Neurological aspects of organophosphate poisoning. Besides their well-known anticholinesterase action resulting in a typical acute cholinergic crisis, organophosphorus (OP) agents are capable of producing several subacute or chronic neurological syndromes. The acute over-stimulation at the neuromuscular junction results in muscle fiber necrosis. The significance of this OP-induced myopathy in human intoxication is unknown. Organophosphate-induced delayed neuropathy (OPIDN) arises 1-3 weeks after exposure to some OP compounds all capable of remarkably inhibiting a distinct esterase called neuropathy target esterase (NTE) during a critical time period. An experimental hen model has been designed to screen new OP compounds as to their delayed neurotoxic effects. The recently described intermediate syndrome emerges 1-4 days after an apparently well-treated cholinergic crisis. It main clinical features are sudden respiratory paralysis, cranial motor nerve palsies, and proximal limb muscle and neck flexor weakness. Whether or not this is a separate entity in OP agent toxicology remains to be seen. Further studies are required to further determine its clinical and paraclinical characteristics and the actual type of underlying neuromuscular dysfunction involved.


Comparative in vitro study of nonoxynol-9: effects on human, bull and boar spermatozoa. Spermicidal activity of different dilutions of nonoxynol-9 (nonyl-phenoxy-polyethoxy-ethanol) on human, bull and boar spermatozoa was studied using the modified Sander-Cramer method. The results obtained showed that differences in spermatozoal resistance among the mentioned species to nonoxynol-9 were statistically very significant (P much less than 0.0005): bull greater than man greater than boar. The results obtained were compared to available literature results.


Effects of gossypol acetate on pituitary-adrenal axis in male albino rats. Histopathological effects of gossypol acetate (GA) on pituitary-adrenal axis in male Wistar rats were investigated. Sexually mature rats of proven fertility were administered orally with 10 and 25 mg/kg for 4 and 5 weeks, respectively. In both experimental groups, corticotrophs (ACTH cells) of anterior pituitary showed progressive regression. At the 10 mg low-dose treatment for 4 weeks, no significant changes were observed in all the zones of the adrenal gland, while at the same dose for 5 weeks of treatment, cells of zona glomerulosa showed hypertrophy and degranulation as compared to that of control. Cells of zona fasciculata and reticularis showed no significant changes. In the medulla, the thickly granulated cells were degranulated and reduced in number. At the 25 mg treatment, the cells of the zona fasciculata showed hypertrophy and degranulation. The possible mechanism of action of GA is discussed.


Effects of culture media on the in vitro susceptibility of selected opportunistic fungi to fluconazole and itraconazole. The sensitivity of 23 isolates of opportunistic fungi, Aspergillus fumigatus (5), A. flavus (5), A. niger (5), Pseudallescheria boydii (5), Alternaria alternata (2) and Xylohypha bantiana (1), was investigated against fluconazole and itraconazole, using Sabouraud's dextrose broth (SD) and a high-resolution (HR) medium (Pfizer, Inc.). The procedure followed was a standard tube dilution (1 ml/tube) method. Candida albicans Y01 09 was included as reference strain to monitor quality and reproducibility. Results indicated that the minimal inhibitory concentrations (MICs) of fluconazole for all Aspergillus spp. and C. albicans were greater than or equal to 100 micrograms/ml in SD medium, whereas, for P. boydi, A. alternata and X. bantiana, the MICs were 50 micrograms/l. The MICs of itraconazole in SD medium were less than 0.195-1.56 micrograms/ml for the fungi tested. In HR medium, the MICs of fluconazole for the Aspergillus spp, were greater than or equal to 100 micrograms/ml, and those for P. boydii, A. alternata and X. bantiana were 0.78, 50 and 50 micrograms/ml, respectively. The MICs of itraconazole for all fungi ranged from less than 0.198 to 0.78 micrograms/ml in the HR medium. The values for the reference strain were 1.56 and 100 micrograms/ml in the HR medium for fluconazole and itraconazole, respectively. The HR medium was more suitable for testing P. boydii against fluconazole. This culture medium did not appear to significantly affect the MICs of itraconazole as compared to those of fluconazole, for the fungi investigated.


Influence of azithromycin and other macrolides on the intracellular killing of Staphylococcus aureus by human polymorphonuclear leucocytes of healthy donors and a patient with Chédiak-Higashi syndrome. A mixture of human blood phagocytes from healthy donors and opsonized staphylococci was incubated in vitro for 30 min. After that time all the bacteria were phagocytosed. The test tubes were further incubated for 2, 4 and 24 h with or without addition of a macrolide (erythromycin, azithromycin, clarithromycin, roxithromycin) and the effect of these drugs on the survival of intracellular staphylococci (Staphylococcus aureus ATCC 25923) was measured. The minimal effective concentration of the antibiotic which killed 80-90% of the bacteria after a 4-hour incubation was 0.1 mg/l for erythromycin, azithromycin and clarithromycin and 1.2 mg/l for roxithromycin. The percentage of surviving bacteria after 2 and 4 h incubation was not significantly different between these macrolides at the minimal effective concentration. Increasing the concentration of each antibiotic above the minimal effective concentration did not alter the killing rate of intracellular staphylococci. The bacterial activity of polymorphonuclear leucocytes (PMNL) from a patient with Chédiak-Higashi syndrome was less in comparison to PMNL from healthy donors, but was improved in vitro by the addition of erythromycin or azithromycin.


