumour suppressor gene product pRB, with a preference for the underphosphorylated, "active" form of pRB. The E7 proteins derived from the "high risk" HPVs bind to pRB with a higher affinity than the E7 proteins from the "low risk" HPVs. The "high risk" HPV E6 proteins can associate with the p53 tumour suppressor protein. This interaction promotes the degradation of p53 in vitro, which presumably accounts for the very low levels of p53 in cervical carcinoma cell lines. The functional inactivation of pRB and p53 by the HPV oncoproteins E7 and E6, respectively, are likely to be important steps in cervical carcinogenesis, since mutations in the RB and p53 genes were detected in HPV negative but not HPV positive cervical carcinoma cell lines. Cytogenetic studies strongly suggest, however, that additional chromosomal changes may be necessary for carcinogenic progression of HPV induced anogenital lesions.


Effect of interferon gamma on the sensitivity of bovine-papilloma-virus(BPV1)-transformed cell lines to cell-mediated cytotoxicity. The effect of interferon gamma (IFN gamma) on the immunogenicity and immunosensitivity of mouse cell lines transformed by bovine papillomavirus type 1 (BPV1) DNA was examined in a syngeneic mouse model. The overnight incubation of BPV1-transformed cell lines with 100 IU/ml IFN gamma did not affect their ability to induce the generation of cytotoxic effector cells but it clearly increased their sensitivity to lysis by interleukin-2-induced lymphokine-activated killer (LAK) cells and by non-specific LAK-type effector cells induced by BPV-1-transformed cell lines. The treatment of two allogeneic lymphoid tumour cell lines, P815X2 and YAC-1, with IFN gamma either decreased or had no effect on their sensitivity to LAK-cell-mediated lysis.


[Bronchoalveolar carcinoma, its diagnosis, therapy and prognosis]. The authors give an account of 18 patients with morphologically confirmed bronchioalveolar carcinoma, hospitalized at the First Clinic for TB and Respiratory Diseases in 1969-1988. Women predominated in the group at a ratio of 2:1, mean age: women 46.5 years, men 63.5 years. In the family-history of six patients a malignant tumour of the digestive tract was recorded. The symptomatology of the disease did not differ from other bronchogenic carcinomas. As to X-ray forms large-node carcinoma was most frequent (8x), there were equal numbers (5x) of pneumonic and disseminated carcinomas. The diagnosis of the disease is difficult. In eight instances it was made only after examination of the resected portion. In disseminated forms a high yield was obtained by open pulmonary biopsy, needle aspiration of the lungs. A small yield was obtained by bronchological examination and examination of sputum. The most favourable prognosis was recorded after resection where from a total of nine patients two survive (19 and 4 years), the remaining 7 patients survived for a mean period of seven years. Patients treated by chemotherapy survive for six months, those with actinotherapy and symptomatic treatment for 11 months.


Chicken "erythroid" cells transformed by the Gag-Myb-Ets-encoding E26 leukemia virus are multipotent. The E26 avian leukemia virus encodes a transcriptional activator-type oncoprotein consisting of Gag, Myb, and Ets domains, and transforms early erythroid cells as well as myeloblasts. Surprisingly, we have found that "early erythroid" transformants obtained in culture are multipotent, since they can be induced to differentiate into myeloblasts and eosinophils after superinfection with retroviruses containing kinase-type or ras oncogenes. In addition, TPA is an efficient inducer that generates predominantly eosinophils at low concentrations and myeloblasts at high concentrations. The determination process involves the complete extinction of erythroid/thrombocytic markers and the subsequent activation of myelomonocytic/eosinophilic properties, including the acquisition of specific growth factor requirements. "Erythroleukemic" cells from virus-infected animals were likewise found to be multipotent, making this a unique system to study the genesis of stem cell leukemias and the molecular basis of lineage commitment during hematopoiesis.


Assembly of the active form of the transposase-Mu DNA complex: a critical control point in Mu transposition. Discovery and characterization of a new intermediate in Mu DNA transposition allowed assembly of the transposition machinery to be separated from the chemical steps of recombination. This stable intermediate, which accumulates in the presence of Ca2+, consists of the two ends of the Mu DNA synapsed by a tetramer of the Mu transposase. Within this stable synaptic complex (SSC), the recombination sites are engaged but not yet cleaved. Thus, the SSC is structurally related to both the cleaved donor and strand transfer complexes, but precedes them on the transposition pathway. Once the active protein-DNA complex is constructed, it is conserved throughout transposition. The participation of internal sequence elements and accessory factors exclusively during SSC assembly allows recombination to be controlled prior to the irreversible chemical steps.


Application of monoclonal antibody panels in the virological and epidemiological review of poliomyelitis in Poland, 1981-1990. Monoclonal antibody panels developed to differentiate vaccine-derived and wild-type strains of polio-viruses were applied to isolates from cases of paralytic poliomyelitis, non-paralytic poliomyelitis, and healthy excreters of poliovirus from Poland. All isolates from poliomyelitis cases were shown to be vaccine-derived, as were most other strains. However, two strains associated with meningitis had wild-type antigenic phenotypes and, as shown by partial genomic sequencing, wild-type genotypes. Correlation of laboratory and epidemiological data suggested that residual cases of paralytic poliomyelitis in Poland between 1981 and 1990 were vaccine-related. Study of the non-paralytic cases, however, helped identify the circulation of endemic wild-type viruses in a well-vaccinated community.


Myeloperoxidase-enhanced formation of (+-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene-DNA adducts in lung tissue in vitro: a role of pulmonary inflammation in the bioactivation of a procarcinogen. Several studies have indicated a correlation between the presence of inflammation and the development of cancer. The aim of our study was to determine if pulmonary neutrophils could transform the proximate respiratory carcinogen (+-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol), to an ultimate carcinogenic metabolite via myeloperoxidase (MPO). To test this hypothesis, virus-free male DBA/2 mice were exposed by inhalation to the Gram-negative bacteria Proteus mirabilis for 1 h. For various time points post-exposure, bronchoalveolar lavage (BAL) was performed to determine total and differential cell counts, cellular MPO activity and production of superoxide. Twelve hours after the exposure, cellular activity of MPO as well as percentage and total number of polymorphonuclear leukocytes peaked and declined thereafter. At this same time point, cells from BAL exhibited increased release of superoxide, as measured by reduction of cytochrome c, after addition of soluble or particulate stimuli, 12-O-tetradecanoylphorbol-13-acetate (TPA) or opsonized zymosan respectively. These cells also elicited biotransformation of B[a]P-7,8-diol as evidenced by enhanced B[a]P-7,8-diol-derived chemiluminescence, tetraol formation and covalently bound adduct formation to exogenous DNA upon addition of TPA or opsonized zymosan. Moreover, the cell-free BAL fluid of infected mice contained substantial MPO activity in comparison to that of uninfected animals. Also, MPO enhanced the binding of B[a]P-7,8-diol to lung DNA in vitro. Unlike previous work emphasizing the potential roles of oxygen free radicals in tumor promotion, our results indicate a role of neutrophilic MPO in the initiation of carcinogenesis.


Electron spin resonance spectroscopy of oxygen radicals generated by synthetic fecapentaene-12 and reduction of fecapentaene mutagenicity to Salmonella typhimurium by hydroxyl radical scavenging. Fecapentaenes form a class of potent fecal mutagens and have been suggested to play an initiating role in colon carcinogenesis. Although several indications have been found that fecapentaenes may induce oxidative DNA damage as well as DNA alkylation, the mechanism of genotoxicity remains unknown. In this study, electron spin resonance spectroscopy with several spin traps has been used in order to determine whether reactive oxygen species can be formed by fecapentaene-12 (FP-12). No specific conditions could be defined that resulted in the direct formation of oxygen radicals from FP-12. However, peroxidation of FP-12 by various peroxidative enzymes has been shown to result in the formation of superoxide adducts of the spin traps alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Addition of superoxide dismutase resulted in a decreased spectrum intensity, whereas the hydroxyl radical scavenger t-butyl alcohol (tBA) appeared of no influence on the signal, both confirming the formation of superoxide. The formation of hydroxyl radical spin adducts has been demonstrated after peroxidation of FP-12 in incubations with the spin-trapping agent 2,2,6,6-tetramethyl-piperidine (TMP). Further, the effects of scavenging hydroxyl radicals with respect to the genotoxic potential of FP-12 in the Salmonella mutagenicity assay has been investigated. It was clearly shown that radical scavenging reduced the number of revertants in Salmonella strains TA100, TA102 and TA104. This mutagenicity-reducing effect was more convincing using both spin traps DMPO and TMP as compared to the effect of hydroxyl radical scavengers tBA and DMSO. Based on these findings, a reaction scheme is proposed that suggests the formation of superoxide after peroxidation of FP-12, which is subsequently converted to hydroxyl radicals by the iron-catalysed Haber-Weiss reaction.


The status of the p53 gene in human papilloma virus positive or negative cervical carcinoma cell lines. We have analyzed p53 gene alterations in five cervical cancer derived cell lines. Two of the five cervical cancer cell lines, HTB31 (C-33A) and 32 (HT-3), harbored missense mutations in codons 273 and 245 respectively, whereas the other three tumor cell lines, HTB33 (ME180), 34 (MS751) and 35 (SIHA), did not reveal any mutation in the p53 coding sequence spanning codons 126-307. Although all the tumor cell lines express comparable levels of p53 RNA, only HTB31 and HTB32 contain high or detectable levels respectively of p53 protein. The other three tumor cell lines, where neither p53 mutation nor the expression of p53 protein could be detected, were found to harbor human papilloma virus (HPV) 16 or 18. The inactivation of the wild-type p53 function resulting from a missense mutation, or the lack of detectable wild-type p53 protein due to the translational/post-translational deregulation of p53 protein levels may be the contributing factor in the tumorigenicity of these five cases of cervical cancer. The lack of detectable p53 protein in HTB33, 34 and 35 associates with the presence of either HPV16 or -18 in these cell lines.


Elevated concentrations of endogenous ouabain in patients with congestive heart failure. An endogenous digitalis-like compound in mammals has long been postulated, but only recently has a substance indistinguishable from ouabain been identified in human plasma. Because of the potential significance of such a substance in patients with congestive heart failure, we sought to evaluate the pathophysiology of endogenous ouabain in these individuals. Using an immunoassay, we determined plasma ouabain concentrations in 51 patients with heart failure and in 19 control subjects. Plasma ouabain concentrations in control subjects ranged from 0.16 to 0.77 nM (mean, 0.44 +/- 0.20 nM). In 19 matched heart failure patients receiving digoxin, the mean ouabain was significantly elevated at 1.59 +/- 2.2 nM (range, 0.17-8.76 nM, p less than 0.05 versus control subjects). The ouabain concentration correlated inversely with both cardiac index (r = -0.62, p less than 0.005) and mean arterial pressure (r = -0.51, p less than 0.05). However, there was no correlation between ouabain and left ventricular filling (r = 0.19, NS) or right atrial pressures (r = 0.20, NS). In 16 heart failure patients not receiving digoxin, the mean ouabain was 1.52 +/- 2.58 nM. No relation between renal function and ouabain was detected. The unanticipated lack of correlation of ouabain with atrial pressures indicates that volume is not the chief determinant of ouabain concentration in patients with congestive heart failure. However, the significant relations of plasma ouabain concentration with cardiac index and mean arterial pressure imply that endogenous ouabain may be an important homeostatic factor in humans.


Cardiac persistence of cardioviral RNA detected by polymerase chain reaction in a murine model of dilated cardiomyopathy. In our model of dilated cardiomyopathy (DCM), cardiac dilatation and hypertrophy developed after inoculation of encephalomyocarditis virus (EMCV), but the infectious virus was isolated only early after infection. In this study, we investigated whether viral RNA could be detected at later times using the polymerase chain reaction (PCR). In the in vitro study, FL (human amnion) cells infected with EMCV were harvested for RNA extraction, and viral cDNA was synthesized by reverse transcription with random hexamers. Using oligonucleotide primers with homology to the 5' noncoding region of EMCV, we enzymatically amplified a 121-base pair band, which was homologous to a probe specific for EMCV as demonstrated by Southern blot hybridization. The sensitivity of this PCR technique was at the level of about 10(2)-10(3) copies of viral RNA genome. In the in vivo study, four-week-old DBA/2 mice were inoculated with EMCV intraperitoneally (10 pfu/mouse) and killed on days 1,2,3,5,7,10,14,18,28,60, and 90. The hearts were divided into three parts for purification of total RNA, histopathological examination, and to culture for infectious virus. The infectious virus was isolated from the heart after the second day but never after the 14th day. The viral genome was detectable by PCR on the second day, when very little mononuclear cell infiltration around the blood vessels was histologically visible. Positive PCR signals were observed in all hearts through day 14. Viral RNA was also detected in four of six 28-day samples, four of six 60-day samples, and two of seven 90-day samples when diffuse myocardial fibrosis was prominent, but myocardial necrosis or cellular infiltration had disappeared. The persistence of EMCV RNA was shown by PCR in the chronic stage of EMCV-induced myocarditis, a time when the inflammatory reaction had largely subsided. The PCR is a potentially useful method to test possible viral etiologies in idiopathic heart muscle disease or DCM.


Low-molecular-weight heparin reduces neointimal proliferation after coronary stent implantation in hypercholesterolemic minipigs. Intracoronary stents have been suggested as a method of reducing the restenosis rate after balloon angioplasty. Proliferation of vascular smooth muscle cells is a major contributing factor to the restenosis process. Heparin and some of its derivatives have been shown to inhibit smooth muscle cell proliferation. We investigated the effect of low-molecular-weight heparin on the proliferative response after implantation of a balloon-expandable tantalum stent in previously deendothelialized coronary artery segments of hypercholesterolemic minipigs. Minipigs were fed a diet containing 2% cholesterol, starting 1 month before balloon denudation of the endothelium in a coronary artery. One month later, a stent was implanted at this site. Animals were killed after 4 weeks (group 1, n = 6) or 3 months (group 2, n = 6). Animals in group 3 (n = 6), also followed for 4 weeks after stenting, received subcutaneous low-molecular-weight heparin at a dose of 200-300 units/kg anti-factor Xa activity per day in addition to the chronic acetylsalicylic acid (100 mg/day) also administered to groups 1 and 2. Eighteen of 22 animals survived to the end of the study. Angiography revealed patent stents in all surviving animals. In group 1, histological analysis showed extensive neointimal proliferation around stent struts. Maximal neointimal thickness seen in group 1 averaged 0.93 +/- 0.11 mm, was lower after 3 months (0.8 +/- 0.14 mm) in group 2, but was significantly reduced (0.44 +/- 0.18 mm, p less than 0.01) in group 3. These data show a significant reduction of the neointimal proliferative response to coronary stent implantation by low-molecular-weight heparin.


Tc-99m uptake in a parathyroid adenoma. Potential pitfall in Tc-99m/Tl-201 subtraction imaging. The authors describe a patient with primary hyperparathyroidism who had a large mediastinal parathyroid adenoma that avidly concentrated both Tc-99m pertechnetate and Tl-201. This unusual finding is presented as another potential reason for false-negative findings in Tc-99m/Tl-201 subtraction scintigraphy in hyperparathyroidism.


Decreased polymorphonuclear leucocyte chemotactic response to leukotriene B4 in cystic fibrosis. Evidence that leukotriene B4 (LTB4) is a significant inflammatory mediator in chronic pseudomonal respiratory disease was sought in adolescents and young adults with cystic fibrosis. Specific chemotaxis of peripheral blood polymorphonuclear leucocytes (PMN) was used as an indirect measure of remote in vivo exposure to LTB4. PMN from 17 patients showed a significant decrease in chemotaxis to 10(-7)-10(-9) M LTB4, but normal responses to 10(-8) M n-formyl-methionyl-leucyl-phenylalanine and 4 mg/ml casein, when compared with 17 healthy age- and sex-matched controls. This result is consistent with chronic production of LTB4, and specific deactivation of circulating PMN receptors for LTB4 in patients with cystic fibrosis. Pharmacologic inhibition of LTB4 production in vivo may help elucidate its role in the pathogenesis of lung damage in cystic fibrosis.


Ocular pharmacokinetics of rufloxacin a new fluoroquinolone antibiotic. Ocular pharmacokinetics of rufloxacin (MF 934), a new monofluorinated quinolone derivative, has been investigated in rabbits. A long half-life, good g.i. absorption and a higher tissue/plasma concentration than that of other quinolones, are its interesting pharmacokinetic properties. However, there is reason to believe that drug accumulation may occur in deep body compartments. We determined plasma, aqueous, and vitreous concentrations of the drug at 1, 4, 8, and 24h after a single 50 mg/kg i.v. administration of rufloxacin. Our data show that rufloxacin, administered by the i.v. route, rapidly reaches chemotherapeutically useful levels in aqueous and vitreous fluids. Although still present in plasma 8 hours after administration, it proved to be undetectable in ocular fluids, signifying that the depletion of the deep compartments occurs well in advance of the next invasion. Due to its antibacterial effectiveness and pharmacokinetic properties rufloxacin may take a relevant place among the quinolone derivatives in the treatment of ocular infections.


Fine-needle aspiration of cystic nephroma (multilocular cyst of the kidney). Cystic nephromas are rare tumors of the kidney most commonly affecting boys or adult females. The fine-needle aspiration cytomorphology has not yet been described. A renal cystic mass in a 56 year old female was aspirated under ultrasound guidance. Papanicolaou stained smears of the cyst fluid revealed markedly atypical cells forming papillary clusters. Subsequent nephrectomy showed a typical cystic nephroma with lining epithelium resembling that seen in the aspirate. The cytomorphology of cystic nephroma has been misdiagnosed as renal cell carcinoma in the literature. Low cellularity, absence of necrosis, and paucity of single cells are features that should raise the possibility of cystic nephroma in a cystic renal mass.