Occupational exposure to asbestos and urinary bladder cancer. By the use of transmission electron microscopy (TEM) and energy dispersion spectrometry the amount (mean value mean = 191 +/- 94 fibers/mg of tissue) and the type (chrysotile and tremolite) of asbestos fibers have been determined in tissue samples of four bladder cancer patients affected by pulmonary asbestosis, working in the same plant producing asbestos-cement pipes and boards. Similar measurements were carried out on samples of bladder cancers of eight control patients not professionally exposed to asbestos. Only five of them also revealed chrysotile fibers (mean = 151 +/- 196 fibers/mg of tissue). The paucity of the study and control cases and the small quantitative difference between them regarding the presence of infraneoplastic asbestos fibers does not consent us to hypothesize a causal relationship between tumor and occupational exposure.


Control of human coagulation by recombinant serine proteases. Blood clotting is activated by recombinant factor XII deleted of five regulatory domains. The availability of engineered serine proteases allows one to study the activation, substrate specificity and regulation of human coagulation and fibrinolytic activities. Human coagulation factor XII is composed of the protease catalytic region at the C-terminus, a hinge proline-rich region and regulatory domains at the N-terminus. From cDNA clones coding for factor XII, two DNA molecules were constructed, one being full length and the other being deleted of exons coding for the regulatory domains. Engineered factor-XII cDNA species were inserted by a homologous recombination technique into vaccinia viruses, which were used to infect the human hepatoma cell line HepG2. Two recombinant proteins were prepared from the culture media and identified by their antigenic properties and electrophoretic mobilities. The recombinant protein of larger size was identified as the full-length factor XII of 80 kDa and its specific activities and activation patterns, determined both by the coagulation and the amidolytic assays, are very similar to these of native human factor XII. The recombinant protein of smaller size was identified as a 319-amino-acid-deleted factor-XII protein of 32 kDa, containing only the entire protease region and part of the proline-rich hinge. This protein was expected to be the 'minimal' portion of factor XII able to sustain protease but unable to recognize substrates and surfaces necessary to activate the contact phase of coagulation. However, this 'minimal' factor-XII protein displays a marked protease activity and, although lacking five regulatory domains of factor XII, is bound and activated by negative charges and promotes coagulation with high efficiency.


Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues. Implications for the CuA site of N2O reductase and cytochrome-c oxidase. The nosZ genes encoding the multicopper enzyme nitrous oxide reductase of Alcaligenes eutrophus H16 and the type strain of Pseudomonas aeruginosa were cloned and sequenced for structural comparison of their gene products with the homologous product of the nosZ gene from Pseudomonas stutzeri [Viebrock, A. & Zumft, W. G. (1988) J. Bacteriol. 170, 4658-4668] and the subunit II of cytochrome-c oxidase (COII). Both types of enzymes possess the CuA binding site. The nosZ genes were identified in cosmid libraries by hybridization with an internal 1.22-kb PstI fragment (NS220) of nosZ from P. stutzeri. The derived amino acid sequences indicate unprocessed gene products of 70084 Da (A. eutrophus) and 70695 Da (P. aeruginosa). The N-terminal sequences of the NosZ proteins have the characteristics of signal peptides for transport. A homologous domain, extending over at least 50 residues, is shared among the three derived NosZ sequences and the CuA binding region of 32 COII sequences. Only three out of nine cysteine residues of the NosZ protein (P. stutzeri) are invariant. Cys618 and Cys622 are assigned to a binuclear center, A, which is thought to represent the CuA site of NosZ and is located close to the C terminus. Two conserved histidines, one methionine, one aspartate, one valine and two aromatic residues are also part of the CuA consensus sequence, which is the domain homologous between the two enzymes. The CuA consensus sequence, however, lacks four strictly conserved residues present in all COII sequences. Cys165 is likely to be a ligand of a second binuclear center, Z, for which we assume mainly histidine coordination. Of 23 histidine residues in NosZ (P. stutzeri), 14 are invariant, 7 of which are in regions with a degree of conservation well above the 50% positional identity between the Alcaligenes and Pseudomonas sequences. Conserved tryptophan residues are located close to several potential copper ligands. Trp615 may contribute to the observed quenching of fluorescence when the CuA site is occupied.


Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation. Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I-sensitive component(s). In the presence of a concentration of Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2- production by Ph2I. The O2(-)-generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2- production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.