Detection of cytomegalovirus DNA in pulmonary specimens: confirmation by in situ hybridization in two cases. Papanicolaou stained bronchial brush and imprint pulmonary smears containing intranuclear and cytoplasmic inclusion bearing alveolar pneumocytes suggestive of cytomegalovirus infection were destained and reprocessed for in situ hybridization using a biotinylated probe for cytomegalovirus DNA. Two cases were processed in this way. A hybridization signal for viral DNA was noted in each case. However, no reddish brown staining reaction products were noted in any of the control samples. This simple and rapid nonradioactive detection system is a valuable supplement to routine pulmonary cytology for the definitive diagnosis of this virus infection, and this technique is also appropriate for retrospective study.


Fine-needle cytology of an eccrine spiradenoma of the breast: diagnosis made by a holistic approach. Eccrine spiradenoma is a rather rare adnexal tumor of the skin. When the clinical presentation is that of a breast neoplasm, diagnosis can be difficult. As cytology was a new procedure for this tumor, the approach of choice appeared to be a holistic one. The cytologic picture showed bland groups of uniformly sized cuboid cells with scant cytoplasm, round to ovoid nuclei, and inconspicuous nucleoli, while in the groups rosettelike structures could be discerned. Only by integrating the data of clinical history, inspection, palpation, reaction on breast puncture, and immunologic findings could the diagnosis be made.


Malignant rhabdoid tumor of soft tissues: a cytopathological and immunohistochemical study. We describe the cytopathological and immunohistochemical features of a primary malignant rhabdoid tumor (MRT) located in the left forehead region of an 8-mo-old female. The cardinal characteristics are hyaline cytoplasmic inclusions, rounded or bean-shaped nucleus, vesicular chromatin, central prominent nucleolus, and poorly defined cytoplasmic profile. Both the cytoplasm and the hyaline inclusion are vimentin and keratin positive, but negative with desmin, S-100 protein, neuron-specific enolase, (NSE) myoglobin, leukocyte common antigen (LCA), and alpha-1-antichymotrypsin. This immunohistochemical pattern, although not exclusive to MRT, is fairly typical of MRT, and when it is assessed in conjunction with cytopathological findings and clinical data permits a confident diagnosis of MRT. We emphasize the utility of immunohistochemical techniques in making the differential diagnosis of MRT against other childhood round-cell tumors.


Cytologic characteristics of congenital mesoblastic nephroma in fine-needle aspiration cytology: a case report. The cytologic features of congenital mesoblastic nephroma (CMN) as recognized in smears of fine-needle aspirated cytology (FNAC) are reported. These included spindle- and tadpole-shaped cells with round to oval nuclei having small nucleoli and a smooth contour. The cytoplasm of these cells was dense and homogeneously stained. The background was composed of mucoid fibrillar material. The findings appear to be different from other types of renal tumors in infancy and specific enough for this tumor to allow diagnosis by FNAC.


Benign metastasizing pleomorphic adenoma of salivary gland: diagnosis of bone lesions by fine-needle aspiration biopsy. Two cases of benign salivary gland pleomorphic adenomas metastatic to bone (benign-metastasizing pleomorphic adenomas) diagnosed by fine-needle aspiration biopsy are presented. Both primary tumors were slightly atypical cytologically but neither case demonstrated features of carcinoma. The metastatic lesions contained benign epithelial, myoepithelial, and stromal components. In both cases the clinical history was either not known by the radiologist or not communicated to the cytopathologist interpreting the case, and a primary tumor of bone was the leading clinical diagnosis. Obtaining pertinent clinical history and comparing the cytomorphology of the bone aspirate with the primary parotid tumor allowed for an accurate diagnosis in both cases. The differential diagnosis with primary bone tumors is discussed and the importance of clinical history is emphasized.


Needle aspiration cytology, immunocytochemistry, and electron microscopy in a rare case of secretory carcinoma of the breast in an elderly woman. Needle aspiration was performed on a breast mass in a 91-year-old woman. The cytologic features in the aspirate were a diffuse, prominent, intracytoplasmic vacuolization and secretion in malignant cells and occasional signet ring-like forms. This was confirmed in a subsequent cell block which was made from the aspirate. Immunocytochemical studies showed a positivity for mucin by alcian blue stain in the vacuolated cells which was periodic acid-Schiff positive and resistant to diastase digestion. Oil-red-O staining was negative and on Colloidal iron stain the tumour cells were positive. Immunopositivity to carcinoembryonic antigen, cytokeratin, and epithelial membrane antigen was found in the malignant cells, while on electron microscopy the tumour cells contained a significant amount of intracytoplasmic secretory material. Secretory carcinoma of the breast is a rare tumour and can be diagnosed and differentiated from other breast carcinomas in view of its characteristic cytologic features.


[Persistent BVD virus infections in a cattle breeding facility--a case report]. Comprehensive serological and virological monitoring for bovine viral diarrhoea (BVD) virus was applied in a dairy herd. Out of 83 calves 26 persistently infected animals were identified. Four viremic calves showed clinical signs of disease, the others displayed no symptoms. Viral isolates from persistently infected animals were homogenous with respect to their antigenicity. The results of virological and serological investigations allowed an almost complete reconstruction of events following the introduction of BVD virus into the herd. This case illustrates the potentially dangerous and damaging effects of unidentified virus carriers in cattle herds. Strategies for the identification of virus-shedding animals and the limitation of economical losses are discussed.


[The significance of bredavirus as a diarrhea agent in calf herds in Lower Saxony]. The objective of this investigation was to determine the distribution of Bredavirus in cattle herds in Lower Saxony and to evaluate its significance as potential cause of diarrhea in calves. Fecal samples and paired blood samples of 119 diarrheic and 46 healthy calves up to two months of age were collected from herds where diarrhea of calves was a problem. Fecal samples were examined for Breda-, rota- and coronavirus by solid phase immune electron microscopy and by ELISA, for K99-positive E. coli and salmonella by microbiological methods, and for cryptosporidia in smears. Antibody titers against Bredavirus, total serum protein and serum gamma globulin content were evaluated in the blood samples. Bredavirus was found in fecal samples from 5% (n = 6) of diarrheic calves which came from four different herds, but not in healthy calves. Rotavirus (31.9%), coronavirus (18.5%) and cryptosporidia (29.9%) were detected more frequently in fecal samples than Bredavirus. In this investigation rotavirus, coronavirus and cryptosporidia were present in addition in all herds where Bredavirus was found. In contrast to the low percentage of fecal samples containing Bredavirus, antibody titers in 75% of calves confirmed the high prevalence of Bredavirus infection in the cattle population of Lower Saxony.


Regulation and role of PDGF receptor alpha-subunit expression during embryogenesis. The platelet-derived growth factor receptor alpha-subunit (PDGFR alpha) is the form of the PDGF receptor that is required for binding of PDGF A-chain. Expression of PDGFR alpha within the early embryo is first detected as the mesoderm forms, and remains characteristic of many mesodermal derivatives during later development. By 9.5 days of development, embryos homozygous for the Patch mutation (a deletion of the PDGFR alpha) display obvious growth retardation and deficiencies in mesodermal structures, resulting in the death of more than half of these embryos. Mutant embryos that survive this first critical period are viable until a new set of defects become apparent in most connective tissues. For example, the skin is missing the dermis and connective tissue components are reduced in many organs. By this stage, expression of PDGFR alpha mRNA is also found in neural crest-derived mesenchyme, and late embryonic defects are associated with both mesodermal and neural crest derivatives. Except for the neural crest, the lens and choroid plexus, PDGFR alpha mRNA is not detected in ectodermal derivatives until late in development in the central nervous system. Expression is not detected in any embryonic endodermal derivative at any stage of development. These results demonstrate that PDGFR alpha is differentially expressed during development and that this expression is necessary for the development of specific tissues.


Polyphosphoinositide metabolism during the fertilization wave in sea urchin eggs. A transient increase in intracellular free calcium is believed to be the signal responsible for the stimulation of the egg metabolism at fertilization and the resumption of the cell cycle. We have studied how the polyphosphoinositides (PPI) turn over at fertilization in sea urchin eggs, in order to determine the relationship between the metabolism of these lipids and the calcium signal. We compare the patterns of PPI turnover that occur during the first minute following fertilization in eggs in which PPI are labelled to steady state with [3H]inositol or [3H]arachidonate with that in which PPI are labelled for a shorter period with [3H]inositol. When eggs are labelled to apparent isotopic equilibrium with either [3H]inositol or [3H]arachidonate, no early increase in [3H]PtdInsP2 occurs while PtdIns decreases slightly. On the contrary, when not labelled to isotopic equilibrium, all [3H]PPI increase during the first 15 seconds following fertilization. We find that, within seconds, fertilization triggers a 600-fold increase in the turnover of PPI, producing an amount of InsP3 apparently sufficient to trigger calcium release. We suggest that phosphoinositidase C and PtdInsP kinase, responsible respectively for the hydrolysis and synthesis of PtdInsP2, are both stimulated to a comparable degree in the first 30 seconds following fertilization and that net changes in the amount of PtdInsP2 at fertilization are very sensitive to the relative levels of activation of the two enzymes. Activating the eggs with the calcium ionophore A23187 showed that both these enzymes are sensitive to calcium, suggesting that calcium-dependent InsP3 production might play a role in the initiation and/or the propagation of the fertilization calcium wave.(ABSTRACT TRUNCATED AT 250 WORDS)


Developmental expression of the alpha receptor for platelet-derived growth factor, which is deleted in the embryonic lethal Patch mutation. The alpha receptor of PDGF (Pdgfra) is expressed in primitive endoderm and mesoderm derivatives throughout embryogenesis. In the early primitive streak stage the gene is transcribed in the visceral and parietal endoderm. Later it is expressed in the presomitic mesoderm, yolk sac and amnion. During somitogenesis its transcription localizes to the heart and the somites. Subsequently, it is transcribed in the dermatome, the sclerotome, the developing limb and in various mesenchymal tissues of visceral organs. Its wild-type expression pattern correlates well with the phenotype of homozygous mutant Patch (Ph) embryos, where the Pdgfra gene is deleted. The Ph phenotype is first detectable at the primitive streak stage with convoluted and hypertrophic visceral yolk sac, deformed neural plate and disorganized or missing mesoderm. Most Ph/Ph embryos die before the 11th day of gestation. Those that survive till early organogenesis are very small, have hypertrophic yolk sacs, small and undifferentiated somites, convoluted neural tubes, large heart and pericardium, rudimentary limb buds and branchial arches. Our observations together suggest that the alpha PDGF receptor may be required for the normal development of visceral endoderm and mesoderm derivatives.