The cation-independent mannose 6-phosphate receptor is not involved in the polarized secretion of lysosomal alpha-glucosidase from Caco-2 cells. In the human adenocarcinoma cell line Caco-2 a substantial amount of a precursor form of the lysosomal enzyme alpha-glucosidase is not segregated into lysosomes, but instead secreted from the apical membrane. In this study we addressed the question whether this process is mediated by mannose 6-phosphate receptors. The subcellular distribution of the cation-independent mannose 6-phosphate receptor was studied by means of electron microscopic immunocytochemistry. The bulk of label was found in the perinuclear region in electron-lucent and dense vesicles, some of the latter bearing a coat. Receptor-containing dense vesicles were also found throughout the cytoplasm. In the apical part of the cells, label for the receptor was present over the surrounding membrane and the interior vesicles of multivesicular bodies, but not over lysosomes. Label on the plasma membrane was mainly restricted to the apical domain. In contrast to alpha-glucosidase, the secreted forms of the lysosomal enzymes cathepsin D, beta-hexosaminidase and beta-glucuronidase are mainly found in the basolateral medium. Enzyme activity measurements and immunoprecipitation of metabolically labeled cells showed that incubation with NH4Cl leads to an enhanced secretion of these enzymes into the basolateral medium, but has no effect on the basolateral secretion of alpha-glucosidase. In addition, NH4Cl caused a minor decrease in the secretion of these enzymes from the apical side and had little or no effect on the secretion of alpha-glucosidase.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of human low density lipoprotein binding proteins on the surface of schistosomula of Schistosoma mansoni. Low density lipoproteins (LDL) bound to the surface of Schistosoma mansoni may protect the parasite from assault by the immune system and provide essential lipids for the parasite in human schistosomiasis. Here we have characterized the LDL binding sites on the surface of schistosomula by comparing the binding of fluorescently labeled LDL to the parasite with LDL binding proteins as seen by ligand blotting before and after enzymatic treatment of viable parasites. Ligand blotting revealed two LDL binding bands, 17.8 +/- 0.8 and 15.7 +/- 0.6 kDa, in intact schistosomula. Trypsinization eliminated all of the specific and approximately two-thirds of the total LDL binding capacity of schistosomula in a time and concentration-dependent manner. LDL did not bind to any bands on blots of trypsinized, viable worms. Specific LDL binding was also eliminated by phosphatidylinositol-specific phospholipase C (PIPLC). PIPLC treatment removed both LDL binding bands from the worms and caused the appearance of an LDL binding band, 16.6 +/- 0.3 kDa, in the culture medium. LDL binding to the parasite recovered within 24 to 48 h after trypsinization but the recovery was inhibited by either monensin or puromycin. Both LDL binding bands reappeared in ligand blots of cultured worms within 24 h; the reappearance was blocked by puromycin but not by monensin. These studies suggest that the specific binding of human LDL to schistosomula is mediated by GPI-linked low molecular weight proteins that are continually synthesized and transported to the parasite surface.


Monoclonal antiidiotypic antibodies against delta opioid receptors as an electron microscopy probe. Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface receptors.


Multianalytic study on cervical HPV lesions. Nowadays new sophisticated techniques of molecular biology based on the principles of hybridization between nucleic acids, allow a correct diagnosis of genital HPV infection. In the present paper, beside traditional diagnostic methods, we used In Situ Hybridization (ISH) and Polymerase Chain Reaction (PCR) to detect the presence of HPV types 6, 11, 16, 18, 31 and 33. We tested ten patients affected by cervical lesions of high histological atypias associated with HPV, who underwent surgical conization. Types 6 and 11, at low risk of evolution, are less frequent than 31 and 33, at medium grade of evolution, and than 16 and 18 which are at high risk of evolution.


Paget's disease of the vulva: advantages of demolitive surgery. Four patients with vulvar Paget's disease were analysed retrospectively. In all cases the clinical manifestation was typical, with pruritus and visible lesion. None of the cases had a palpable cutaneous or subcutaneous mass but at the histologic exam a minimally invasive Paget's disease was detected. Treatment consisted of total vulvectomy and subsequent new reconstructive time; only one patient with clinically localized disease was treated with simple vulvectomy. Postoperative complications were minimal, thanks to the reconstructive time that assured a rapid recovery of the surgical injury and reconstruction of the urethral and vaginal meatuses. In all cases we obtained the complete removal of the lesion (negativity of resection margins), except in one case that was also the only one to have a recurrence after 40 months. The patients have been followed for an average of 58 months. Considering the results, we suggest demolitive surgery with subsequent reconstructive time in the treatment of vulvar Paget's disease.


Metastatic involvement of the stomach secondary to breast carcinoma. A case report. The Authors describe a case of gastric metastases from breast cancer. The staging procedures in breast cancer do not usually include the endoscopic examination of the gastroenteric tract; nevertheless the complaint of persistent gastric disorders in patients affected by metastatic breast cancers must not be underestimated. In this case the finding of gastric neoplastic involvement led to effective treatment of the above mentioned disorders and consequently to an improvement in the patient's quality of life. Moreover, the evaluation of this clinical case shows that chemotherapeutic treatment of this metastatic localisation does not differ from that of metastases in other sites.


Growth factor responsiveness: role of MyoD and myogenin. A differentiation-defective variant (DD-1) of the MM14 myoblasts acquired the ability to synthesize DNA in response to treatment with epidermal growth factor (EGF) (R. W. Lim and S. D. Hauschka, 1984, Dev. Biol. 105, 48) and no longer expressed myogenic determinant genes (i.e., MyoD and myogenin) (P.R. Mueller, and B. Wold, 1989, Science 246, 780). To determine the effect of expression of MyoD on EGF responsiveness, DD-1 cells were cotransfected with a MyoD expression vector and with pRSVneo. A clone, MyoDD-1 cells, which was G418 resistant, formed multinuclear syncitia, and also expressed MyoD and myogenin, was further characterized. EGF responsiveness, as assessed by DNA synthesis, was decreased 5- to 10-fold in the MyoDD-1 cells from that in G418-resistant control DD-1 cells, despite similar EGF receptor numbers and binding affinities of the receptors. Responsiveness of MyoDD-1 cells to fibroblast growth factor (FGF) was also diminished although to a lesser extent. To determine the effects of decreased myogenic determinant gene expression on mitogen responsiveness, MM14 myoblasts were grown in medium supplemented with 5 microM 5-bromo-2'-deoxyuridine (BUdR-MM14). BUdR-MM14 cells had decreased expression of MyoD and myogenin, did not fuse, and had an altered morphology, from round to flat. The BUdR effect on fusion and cell shape was reversed by growth in control medium. BUdR-MM14 cells were responsive to EGF and had enhanced responsiveness to FGF. The combined studies support the view that expression of MyoD and/or myogenin contributes to negative regulation of mitogen responsiveness.


Regulation of urokinase plasminogen activator localization in keratinocytes by calcium ion and E-cadherin. In keratinocyte culture, the cellular distribution of many adhesion markers and the organization of intercellular junctions are controlled by the calcium ion concentration of the medium. We show in the present study that urokinase plasminogen activator (uPA) localization in the human keratinocyte is similarly dependent upon calcium concentration. At 30 microM calcium, uPA is present throughout the cell, often with a perinuclear concentration. Upon calcium elevation to 1.0 mM, uPA is concentrated along the cell-cell borders, where it colocalizes (at the light microscope level) with E-cadherin. Blocking antibody to E-cadherin delays the calcium-induced redistribution of uPA, in a manner very similar to the previously observed delay in redistribution of several adhesion-related markers, including vinculin, desmoplakin, and beta 1 integrin. These data suggest a link between the redistribution of uPA to the cell-cell borders and the calcium-induced organization of intercellular junctions in the human keratinocyte. The presence of uPA along the intercellular borders suggests that this enzyme may be involved in regulation of epidermal adhesion through proteolysis.


Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression. To study the effect of SV40 T-antigen in mammary epithelial cells, a rat beta-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.


Molecular dissection of a specific nuclear domain: the chromatin region of the ribosomal gene cluster in Xenopus laevis. Molecular dissection of the nuclear domain corresponding to the ribosomal chromatin cluster was investigated. The experimental scheme was based on the ability of restriction enzymes to digest the whole genome without affecting this region (several megabases in length). Such a strategy involved the judicious choice of restriction enzymes, which is possible in Xenopus laevis, where the rDNA sequence is known and the repeated units are organized into one unique cluster. SalI, XhoI, and EcoRV digestion produced frequent cutting of the genome leaving the ribosomal cluster intact. Isolation of the rDNA cluster was confirmed by separation of the digested DNA by pulsed-field electrophoresis. When applied to purified nuclei, this approach allowed the isolation of the ribosomal chromatin cluster under very mild conditions: no cleavages (either enzymatic or mechanical) were detectable. Since the purification scheme depends only on the DNA sequence outside of the rDNA cluster, it permits the obtention of this domain in different functional states. Electron microscopic analysis demonstrated that the domain organization is substantially preserved and maintains its looped organization (the size and the full number of loops were preserved). This purification scheme provides a powerful tool for studying the structure-function relationships within the ribosomal nuclear domain.


Ca2+/calmodulin-activated protein phosphatase (PP2B) of Saccharomyces cerevisiae. PP2B activity is not essential for growth. Protein phosphatase (PP2B) whose activity is stimulated 12-20-fold by Ca2+/calmodulin (CaM) was partially purified by CaM-Sepharose and heparin-agarose chromatographies from cell extract of the yeast Saccharomyces cerevisiae. PP2B activity was not detectable in a mutant in which two genes (CMP1 and CMP2) encoding homologs of mammalian PP2B catalytic subunit were disrupted. We have previously shown that the double gene disruption has no significant effect on the growth of yeast [1991, Mol. Gen. Genet. 227, 52-59]. The results indicated that CMP1 and CMP2 are the only genes that encode the PP2B catalytic polypeptide in S. cerevisiae, and PP2B activity is not essential for the growth of the yeast under normal conditions.


Induction of the peroxisome proliferator activated receptor by fenofibrate in rat liver. The process of peroxisome proliferation in rodent liver by hypolipidemic compounds and related substances has recently been shown to be receptor-mediated. In the present study, we have examined the effect of oral administration of the strong peroxisome proliferator fenofibrate on the hepatic expression level of the peroxisome proliferator activated receptor (PPAR) in rats. Immunoblots of rat liver cytosols and nuclear extracts using antibodies raised against recombinant PPAR/beta-galactosidase fusion proteins revealed a pronounced increase in the amount of PPAR protein in response to fenofibrate treatment. This induction could also be confirmed at the level of RNA by Northern blotting. A time-course investigation showed a delayed accumulation of mRNA in response to the treatment, starting on day 2 after a latency period of at least one day. Thus, induction of the PPAR as a response to peroxisome proliferators represents one important dimension of the pleiotropic effects of peroxisome proliferators.


Synthetic peptides of the effector-binding domain of rab enhance secretion from digitonin-permeabilized chromaffin cells. There is evidence that the rab class of low molecular weight GTP-binding proteins is involved in vesicular transfer from endoplasmic reticulum to Golgi and between Golgi cisternae. To determine whether similar proteins play a role in regulated exocytosis, the effects of synthetic peptides derived from low molecular weight GTP-binding proteins on catecholamine secretion from digitonin-permeabilized chromaffin cells were investigated. The synthetic peptides represent the putative effector-binding domains of the rab, ras and ral classes of low molecular weight GTP-binding proteins and correspond to ras(33-48). Two rab peptides but neither a ras nor a ral peptide enhanced Ca(2+)-dependent secretion by approximately 30%. Maximal secretion in response to Ca2+ was increased. The enhancement was not blocked by the pseudosubstrate inhibitor of protein kinase C, PKC(19-31), thus indicating that activation of protein kinase C was not responsible for the enhancement of secretion. Similarly a rab peptide but neither a ras nor a ral peptide enhanced GppNHp-induced secretion 30-70%. The peptides had little or no effect in the absence of Ca2+ or GppNHp. The data are consistent with a protein of the rab class playing a role in regulated exocytosis.