Role of the carboxyl-terminal half of the extracellular domain of the human thyrotropin receptor in signal transduction. We studied the role of the carboxyl-terminus of the extracellular region of the human TSH receptor in signal transduction (cAMP generation). For this purpose, we introduced homologous substitutions of smaller segments within amino acids 261-418 (domains D and E) of the TSH receptor with the corresponding amino acids of the rat LH/CG receptor. Amino acids 317-366 were not investigated in view of previous data indicating their noninvolvement. Mutant TSH receptor cDNAs, in a eukaryotic expression vector, were stably transfected into Chinese hamster ovary cells. Eight of nine plasmid constructs expressed TSH receptors that could be detected by radiolabeled TSH binding; six of these were of high affinity similar to the wild-type receptor and, therefore, provided informative data on signal transduction. Despite high affinity TSH binding, five of six TSH receptor mutants displayed a diminished cAMP response to TSH stimulation, suggesting the involvement of broad segments of domains DE in signal transduction. Amino acids 270-278 and 287-297 were particularly important in this respect. The conformation conferred by these segments of the TSH receptor, therefore, appears to be involved in transducing a signal from the extracellular to the intracellular region of the receptor.


N-acetylation is required for the lactotrope recruitment activity of alpha-melanocyte-stimulating hormone and beta-endorphin. Pituitary neurointermediate lobe (NIL) cells obtained from ovariectomized rats and exposed to 17 beta-estradiol in vitro have been shown to release an activity that induces acute recruitment of additional PRL-secreting cells. We have recently reported that alpha MSH, a major secretory product of the NIL, can substitute for this lactotrope-recruiting factor released by NIL cells in response to 17 beta-estradiol. beta-Endorphin (beta END) was not effective in this regard. Inasmuch as the degree of acetylation is critical to the activities of both of these molecules in other systems, we decided to assess its importance to lactotrope-recruiting activity in the present study. Anterior pituitary cells from ovariectomized rats were cultured overnight, exposed to various treatments for 3 h, and then subjected to a reverse hemolytic plaque assay for PRL. Exposure to mono- and diacetylated alpha MSH (N-ac-alpha MSH and di-ac-alpha MSH, respectively) or N-acetylated beta END (N-ac-beta END) caused a significant increase in the fraction of anterior pituitary cells that released PRL. In contrast, the Des-acetylated variants of both molecules had no lactotrope-recruiting activity. In a dose-response study, maximally effective doses of di-ac-alpha MSH and N-ac-beta END were equally effective with respect to their function as lactotrope-recruiting factors. Furthermore, the two peptides acted cooperatively when simultaneously applied in submaximal concentrations. Taken together, these results demonstrate that 1) N-acetylation is an essential requirement for the lactotrope-recruiting activity of alpha MSH and beta END in vitro; and 2) di-ac-alpha MSH and N-ac-beta END can act in a cooperative fashion to recruit additional cells into the PRL-secreting population.


Dissociation of antiproliferative and antihormonal effects of the somatostatin analog octreotide on 7315b pituitary tumor cells. The somatostatin (SS) analog octreotide has been successfully used in the treatment of (neuro)endocrine tumors. The mechanism of action of the tumor (growth) inhibitory action by octreotide is not fully understood. We have investigated the effect of octreotide on 7315b rat pituitary tumor cell growth, PRL release, and intracellular PRL concentrations in vitro. When cultured in medium with 10% fetal calf serum, the number of high affinity SS receptors increased with increasing culture time. On days 7, 14, and 21 of culture, the number of SS receptors amounted to 978 +/- 217, 3588 +/- 705, and 5865 +/- 3332 fmol/mg protein, respectively, whereas they were not measurable on day 0. From days 0-7, 7-14, and 14-21 of culture, octreotide (1 pM to 1 microM) inhibited PRL release and the intracellular PRL concentration, with IC50 values in the nanomolar range. However, no inhibition of cell growth was observed by these octreotide concentrations from day 0-7 of culture, while octreotide inhibited cell growth in a dose-dependent fashion from days 7-14 and 14-21 of culture (maximal inhibition by 25% and 26%, respectively). In a series of nine consecutive experiments we found a significant positive correlation between the percent inhibition of cell growth induced by 1 microM octreotide and the number of SS receptors on 7315b cells (r = 0.7865; P = 0.012). Inhibition of PRL release did not correlate with SS receptor numbers. Octreotide (1 microM) inhibited forskolin (0.5 microM)-stimulated cell growth and intracellular PRL concentrations, while in the presence of a high concentration of forskolin (10 microM), octreotide had no effect on forskolin-stimulated cell growth and intracellular PRL concentrations. In addition, its PRL release inhibitory effect was significantly lower in forskolin-stimulated cultures. Pretreatment of the cells with pertussis toxin (10 micrograms/liter) completely prevented the inhibition of cell growth by octreotide and diminished the inhibitory effect of octreotide on PRL release. Finally, 1 microM octreotide significantly inhibited forskolin-stimulated cAMP production (by 29% and 53% on days 7 and 14 of culture, respectively). We conclude that 1) octreotide inhibits 7315b rat pituitary tumor cell proliferation via a pertussis toxin-sensitive GTP-binding protein- and adenylate cyclase-dependent mechanism; and 2) the number of SS receptors on 7315b pituitary tumor cells may determine whether octreotide exerts a direct antiproliferative effect, whereas its antihormonal effect occurs in the presence of relatively low numbers of SS receptors. This suggests a dissociation of the antiproliferative and antihormonal effects induced by octreotide.


Feedback sensitivity of the rat hypothalamo-pituitary-adrenal axis and its capacity to adjust to exogenous corticosterone. Chronic stress causing elevated morning (AM) corticosterone (B) concentrations of 2-8 micrograms B/dl does not appear to inhibit subsequent activity in the hypothalamic-pituitary-adrenal (HPA) axis, a surprising finding in view of the known depression in AM basal ACTH by only 3 micrograms B/dl in adrenalectomized rats. To distinguish between the possibilities that either intact rats are less sensitive to B feedback than adrenalectomized rats, or that chronic stress facilitates responses in the HPA axis, we elevated basal B levels in young male rats with slow-release B pellets in the absence of stress. Between 4-6 days after implantation of B pellets at three doses that elevated basal AM (diurnal trough) plasma B to approximately 1.2, 4, and 10 micrograms/dl, we studied basal ACTH and B at trough (AM) and peak evening (PM) times of the diurnal cycle, as well as the responses to the stress of restraint and blood collection from the tail at each time of day. We also determined mean daily plasma B, insulin, and glucose from samples collected at six intervals during the day. Adrenal, thymus, and body wts were measured as were transcortin (CBG) and adrenal phenylethanolamine-N-methyl transferase activity. Compared to controls implanted with wax pellets, all doses of B inhibited adrenal wt and AM stress responses and tended to inhibit pituitary ACTH content and adrenal phenylethanolamine-N-methyl transferase activity. Inhibition with the middle dose B pellet was close to maximally effective for these endpoints. Plasma glucose and thymus wt were significantly decreased and insulin was significantly increased in the middle and highest B pellet groups, with significantly greater effects at the highest dose. The gain in body wt and transcortin concentrations were significantly decreased only in the highest dose groups, in which mean daily plasma B was approximately 10 micrograms/dl, a level that clearly overwhelmed the capacity of the adrenocortical system to respond to any stimulus tested. By contrast, rats with low and middle dose B pellets appeared to adjust HPA axis function by decreasing the peak diurnal increase in B, so that 24-h mean B levels did not differ from control, and were maintained at approximately 5 micrograms/dl. Both of these groups also had inhibited ACTH responses to stress applied during the diurnal trough (AM). By contrast, neither group had inhibited ACTH responses to stress applied during the diurnal peak (PM). We conclude that: 1) The HPA axis of intact rats is extremely sensitive to exogenous B.(ABSTRACT TRUNCATED AT 400 WORDS)