Interaction of plasma gelsolin with tropomyosin. Horse plasma gelsolin labelled with benzophenone-4-isothiocyanate can be photochemically cross-linked to rabbit cardiac tropomyosin. The cross-linking proceeds with greater efficiency in calcium-containing buffers. Further evidence for interaction between these proteins is provided by retention of fluorescently labelled gelsolin on tropomyosin-agarose affinity columns and by the ability of tropomyosin to cause an increase in the fluorescence intensity of gelsolin labelled with fluorescein-5-isothiocyanate. Both of these effects require the presence of calcium ions.


Properties of the tungsten-substituted molybdenum formylmethanofuran dehydrogenase from Methanobacterium wolfei. In Methanobacterium wolfei two formylmethanofuran dehydrogenases are present, one of which is a molybdenum- and the other a tungsten enzyme. We report here that also the 'molybdenum' enzyme contained tungsten when the archaeon was grown on molybdenum-deprived medium supplemented with tungstate (1 microM). Unexpectedly the tungsten-substituted molybdenum enzyme was catalytically active and displayed a rhombic EPR signal which was attributed to tungsten by the characteristic 183W splitting.


Copper(II)-substituted horse liver alcohol dehydrogenase: structure of the minor species. Oxygen treatment of horse liver alcohol dehydrogenase EE isozyme substituted with Cu(II) at the catalytic site leads to bleaching with concomitant reduction to Cu(I) of approximately 90% of total Cu(II). The Cu(II) of the remaining 'minor species' cannot be reduced nor does it interact with exogenous ligands, e.g. 2-mercaptoethanol, imidazole, pyrazole, or azide ions. The EPR spectrum is axial with a super-hyperfine splitting of 15.6 G indicating binding of one nitrogen atom to Cu(II). These data as well as the energies and intensities of the absorption and CD spectra suggest the Cu(II) ion of the minor species to be located in the catalytic site of HLADH in a position and geometry different from that of the major species.


Vector methylation inhibits transcription from the SV40 early promoter. Methylation of a plasmid containing the SV40 promoter linked to the chloramphenicol acetyl transferase (CAT) gene, with either murine DNA methylase or methylase SssI results in inhibition of the expression of the reporter gene after transfection into cultured cells. Methylation of the plasmid with the methylases HhaI and HpaII has no effect on the expression of this gene. Protein-DNA interactions in the SV40 promoter are not affected by the presence of methylcytosine suggesting that inactivation results from the formation of an inactive chromatin structure that is dependent on the high CG content of the plasmid.


Protein kinase C stimulatory activity in the pseudopregnant rat ovary. Ovarian cytosol from pseudopregnant rats was heated to 80-90 degrees C for 2 min and precipitated proteins removed by centrifugation. The supernatant of the heated ovarian cytosol contained no protein kinase C activity but when added to a control preparation containing protein kinase C, enzyme activity was increased to 200% of control. The stimulatory activity was stable to heating for 10 min, was retained on a centrifugal filtration device with a 100,000 M(r) cut-off, did not affect cAMP-dependent protein kinase, was not extractable in petroleum ether or chloroform/methanol (2:1), and enhanced the phosphorylation of protein kinase C-specific peptide substrates. The stimulatory factor was calcium-dependent and could substitute for phosphatidylserine and diacylglycerol in the protein kinase C assay. This stimulatory factor may provide a mechanism whereby the response of protein kinase C to hormonal activation could be regulated by the cell.


Interferon regulatory factor-1 is inducible by prolactin, interleukin-2 and concanavalin A in T cells. Interferon regulatory factor-1 (IRF-1) gene expression is rapidly upregulated in the prolactin (PRL)-activated Nb2 rat T lymphoma cell line. To further elucidate its role as a T cell activation molecule, IRF-1 gene expression in response to various T cell stimuli was examined. In Nb2 T cells, PRL induced two peaks of IRF-1 gene expression: a rapid, transient peak at 1 h and a sustained peak at 12 h. PRL subsequently induced interferon-gamma (IFN-gamma) gene expression at 3-6 h. However, the early induction of IRF-1 and IFN-gamma does not appear to be interdependent. Interleukin-2 (IL-2) also induced IRF-1 gene expression in Nb2 T cells but only one broad peak at 10 h was observed. In primary mouse splenocytes, concanavalin A induced rapid and transient expression of the IRF-1 gene; maximal expression occurred by 6 h, and then returned to basal levels by 12-15 h. These results provide additional evidence for the importance of IRF-1 in T cell activation.