Epidermal growth factor, a phorbol ester, and 3',5'-cyclic adenosine monophosphate decrease the transcription of the luteinizing hormone/chorionic gonadotropin receptor gene in MA-10 Leydig tumor cells. In a recent series of experiments we have shown that the previously recognized ability of mouse epidermal growth factor (mEGF), cAMP, or phorbol 12-myristate 13-acetate (PMA) to reduce the density of LH/CG receptors in MA-10 cells is secondary to a reduction in receptor messenger RNA (mRNA). As a follow-up to these studies we now present experiments designed to determine if the reduction in LH/CG receptor mRNA is due to a decrease in transcription of the receptor gene and/or an increase in the rate of degradation of the mRNA. The potential effects of mEGF, cAMP, or PMA on the degradation of the LH/CG receptor mRNA were measured in MA-10 cells treated with Actinomycin D or in kidney cells permanently transfected with the LH/CG receptor complementary DNA driven by a heterologous promoter. Both experimental strategies revealed that none of these compounds increase the rate of degradation of the receptor mRNA. If anything, a stabilizing effect was noted. The potential effects of mEGF, cAMP, or PMA on the transcription of the LH/CG receptor gene in MA-10 cells were measured using nuclear run-off assays. All three compounds induced a rapid decrease in the transcription of the LH/CG receptor gene. The time course of these effects is similar, and by 2 h all of the stimuli had decreased transcription to 15-30% of control. Our studies show that the mEGF-, cAMP- and PMA-induced down-regulation of the LH/CG receptor in MA-10 cells is primarily (if not entirely) due to a decrease in the transcription of the receptor gene.


Transforming growth factor-beta 1 inhibits aldosterone and stimulates adrenal renin in cultured bovine zona glomerulosa cells. Transforming growth factors-beta (TFG beta s) are multifunctional peptides that affect proliferation, differentiation, and many other functions in a variety of cell types. In this study we examined the effect of TGF beta 1 on aldosterone and adrenal renin production using cultured bovine adrenal zona glomerulosa cells. Collagenase-dispersed zona glomerulosa cells were incubated in PFMR-4 medium containing 10% fetal calf serum for 72 h, and the medium was replaced with serum-free medium for the next 24 h. The cells during this 24-h period were exposed to TGF beta 1, ACTH, and (Bu)2cAMP (dbcAMP). It was observed that TGF beta 1 at 1 nM 1) inhibited basal aldosterone secretion from 680.0 +/- 40.0 to 270.0 +/- 10.0 pg/10(6) cells.h, 2) inhibited ACTH- and dbcAMP-stimulated aldosterone production, 3) increased levels of active renin in the cells from 17.8 +/- 2.5 to 70.7 +/- 4.4 pg angiotensin-I/10(6) cells.h and prorenin from 270.0 +/- 5.0 to 970.0 +/- 90 pg angiotensin-I/10(6) cells.h, 4) stimulated prorenin in the medium synergistically in combination with ACTH and dbcAMP, and 5) had no significant effect on basal cAMP production, but significantly inhibited the ACTH-stimulated production of cAMP. These observations show that TGF beta 1 is a potent inhibitor of basal and ACTH- and cAMP-stimulated aldosterone production and inhibits ACTH-stimulated cAMP production. Contrary to its effect on aldosterone, TGF beta 1 stimulates the synthesis and release of adrenal renin and prorenin. TGF beta 1 may act as an autocrine or paracrine regulator of aldosterone production.


Biochemical mechanisms involved in monomethyl succinate-induced insulin secretion. Esters of succinic acid stimulate insulin secretion from pancreatic beta-cells. Using collagenase-isolated rat islets, the transduction mechanisms involved were investigated. In freshly isolated perifused islets, monomethyl succinate (MMSucc), in the presence of basal (2.75 mM) glucose, stimulated insulin release in a biphasic pattern. This secretory response was dependent on extracellular calcium movement into the beta-cell, since the calcium channel blocker nitrendipine (5 microM) abolished it. The glucokinase inhibitor mannoheptulose (20 mM) had no effect on its secretory action, while the protein kinase-C inhibitor staurosporine (20 nM) reduced secretion to MMSucc. In addition, while ineffective alone, the diacylglycerol kinase inhibitor monooleoylglycerol (25 microM) potentiated MMSucc-induced insulin release. A similarly amplified response occurred in the presence of forskolin (0.25 microM), a compound that elevates islet cAMP levels. The sodium salt of succinic acid (20 mM) had no effect on insulin release in the presence or absence of forskolin. Prior treatment with MMSucc in the presence of 2.75 mM glucose sensitized islets to the usually weak insulin secretory effect of 7.5 mM glucose. Other groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide pools. These islets were subsequently stimulated, and the kinetics of [3H]inositol efflux and insulin secretion were measured. MMSucc induced a rapid and sustained dose-dependent increase in [3H]inositol efflux rates. In batch-incubated islets, MMSucc increased inositol phosphate levels. Finally, MMSucc (20 mM), in the presence of 8 mM glucose, did not influence the detritiation of [5-3H]glucose, but reduced the oxidation of [U-14C] glucose. These results support the following conclusions. First, MMSucc is a potent activator of islet phosphoinositide hydrolysis. Second, the activation of protein kinase-C appears to contribute to the acute insulin secretory effect of MMSucc. Third, MMSucc-induced increases in phosphoinositide hydrolysis contribute at least in part to its ability to acutely stimulate insulin release and prime the beta-cell to subsequent stimulation. Finally, mitochondrial events associated with the oxidative metabolism of MMSucc may underlie its insulinotropic action.


Pituitary corticotroph hyperplasia in rats implanted with a medullary thyroid carcinoma cell line transfected with a corticotropin-releasing hormone complementary deoxyribonucleic acid expression vector. CRH stimulates both the synthesis and release of ACTH and other derivatives of POMC by the adenohypophysis. It is uncertain, however, whether it also causes proliferation of corticotrophs. Patients with CRH-producing tumors develop Cushing's syndrome, and some have been reported to have pituitary corticotroph hyperplasia. We now report an animal model that accurately reproduces the human disorder of ectopic production of CRH by a neoplasm. Prolonged CRH secretion by a transplanted medullary thyroid carcinoma cell line stably transfected with a CRH cDNA under transcriptional control of a cytomegalovirus promoter resulted in corticotroph hyperplasia and hypertrophy; the percentage of ACTH-containing cells in animals bearing W2CRH tumors was increased at 9.8 +/- 0.5% (controls, 6.2 +/- 0.3%; W2 implanted tumors, 7.7 +/- 0.4%). Occasional mitotic figures were identified, and the cells were larger, with abundant cytoplasm but generally less intense immunohistochemical staining for ACTH due to relative degranulation compared to controls. Melanotrophs of the intermediate lobe were also increased in number and were larger, with abundant cytoplasm. No corticotroph adenomas were found. Our experiment accurately reproduces the gradually increasing CRH levels in the general circulation produced by a growing tumor, as found in the human ectopic CRH syndrome, and confirms that long term exposure to CRH excess, as produced by a tumor, results in an increased number of corticotrophs in the adenohypophysis.


Dissociation of actin polymerization and enzyme inactivation in the hormonal regulation of type II iodothyronine 5'-deiodinase activity in astrocytes. T4 dynamically regulates the levels of type II iodothyronine 5'-deiodinase in the brain. Using an astrocyte cell culture model, we have shown that thyroxine increases inactivation of this enzyme through a mechanism using the actin cytoskeleton. In the absence of T4, the filamentous actin (F-actin) stress fibers are absent, and deiodinase inactivation is relatively slow. T4 increases inactivation of type II 5'-deiodinase by 1) restoring the F-actin stress fibers, 2) promoting the binding of the enzyme to F-actin, and 3) stimulating enzyme internalization. To determine whether inactivation of the deiodinase is due solely to the restoration of stress fibers by T4 or also involves direct thyroxine-mediated enzyme-F-actin interactions, we examined the effects of retinoids on both actin polymerization and type II 5'-deiodinase activity in cultured astrocytes, as these hormones have been shown to alter cytoskeletal organization in other tissues. In thyroid hormone-deficient astrocytes, retinoic acid increased F-actin levels, with no change in total cell actin. The F-actin content increased approximately 40% within 30 min after the addition of retinoic acid. After a plateau of 6-8 h, the F-actin content increased further to approximately 90% of the total cell actin and was associated with the reappearance of stress fibers. Only this latter retinoid-stimulated increase in F-actin content was blocked by actinomycin-D. Restoration of the F-actin stress fibers by retinoids did not increase the turnover of the type II 5'-deiodinase (t1/2, 1.99 h-1) or promote binding of the enzyme to F-actin in the absence of T4. Similarly, retinoids did not affect the rapid T4-mediated turnover (t1/2, 0.18 h-1) of type II 5'-deiodinase. These data show that an intact F-actin cytoskeleton in the absence of T4 is inadequate to alter the inactivation of type II 5'-deiodinase and that specific T4-enzyme-F-actin interactions are necessary to initiate the rapid inactivation/internalization of this enzyme.