Regulatory effect of 1,25-dihydroxycholecalciferol on calcium fluxes in thyroid FRTL-5 cells. The aim of the present study was to investigate the effect of 1,25-dihydroxycholecalciferol (1,25(OH)2-D3) on the regulation of calcium fluxes in rat thyroid FRTL-5 cells. The ATP-induced uptake of 45Ca2+ was decreased in cells pretreated with 1,25(OH)2D3 for 48 h. No effect was seen on basal uptake of 45Ca2+. At least a 24 h incubation period was required for the effect of 1,25(OH)2D3 to be expressed. Pretreatment with 1,25(OH)2D3 for 48 h did not change resting intracellular Ca2+ ([Ca2+]i) in fura-2-loaded FRTL-5 cells. However, the ATP-induced increase in [Ca2+]i was significantly enhanced in cells preincubated with 1,25(OH)2D3. The effect of 1,25(OH)2D3 was abolished in Ca(2+)-free buffer. No difference in the ionomycin-induced increase in [Ca2+]i was observed between control cells and cells pretreated with 1,25(OH)2D3. However, in Ca(2+)-free buffer the ionomycin response was decreased in cells incubated with 1,25(OH)2D3. The ATP-induced change in [Ca2+]i was decreased when ATP was added after ionomycin to cells treated with 1,25(OH)2D3. The results suggest that 1,25(OH)2D3 has a regulatory effect on Ca2+ fluxes in FRTL-5 cells, possibly by acting on Ca2+ sequestration.


Lactate transport in insulin-secreting beta-cells: contrast between rat islets and HIT-T15 insulinoma cells. The transport of L- and D-lactate into rat pancreatic islets and HIT-T15 insulinoma cells was studied by measuring uptake of 14C-labelled substrate at room temperature and by following changes in intracellular pH (pHi) in islets and HIT-T15 cells loaded with 2',7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF). Uptake of L-lactate into HIT-T15 cells was rapid, reaching equilibrium after 5 min with an apparent Km value of 4.8 mM. Transport was markedly inhibited by alpha-cyano-4-hydroxycinnamate, alpha-fluorocinnamate, quercetin and p-chloromercuribenzenesulphonate (pCMBS), and was enhanced in citrate medium. Uptake of D-lactate was less rapid, apparent equilibrium not being reached within 10 min. In contrast to HIT-T15 cells, rat pancreatic islets showed greatly reduced rates of transport of L- and D-lactate together with a correspondingly lower degree of inhibition by alpha-cyano-4-hydroxycinnamate. The addition of L- or D-lactate to HIT-T15 cells, but not dispersed islet cells, resulted in a marked and rapid intracellular acidification followed by a gradual recovery. In both HIT-T15 cells and isolated islets, the rates of transport of both L- and D-lactate in the presence of alpha-cyano-4-hydroxycinnamate were significantly greater in a depolarising K+ medium compared to the normal Na+ medium. These observations suggest that native rat islet cells have considerably reduced activity of the lactate-/H+ transport system compared to HIT-T15 insulinoma cells. There is evidence in both cell types of an additional electrogenic pathway for lactate which might play a role in coupling lactate efflux to beta-cell depolarisation.


Effect of 3,3'-diiodothyronine and 3,5-diiodothyronine on rat liver oxidative capacity. We report that 3,5,3'-triiodothyronine (T3) as well as two other iodothyronines (3,3'-diiodothyronine and 3,5-diiodothyronine (T2s)) stimulate rat liver oxidative capacity (measured as cytochrome oxidase activity (COX)). In hypothyroid rats COX activity and mitochondrial protein content are significantly lower than in normal control animals. The administration of both T3 and T2s to hypothyroid rats significantly enhances hepatic COX activity with T3 having the greatest effect (+60%); moreover, T3 restores the mitochondrial protein content whereas the T2s are ineffective. Administration of T2s results in a faster stimulation (already significant 1 h after the injection) of hepatic COX activity than T3 injection. Our results suggest that T3 acts on the protein synthesis mechanism involved in the regulation of the mitochondrial mass while T2s would act directly at the mitochondrial level.


Glucagon-like peptide-1, a new hormone of the entero-insular axis. The post-translational processing of proglucagon in the small intestine gives rise to glucagon-like peptide-1 (PG 78-107 amide) which has profound effects on the endocrine pancreas, and in many species also on the stomach. Glucagon-like peptide-1 (PG 78-107 amide) is secreted in man in response to physiological stimuli e.g. a mixed meal. Glucagon-like peptide-1, in concentrations corresponding to those observed in response to meals, strongly stimulates insulin secretion, in all mammals studied, even more potently than the gastric inhibitory peptide. Thus, glucagon-like peptide-1 fulfills the classic criteria for being a hormone and is likely to be a new incretin. The glucagon inhibitory effect of glucagon-like peptide-1 (PG 78-107 amide) probably further potentiates the effect of glucagon-like peptide-1 on glucose metabolism and distinguished this peptide from other intestinal peptides which have been proposed as incretins. Glucagon-like peptide-1 also inhibits gastric acid secretion and gastric emptying in man. The latter delays nutrient entry to the intestine and thereby diminishes meal-induced glucose excursions. Elevated plasma concentrations of immunoreactive glucagon-like peptide-1 have been reported in Type 2 (noninsulin-dependent) diabetic patients, however, the consequences of the elevation are not yet known. However, elevated levels of glucagon-like peptide-1 in patients with increased gastric emptying rate (post-gastrectomy syndromes) may be responsible for the exaggerated insulin secretion seen in these patients.