Modulation of luteinizing hormone-stimulated inositol phosphate accumulation by phorbol esters in bovine luteal cells. The present studies were conducted to evaluate the effects of protein kinase C activators on the inositol phospholipid-phospholipase C second messenger system in isolated bovine luteal cells. This report describes the effects of phorbol esters on inositol phosphate accumulation in LH- and prostaglandin F2 alpha (PGF2 alpha)-stimulated bovine luteal cells. Corpora lutea of early pregnancy were dispersed with collagenase and luteal cells were prelabelled for 3 h with [3H]inositol. Inositol phosphates produced in response to LH or PGF2 alpha were analyzed by ion exchange column chromatography. The tumor promoter and protein kinase C activator 12-O-tetradecanolyphorbol 13-acetate (TPA) had no effect on basal levels of inositol phosphates but inhibited LH-stimulated accumulation of inositol mono-, bis-, and trisphosphates by 72%, 68%, and 65%, respectively. TPA reduced the response to maximally effective concentrations of LH and tripled the concentrations of LH required to evoke half-maximal accumulation of inositol mono-, bis-, trisphosphates. The inhibitory effects of TPA were rapid (5 min) whether added before or after treatment with LH. Treatment with TPA also reduced (58%) the initial phase of intracellular calcium mobilization in LH-treated cells. The inhibitory effects of TPA were not associated with acute reductions in [3H]inositol incorporation, [3H]inositol phospholipid levels, cAMP levels, or progesterone accumulation in control or LH-stimulated luteal cells. The effects of phorbol esters were concentration dependent and specific for active tumor promoters with 10-50 nM TPA producing maximal inhibitory effects. A synthetic diacylglycerol, 1-oleyl-2-acetylglycerol, mimicked the inhibitory effects of TPA. In contrast, pretreatment with a physiological activator of protein kinase C, PGF2 alpha, had no effect on LH-stimulated inositol phosphate accumulation. The inhibitory effects of TPA could not be explained by a generalized inhibition of phospholipase C or G-proteins since the accumulation of inositol phosphates in PGF2 alpha- and NaF-treated cells was not inhibited by TPA. These results demonstrate that tumor promoting phorbol esters modulate the inositol phospholipid-phospholipase C transmembrane signaling system in LH-stimulated bovine luteal cells. The results suggest that phorbol esters may alter the coupling of the LH-receptor complex to phospholipase C. These findings implicate protein kinase C in the regulation of transmembrane signaling in the bovine corpus luteum.


A certain step of proteolytic processing of proopiomelanocortin occurs during the transition between two distinct stages of secretory granule maturation in rat anterior pituitary corticotrophs. To obtain antibodies specific for precursor POMC and its cleaved peptides, we prepared synthetic peptides corresponding to the cleavage site (ST-1) between ACTH and beta-lipotropic hormone moieties of POMC and to amidated joining peptide (JP; ST-3) and generated their polyclonal antibodies in rabbits. The anti-ST-1 serum immunoprecipitated only POMC in the extract of AtT-20 cells. In addition to a similar immunoprecipitation examination, an immunoabsorption test showed that anti-ST-3 serum recognizes cleaved peptides, 16K amino-terminal peptide, and JP, but not POMC precursor. Double immunolabeling with gold particles of different sizes for the precursor POMC and its cleaved peptides revealed that the former was localized in electron-dense secretory granules, whereas the latter was found in the electron-lucent secretory granules in the same ultrathin cryosections. Colocalization of POMC and its cleaved peptides in the same secretory granules was rarely observed. Only POMC immunolabeling was detected in the Golgi cisternae. These results suggest that a certain step of proteolytic processing of POMC occurs in the secretory granules during the transition between the two distinct stages of secretory granule maturation in the rat anterior pituitary corticotroph.


Expression of recombinant human follicle-stimulating hormone receptor: species-specific ligand binding, signal transduction, and identification of multiple ovarian messenger ribonucleic acid transcripts. The ligand specificity and biochemical properties of the human (h) FSH receptor are poorly characterized due to the low abundance of these receptors and the limited availability of human tissues. Using a fragment of rat FSH receptor cDNA, we screened a human testicular cDNA library and obtained a FSH receptor cDNA covering the entire amino acid-coding region. After transfection of a human fetal kidney cell line (293) with the hFSH receptor cDNA, radioligand receptor analysis revealed the presence of high affinity (Kd, 1.7 x 10(-9) M) FSH-binding sites on the plasma membrane. Both recombinant and wild-type hFSH displaced [125I]hFSH binding, with ED50 values of 25 and 70 ng/ml, respectively, whereas hLH, hCG, and hTSH were ineffective. Although human, rat(r), and ovine FSH as well as equine CG competed for rat testicular FSH receptor binding, only hFSH and rFSH interacted effectively with the recombinant hFSH receptor, suggesting that species-specific ligand recognition exists between human and rodent receptors. After incubation of transfected cells with hFSH, but not recombinant hLH or hCG, a dose-dependent increase (ED50, 10 ng/ml) in extracellular cAMP accumulation was observed, indicating a functional coupling of the expressed human receptor with the endogenous adenyl cyclase. In cells cotransfected with the FSH receptor expression plasmid and a luciferase reporter gene driven by the promoter of a cAMP-responsive gene, treatment with hFSH, but not hCG, resulted in a dose-dependent increase in luciferase activity. Northern blot analysis using a cRNA probe derived from the human receptor cDNA indicated the presence of multiple FSH receptor mRNA transcripts (7.0, 4.2, and 2.5 kilobases) in RNA prepared from human follicular phase ovary, but not from human corpus luteum or placenta. Additionally, two FSH-binding sites of 76 and 112 kilodaltons were detected in transfected 293 cells after ligand/receptor cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These results demonstrate the expression of functional hFSH receptor with unique ligand specificity and provide new data on the biochemical properties of the human receptor at the mRNA and protein levels.


The role of the adrenal gland in hypertensive transgenic rat TGR(mREN2)27. The TGR(mREN2)27 is a new monogenetic rat model in hypertension research. As the mouse Ren-2d renin gene is integrated into their genome, they develop fulminant hypertension between 5 and 15 weeks of age, with blood pressure maxima of 300 mm Hg. Their plasma renin-angiotensin system (RAS) is suppressed, but the transgene is highly expressed in the adrenal gland, so we investigated its possible role in steroid metabolism and the pathogenesis of hypertension. During the phase of hypertension development (between 6-18 weeks), the urinary excretion of deoxycorticosterone (DOC), corticosterone (B), 18-hydroxycorticosterone, and aldosterone is 1.5- to 2.5-fold elevated compared with that in Sprague-Dawley (SD) rats (P less than 0.0005) despite the suppressed plasma RAS. Moreover, the adrenal gland in TGR(mREN2)27 shows an increased maximal response to ACTH stimulation in regard to urinary excretion of DOC (after ACTH, 244 +/- 42 ng/24 h in TGR; 62 +/- 10 ng/24 h in SD; P less than 0.0005) and B (after ACTH, 5144 +/- 346 ng/24 h in TGR; 2607 +/- 324 ng/24 h in SD; P less than 0.0005). Additionally, plasma prorenin in TGR was stimulated more than 10-fold, indicating transgene regulation by ACTH. Since spironolactone treatment did not lower the blood pressure in TGR, hypertension solely due to hypermineralocorticoism is unlikely. Our results indicate that the adrenal steroid metabolism is markedly stimulated in young TGR, and the absolute increase in urinary DOC and B after ACTH injections is enhanced, possibly due to a stimulated local intraadrenal RAS.


Chronic psychosocial stress enhances vasopressin, but not corticotropin-releasing factor, in the external zone of the median eminence of male rats: relationship to subordinate status. Male Wistar rats living in hierarchically structure male/female colonies were used to investigate the effects of chronic psychosocial stress on the hypothalamus-pituitary-adrenal system. Colony-housed subordinates were compared to control rats housed in male-female pairs. Classical parameters of chronic stress (thymus involution, impaired somatic growth, and elevated resting plasma corticosterone level) were found in all subordinate rats. Changes in vasopressin (AVP) and CRF stored in the external zone of the median eminence (ZEME) were measured by quantitative immunocytochemistry. Chronic psychosocial stress for 19-28 days increased AVP immunostaining in the ZEME to 160-190% of that in pair-housed controls, whereas CRF immunostaining in the ZEME remained unchanged. Within colonies, subordinates differed in avoidance behavior and aggression received (subordinate status). This intracolony subordination rank was correlated with AVP in the ZEME (P less than 0.01). Although resting corticosterone was elevated in subordinate rats (P less than 0.01), the increase in AVP was not associated with detectable secretion of AVP and/or CRF from the ZEME, as measured after blockade of axonal transport. In control rats, interaction with a dominant male increased plasma ACTH and corticosterone levels and caused depletion of AVP, but not CRF, from the ZEME. Subordinates showed suppressed hypothalamic (AVP depletion), pituitary (plasma ACTH) and adrenal (plasma corticosterone) responses to interaction with the dominant male, which may reflect suppressive actions of elevated corticosterone on CRF neurons or suprahypothalamic centers.


Aging of FRTL-5 rat thyroid cells causes sensitivity to cytotoxicity induced by tumor necrosis factor-alpha. While investigating the modulation of the growth and function of the FRTL-5 rat thyroid cell line by recombinant human tumor necrosis factor-alpha (TNF alpha), we noticed that pronounced changes in several response parameters occurred with increasing passage number. For young cells (passage less than 20), TNF alpha by itself slightly increased [3H]thymidine incorporation and DNA content, and had a minimal effect on basal 125I uptake. When combined with TSH, TNF alpha had no influence on TSH-stimulated [3H]thymidine incorporation, but significantly inhibited TSH-stimulated 125I uptake. Compared with young cells, aged cells (passage greater than 40), in contrast, developed a high sensitivity to TNF alpha. TNF alpha markedly stimulated [3H]thymidine incorporation into DNA, inhibited TSH-stimulated 125I uptake per micrograms DNA, but dramatically decreased the total DNA content and cell number. TSH augmented the TNF alpha effect in aged cells, resulting in a further reduction of DNA content. Aphidicolin, a specific inhibitor of DNA polymerase-alpha which is associated with DNA replication, dramatically inhibited TNF alpha-induced [3H]thymidine incorporation in both young and aged cells; this suggested that the effect of TNF alpha on FRTL-5 cell growth is related to DNA replication, rather than DNA repair. 51Cr release from FRTL-5 cells, a measure of cytotoxicity, increased 2-fold over baseline in aged cells at a dose of 400 ng/ml TNF alpha and decreased to 70% of baseline in young cells at this same dose. The protein kinase-A (PKA) and protein kinase-C (PKC) signal transduction mechanisms of TNF alpha in aged cells (passage greater than 40) were also studied. TNF alpha increased cAMP and also increased relative PKA and PKC activity in 1-40 min. Phorbol myristate acetate (PMA), an activator of PKC, increased [3H]thymidine incorporation and DNA content. PMA did not affect the TNF alpha-induced increase in [3H]thymidine incorporation or its reduction of DNA content. When the cells were pretreated with a high concentration of PMA (1 microM/24 h) to down-regulate PKC, the TNF alpha dose-dependent increase in [3H]thymidine incorporation and decrease in DNA content were only slightly inhibited, suggesting that the main effects of TNF alpha are independent of PKC. We conclude that the sensitivity of FRTL-5 cells to the cytotoxic effect of TNF alpha increases with aging.


Structure-function relationships for the effects of various aminoglycoside antibiotics on dispersed bovine parathyroid cells. We previously showed that the polycationic aminoglycoside antibiotic neomycin mimics the effects of high extracellular calcium (Ca2+) concentrations on several aspects of parathyroid function. In the present studies we examined the actions of several additional aminoglycosides on dispersed bovine parathyroid cells to investigate the relationship between antibiotic structure and function in eliciting Ca(2+)-like effects on intracellular second messengers and PTH release. Of the antibiotics tested, those with six amino groups (neomycin-B and -C) were most potent in inhibiting dopamine-stimulated cAMP accumulation, showing IC50 values (the concentration producing a half-maximal inhibitory effect) of 7.7 x 10(-5) and 1.5 x 10(-4) M. Gentamicin-C, paromomycin, and tobramycin, which have five amino groups, were less potent, with IC50 values of 4 x 10(-4), 10(-3), and 3.3 x 10(-4) M, respectively, while gentamicin-B, kanamycin, and ribostamycin, with four amino groups, were least potent (respective IC50 values, 2.0, 2.9, and 3 x 10(-3) M). These antibiotics showed a similar order of potency for inhibiting PTH release, with a close correlation between their IC50 values for modulating cAMP accumulation and PTH release (r = 0.98; P less than 0.001). Finally, they showed qualitatively similar potencies for eliciting transient increases in the cytosolic free Ca2+ concentration arising from the release of Ca2+ from intracellular stores. Neomycin-B and -C both acted at 10(-4) M; gentamicin-C, paromomycin, and tobramycin evoked free intracellular Ca2+ spikes at 1.4 x 10(-4) to 6.3 x 10(-4) M; and gentamicin-B, kanamycin, and ribostamycin had little or no effect at 7 x 10(-4) M, the highest concentration tested. Thus, a variety of aminoglycoside antibiotics mimic the effects of Ca2+ and other polyvalent cations on parathyroid function. Their relative potencies are closely related to the total number of amino groups on the molecule, with a 5- to 6-fold increase in potency for each additional amino group between four and six.


Sodium-dependent effects of melatonin on membrane potential of neonatal rat pituitary cells. Melatonin inhibits GnRH-stimulated release of LH from neonatal rat pituitary cells, probably by inhibiting GnRH-induced elevation of intracellular Ca2+. This effect of melatonin seems to involve inhibition of Ca2+ influx through voltage-sensitive channels. Accordingly, it is possible that melatonin could act by hyperpolarizing pituitary cells, which would close these channels. This issue was addressed here by determining if melatonin influences membrane potential. Membrane potential and intracellular Ca2+ were studied in neonatal rat pituitary cells in suspension, using bis-oxonol and Fluo-3 as fluorescent indicators, respectively. It was found that treatment with melatonin alone causes membrane hyperpolarization and that it has a repolarizing effect after GnRH-induced membrane depolarization. This effect on membrane potential appears to be mediated by high affinity melatonin receptors and a pertussis toxin-sensitive Na(+)-dependent mechanism; it is not dependent upon Ca2+, Cl-, or bicarbonate. This may be the molecular basis of action of melatonin in other tissues with high affinity melatonin receptors.


The biosynthesis of membrane and soluble plastidic c-type cytochromes of Chlamydomonas reinhardtii is dependent on multiple common gene products. Cytochrome c6 functions in the thylakoid lumen to catalyze electron transfer from reduced cytochrome f of the cytochrome b6f complex to P700+ of photosystem I. The biogenesis of mature cyt c6 from cytosolically translated pre-apocytochrome c6 involves numerous post-translational modifications including the proteolytic removal of a transit sequence and the covalent attachment of heme to two cysteinyl thiols on the apoprotein. Here, we report on the characterization of a previously unrecognized class of non-allelic mutants of Chlamydomonas reinhardtii that are blocked at the conversion of apocyt c6 to holocyt c6. The mutants are acetate requiring since they are also deficient in cyt f, cyt b and the Rieske FeS protein. Pulse-chase studies indicate that heme attachment is not required for the two-step processing of pre-apocytochrome c6 to apocyt c6, but is required for the stability of the mature protein. This is in contrast to the biosynthesis of mitochondrial cyt c1 where heme attachment is required for the second processing step. We propose that the assembly of both holocytochrome c6 and the cytochrome b6f complex are dependent on common gene products, possibly those involved in heme delivery or metabolism. This is the first suggestion that multiple loci are involved in the biosynthesis of both plastidic c-type cytochromes.


Rat liver mitochondrial intermediate peptidase (MIP): purification and initial characterization. A number of nuclearly encoded mitochondrial protein precursors that are transported into the matrix and inner membrane are cleaved in two sequential steps by two distinct matrix peptidases, mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). We have isolated and purified MIP from rat liver mitochondrial matrix. The enzyme, purified 2250-fold, is a monomer of 75 kDa and cleaves all tested mitochondrial intermediate proteins to their mature forms. About 20% of the final MIP preparation consists of equimolar amounts of two peptides of 47 kDa and 28 kDa, which are apparently the products of a single cleavage of the 75 kDa protein. These peptides are not separable from the 75 kDa protein, nor from each other, under any conditions used in the purification. The peptidase has a broad pH optimum between pH 6.6 and 8.9 and is inactivated by N-ethylmaleimide (NEM) and other sulfhydryl group reagents. The processing activity is divalent cation-dependent; it is stimulated by manganese, magnesium or calcium ions and reversibly inhibited by EDTA. Zinc, cobalt and iron strongly inhibit MIP activity. This pattern of cation dependence and inhibition is not clearly consistent with that of any known family of proteases.


DIF-1 induces its own breakdown in Dictyostelium. DIF-1 is a novel chlorinated alkyl phenone which induces differentiation of prestalk cells in Dictyostelium discoideum. It is broken down and inactivated by a cytoplasmic enzyme, DIF-1 3(5)-dechlorinase (hereafter referred to as DIF-1 dechlorinase), which is found only in prestalk cells. We show that DIF-1 dechlorinase levels are induced at least 50-fold when cells are treated with DIF-1. This response is rapid--enzyme activity doubles within 15 min and is fully induced within an hour--and occurs early in development, before other prestalk markers can be induced by DIF-1. Maximum inducibility is seen towards the end of aggregation, when DIF-1 dechlorinase is barely detectable in uninduced cells. The dose-dependence reveals a threshold concentration of DIF-1 (15 nM) below which almost no response is seen. Cyclic AMP, which is the chemoattractant during aggregation and plays a key role in later development, suppresses the induction of DIF-1 dechlorinase by DIF-1. We conclude that induction of DIF-1 dechlorinase is one of the first steps on the developmental pathway which leads to prestalk cell differentiation, and suggest that the resulting negative feedback on DIF-1 levels is an important part of the mechanism by which cells decide whether to become prestalk or prespore cells.


Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation. Xenopus MAP kinase activator, a 45 kDa protein, has been shown to function as a direct upstream factor sufficient for full activation and both tyrosine and serine/threonine phosphorylation of inactive MAP kinase. We have now shown by using an anti-MAP kinase activator antiserum that MAP kinase activator is ubiquitous in tissues and is regulated post-translationally. Activation of MAP kinase activator is correlated precisely with its threonine phosphorylation during the oocyte maturation process. It is a key question whether MAP kinase activator is a kinase or not. We have shown that Xenopus MAP kinase activator purified from mature oocytes is capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. Dephosphorylation of purified activator by protein phosphatase 2A treatment inactivates its autophosphorylation activity as well as its activator activity. Thus, Xenopus MAP kinase activator is a protein kinase with specificity for both serine/threonine and tyrosine. Partial protein sequencing of purified activator indicates that it contains a sequence homologous to kinase subdomains VI and VII of two yeast protein kinases, STE7 and byrl.


The DNA-binding protein E12 co-operates with XMyoD in the activation of muscle-specific gene expression in Xenopus embryos. Two alternatively spliced products of the human E2A gene, E12 and E47, encode helix-loop-helix DNA-binding proteins. Here we describe the isolation of two Xenopus cDNAs; one, XE12, is structurally similar to human E12 and the other contains a sequence similar to E47. Transcripts of both cDNAs are present at all the stages of Xenopus development tested and in all regions of the embryo. The DNA binding properties of in vitro translated XE12 are indistinguishable from those of human E12. We have shown previously that an embryonic muscle DNA-binding activity, EMF1, that binds to a promoter sequence required for the expression of the cardiac actin gene, contains the Xenopus myogenic factor XMyoD. Here we show that it also contains protein that interacts with an anti-E12 antiserum, suggesting that XE12 and XMyoD proteins, or very similar ones, are present in EMF1. We have addressed the functional role of XE12 in muscle gene transcription in Xenopus embryos by injecting in vitro synthesized RNA into the two cell embryo. Overexpression of XE12 and XMyoD augments by greater than 10-fold the ectopic activation of the endogenous cardiac actin gene that can be produced by XMyoD alone. Our DNA binding results strongly suggest that this effect is mediated through a direct interaction of the XE12-XMyoD complex with specific sites in the cardiac actin promoter. We suggest that XE12 is functionally important in muscle gene activation in embryonic development.


Human immunodeficiency virus infection of cells arrested in the cell cycle. Cell proliferation is necessary for proviral integration and productive infection of most retroviruses. Nevertheless, the human immunodeficiency virus (HIV) can infect non-dividing macrophages. This ability to grow in non-dividing cells is not specific to macrophages because, as we show here, CD4+ HeLa cells arrested at stage G2 of the cell cycle can be infected by HIV-1. Proliferation is necessary for these same cells to be infected by a murine retrovirus, MuLV. HIV-1 integrates into the arrested cell DNA and produces viral RNA and protein in a pattern similar to that in normal cells. In addition, our data suggest that the ability to infect non-dividing cells is due to one of the HIV-1 core virion proteins. HIV infection of non-dividing cells distinguishes lentiviruses from other retroviruses and is likely to be important in the natural history of HIV infection.


In vivo function of the proteasome in the ubiquitin pathway. A major eukaryotic proteolytic system is known to require the covalent attachment of ubiquitin to substrates prior to their degradation, yet the proteinase involved remains poorly defined. The proteasome, a large conserved multi-subunit protein complex of the cytosol and the nucleus, has been implicated in a variety of cellular functions. It is shown here that a yeast mutant with a defective proteasome fails to degrade proteins which are subject to ubiquitin-dependent proteolysis in wild-type cells. Thus, the proteasome is part of the ubiquitin system and mediates the degradation of ubiquitin-protein conjugates in vivo.


Individual dorsal morphogen binding sites mediate activation and repression in the Drosophila embryo. The dorsal (dl) morphogen gradient is responsible for initiating the differentiation of the mesoderm, neuroectoderm and dorsal ectoderm in the Drosophila embryo. dl encodes a sequence-specific DNA binding protein that belongs to the Rel family of transcription factors. Previous studies have shown that dl activates the mesoderm determinant twist (twi); here we use a combination of site-directed mutagenesis and P-transformation assays to demonstrate that it also functions as a direct transcriptional repressor of a second target gene, zerknüllt (zen). By exchanging dl binding sites between the promoters we show that activator sites from twi can mediate repression when placed in the context of the zen promoter, and that repressor sites from zen can mediate activation in the context of the twi promoter. This represents the first demonstration that common binding sites for any DNA binding protein can mediate both activation and repression in a developing embryo. Evidence is also presented that the affinities of dl binding sites are important for the efficiency of repression, but are not the sole determinants of the threshold response to the dl gradient.


Hormone-sensitive carbohydrate metabolism in rat hepatocyte-hepatoma hybrid cells. Hepatocyte-hepatoma hybrid cells were obtained by fusion of hepatocytes from adult rats and Fao hepatoma cells in the presence of polyethylene glycol. These hybrids were called hepatocytoma cells. The preservation of liver-specific enzyme activities and metabolic functions was studied in the hybrid clone 1E3. 1) The proliferating hepatocytoma cells formed monolayers presenting morphological similarity to primary cultures of hepatocytes. 2) In contrast to Fao hepatoma cells, activities of all gluconeogenic key enzymes were preserved at normal or reduced levels. 3) Lactate-dependent glucose formation was maintained at a state reduced to 36% of the gluconeogenesis in hepatocytes; no glucose formation was detected in Fao hepatoma cells. 4) The activity of the liver-specific glucokinase was reduced in hepatocytoma cells, but it was still present in contrast to Fao cells. The liver-specific isoenzyme pyruvate kinase type L was replaced by the isoenzyme type M2. 5) Gluconeogenic and glycolytic enzyme activities were regulated in hepatocytoma cells by glucagon (0.1 microM) and by insulin (0.1 microM). 6) The genome of hepatocytoma cells and its expression were stable for at least one year, when spontaneously dedifferentiating cells were removed by recloning in hypoxanthine-aminopterine-thymidine (HAT) medium.


Different concentrations of thyroid peroxidase and thyroglobulin in the nuclear envelope and the endoplasmic reticulum throughout the cytoplasm. Thyroid peroxidase (TPO) and thyroglobulin (TG) represent two major glycoproteins of thyroid follicular cells performing biological functions such as iodination, transcytosis of thyroglobulin, and formation of thyroid hormones. They are involved in thyroid autoimmunity and thyroid inborn metabolic disorders. Studying these processes at a molecular level includes the determination of their precise intracellular distribution. An evaluation of the relative concentrations of TG and TPO in different subcellular compartments was carried out in stimulated human follicular cells using thin-frozen sections and the immunogold technique. It is documented that TG is transported from the endoplasmic reticulum and the Golgi apparatus to the follicular lumen by transport vesicles; most of it being present in the expanded endoplasmic reticulum throughout the cytoplasm. On the other hand, gold particles indicating TPO are adjacent to the membranes of the exocytotic pathway. They do not label the basolateral membrane but show the strongest density in the nuclear envelope and the apical membrane. The labeling density of TPO is about four times higher in the nuclear envelope than in the endoplasmic reticulum throughout the cytoplasm. In contrast, TG is concentrated three times higher in the rough endoplasmic reticulum throughout the cytoplasm than in the nuclear cisternae. Our results give the first quantitative evidence that TPO and TG are concentrated in different subcompartments of the endoplasmic reticulum. Because previous studies demonstrated the nuclear envelope as the site where the synthesis of endogenous peroxidase (Brökelmann, J., D. W. Fawcett, Biol. Reprod. 1, 59-71 (1969)) begins, we suggest that synthesis of these functionally related proteins happens in specialized parts of the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)


Connexin43 in MDCK cells: regulation by a tumor-promoting phorbol ester and Ca2+. Prior to confluence, cultures of Madin Darby canine kidney (MDCK) cells expressed gap junctional communication, as assessed by fluorescent dye transfer, as well as relatively high levels of an anti-connexin43 immunoreactive component referred to as connexin43 (Cx43). After confluence, dye coupling and levels of Cx43 were dramatically reduced. Immunofluorescence analysis of the distribution of Cx43 in subconfluent cultures showed punctate labeling on the plasma membrane at regions of cell apposition and a more diffuse labeling in perinuclear regions. Western blots of total cell homogenates showed that the dephosphorylated form of Cx43 was more abundant than the phosphorylated forms. Phosphorylation of Cx43 was not significantly affected by 8-Bromo-cAMP or 8-Bromo-cGMP. However, 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited dye coupling and induced an increase in the amount of phosphorylated forms of Cx43 at the expense of the dephosphorylated form. This effect occurred as rapidly as 5 min after TPA treatment without apparent changes in distribution of Cx43 or cell morphology. These results suggest that second messenger pathways involving protein kinase C, but not cAMP- or cGMP-dependent protein kinase, led to changes in electrophoretic mobility of Cx43, revealed by Western blot, consistent with an alteration in the state of phosphorylation of the gap junction protein. Treatments with staurosporine, a protein kinase inhibitor, or okadaic acid, a protein phosphatase inhibitor, either alone or in combination with TPA, indicated that the abundance of the dephosphorylated form of Cx43 in MDCK cells was due to low kinase activity. It was also found that lowering the concentration of extracellular Ca2+, which reduced cell contact, did not affect the abundance, the state of phosphorylation, or the TPA-induced phosphorylation of Cx43. These results suggest that neither extracellular Ca2+ nor cell contact is required for basal or TPA-induced phosphorylation of Cx43.