Normal insulin receptor tyrosine kinase activity and glucose transporter (GLUT 4) levels in the skeletal muscle of hyperinsulinaemic hypertensive rats. The spontaneous hypertensive rat is an animal model characterized by a syndrome of hypertension, insulin resistance and hyperinsulinaemia. To elucidate whether in analogy to other insulin resistant animal models an inactivity of the insulin receptor kinase or an alteration of the glucose transporter (GLUT 4) level in the skeletal muscle might contribute to the pathogenesis of insulin resistance we determined insulin receptor kinase activity and GLUT 4 level in the hindlimbs of spontaneous hypertensive rats and normotensive control rats. Normotensive normoinsulinaemic Lewis and Wistar rats were used as insulin sensitive controls, obese Zucker rats were used as an insulin resistant control with known reduced skeletal muscle insulin receptor kinase activity. Binding of 125I-insulin, crosslinking of 125I-B26-insulin, autophosphorylation in vitro with 32P-ATP and phosphorylation of the synthetic substrate Poly (Glu 4: Tyr 1) were performed after partial purification of solubilized receptors on wheat germ agglutinin columns. GLUT 4 levels were determined by Western blotting of subcellular muscle membranes. Insulin receptors from spontaneous hypertensive rats compared to those from Lewis and Wistar rats showed no difference of the binding characteristics or the in vitro auto- and substrate phosphorylation activity of the receptor, while in the Zucker rats the earlier described insulin receptor kinase defect was clearly evident. Western blots of subcellular muscle membrane fractions with antibodies against GLUT 4 revealed no difference in transporter levels. These data suggest that insulin resistance in spontaneous hypertensive rats is caused neither by an insulin receptor inactivity nor by a decreased number of glucose transporters in the skeletal muscle.


Mechanisms in rabbit aorta for hyperglycaemia-induced alterations in angiotensin II and norepinephrine effects. The (Na+,K+)-ATPase activity operative in rabbit aortic intima-media incubated with normal plasma levels of glucose and myo-inositol (70 mumol/l) is decreased when the glucose content of the medium is raised from 5 to 10 mmol/l or higher; this effect is prevented by aldose reductase inhibitors and by raising the myo-inositol content of the medium to 500 mumol/l. The decrease in (Na+,K+)-ATPase activity results from the loss of a component normally regulated (stimulated) by endogenously released adenosine through a receptor that stimulates phosphatidylinositol turnover in a discrete pool. The replenishment of this phosphatidylinositol pool selectively requires myo-inositol transport and is inhibited when increased polyol pathway activity impairs myo-inositol transport at a normal plasma level. Adenosine is a vasodilator, some endothelium-released vasodilators modulate the responses to vasoconstrictors by stimulating an increase in (Na+,K+)-ATPase activity in vascular smooth muscle. Whether adenosine mediates this effect in angiotensin II or norepinephrine-stimulated aorta was examined. Angiotensin II (100 nmol/l) and norepinephrine (1 mumol/l) evoked marked increases in (Na+,K+)-ATPase activity in aortic intima-media incubated with 5 mmol/l glucose and 70 mumol/l myo-inositol, which were inhibited when adenosine deaminase was added or the medium myo-inositol omitted to inhibit myo-inositol transport. Raising the medium glucose to 30 mmol/l inhibited the angiotensin II and norepinephrine-evoked increases in (Na+,K+)-ATPase activity, and this was prevented when tolrestat (10 mumol/l) was added or the myo-inositol content of the medium was raised from 70 to 500 mumol/l.(ABSTRACT TRUNCATED AT 250 WORDS)


An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation. Crude Clostridium histolyticum collagenase is widely used for the enzymatic degradation of pancreatic extracellular matrix in order to isolate the islets of Langerhans. The variable enzymatic composition of crude collagenases is a critical issue which contributes to the poor reproducibility of islet isolation procedures. In this study, the separate contributions of collagenase and protease to the islet isolation process were analysed by testing various combinations of purified collagenase and purified protease in rat pancreas dissociations under conditions which eliminated all other proteolytic activity. Under these conditions, complete tissue dissociation by purified collagenase required 99 +/- 10 min, whereas increasing amounts of protease progressively reduced this time to a minimum of 36 +/- 1 min. Histochemical analysis of the dissociation process showed that protease enhanced the degradation of all four major components of the extracellular matrix: collagen was degraded more completely, while proteoglycans, glycoproteins and elastin were degraded at a higher rate. Pancreas dissociation under the present, strictly controlled conditions resulted in a high yield of viable islets: 4.2-5.0 microliters islet tissue volume (3,300-3,800 islets) were isolated per g pancreas in the presence of a high or low protease concentration, respectively. Prolonged dissociation in the presence of protease resulted in a dramatic decrease in islet yield which correlated with the observation that the enzyme accelerated islet disintegration. It is concluded that the collagenase-induced dissociation of the extracellular matrix is facilitated by protease. Our study shows that high yields of viable islets can be obtained under controlled enzymatic conditions, provided that the exposure of islets to protease is limited.


GABAB-mediated modulation of the voltage-gated Ca2+ channels. 1. The amino acid, gamma-aminobutyric acid (GABA), activates two different receptor types (Bowery et al., 1980; reviewed by Ogata, 1990a). 2. GABAA receptors are bicuculline-sensitive and are coupled to Cl- channels, while activation of bicuculline-insensitive GABAB receptors has been implicated in the modulation of Ca2+ (Dunlap and Fischbach, 1981) and K+ (Gahwiler and Brown, 1985; Inoue et al., 1985a,b; reviewed by Ogata, 1990b) channels. 3. Baclofen is a specific agonist for GABAB receptors (Bowery et al., 1980). In rat sensory neurones, baclofen suppresses the membrane Ca2+ current (ICa) by a mechanism involving a partussis toxin-sensitive G protein (Holz et al., 1986; Scott and Dolphin, 1986). 4. It has been shown that the inhibitory effect of baclofen is more potent on the early portion of ICa than on the later portion and consequently the rate of ICa activation is slowed (Deisz and Lux, 1985; Dolphin and Scott, 1986). 5. The mechanisms underlying these GABAB-mediated modulation of ICa is not fully understood. This article reviews the inhibitory action of baclofen on ICa in sensory neurones.


Specific binding sites for [3H]Ro 5-4864 in rat prostate and seminal vesicle. 1. The peripheral-type benzodiazepine receptor was characterized in rat prostate and seminal vesicle using [3H]Ro 5-4864 as radioligand. 2. The affinity of this radioligand for this receptor was higher in rat prostate (KD = 4.36 +/- 1.02 nM) than in seminal vesicle (KD = 8.45 +/- 1.34 nM). 3. The density of binding sites obtained in these two tissues was Bmax = 4164 +/- 873 fmol/mg in prostate and 5978 +/- 1022 fmol/mg in seminal vesicle. 4. The [3H]Ro 5-4864 binding was inhibited non-competitively by atractyloside and alpha,beta-methylene-ATP, suggesting a modulation by the ADP/ATP mitochondrial carrier. 5. Flutamide was able to displace bound [3H]Ro 5-4864 with an IC50 similar to protoporphyrin IX.


Contractile response of guinea pig ileum by repetitive application of morphine. 1. The repetitive application of morphine gradually induced a contracture in the isolated guinea pig ileum. 2. The optimum conditions for induction of the contracture were as follows: the concentration, incubation time and washout time of morphine were 1 or 10 microM, 2 and 3 min, respectively. 3. Preincubation with naloxone or TTX blocked this morphine-induced contracture. 4. Among twitch-inhibiting drugs, only clonidine induced a contracture similar to that induced by morphine, while tetrodotoxin (TTX) and adenosine did not. 5. The contracture was also observed in the longitudinal muscle-myenteric plexus preparations. 6. These findings indicate that morphine has a dual inhibitory and excitatory action on the guinea pig ileum and that its repetitive application preferentially diminish the inhibitory one.


Marked dependence of the cardiac effects of gallopamil on the extracellular K(+)-concentration. 1. In guinea-pig Langendorff hearts, the negative inotropic effect of the calcium antagonist gallopamil is shifted by 15-fold to the left, when the extracellular K(+)-concentration is raised from 2.7 to 8.1 mM. 2. In papillary muscles, the ability of gallopamil to shorten the action potential (AP) markedly depends on K+: 100-fold lower gallopamil concentrations were required at 10.8 mM, compared to 2.7 mM. 3. In isolated myocytes, a change in the holding potential from -90 to -70 mV displaces the gallopamil dose-response curve to block Ca2+ currents leftward by only 6-fold. 4. Tetraethylammonium (TEA, 10 mM) mimics the mitigating effect of low K+ on the gallopamil-induced AP-shortening. Hence, the K(+)-dependence of gallopamil may be comprised of modulation of Ca(2+)-channel and K(+)-channel blocking effects.


Endothelin-1-induced contractions of isolated pig detrusor and vesical arterial smooth muscle: calcium dependence and phosphoinositide hydrolysis. 1. In isolated pig detrusor and vesical arterial smooth muscle preparations, endothelin-1 (ET-1) caused concentration-dependent contractions. Nifedipine (10(-6) M) did not significantly affect the action of ET-1 in the vessels, but almost abolished its effect in the detrusor. Incubation for 30 min in Ca(2+)-free solution markedly reduced the ET-1-induced contractions in both detrusor and vesical arteries. 2. The protein kinase C inhibitor H-7 (3 x 10(-5) M), reduced the response to ET-1 in detrusor muscle as well as in vessels, and abolished the contractions evoked by ET-1 in Ca(2+)-free solution. 3. ET-1 caused an increase in the accumulation of inositol phosphates (IPs) in preparations prelabelled with myo-[3H]inositol. After exposure to ET-1 (10(-7) M) for 60 min, an approx. 4-fold increase in IPs levels were demonstrated, compared to untreated controls, in both detrusor and vessel preparations. Pretreatment with nifedipine (10(-6) M) did not reduce IPs formation. In contrast, no increase in IPs formation was demonstrated in Ca(2+)-free medium. 4. The increase in accumulation of IPs was slow in onset in both detrusor and vesical arteries, with no significant accumulation demonstrable during the first 30 min. Time-course studies of tension development for ET-1 revealed that maximum tension was reached before significant levels of IPs could be detected.


Effects of ageing on responses of rabbit iris smooth muscles to agonists and field stimulation. 1. The pharmacological properties of contractile responses of sphincter and dilator to field stimulation did not change with age, so that innervation of both these muscles does not change apparently with age. 2. In the sphincter, no age-related change was observed in muscarinic cholinoceptor mechanisms. Tension induced by field stimulation increased with age from 5 to 13 weeks, decreased from 40 to 125 weeks and did not change thereafter. Age-related change in tension is due to change in the amount of acetylcholine released by stimulation. 3. In the dilator, the pD2 value of norepinephrine increased with age from 5 to 13 weeks, decreased from 13 to 125 weeks and did not change thereafter. The pD2 value of norepinephrine was proportional to the receptor reserve, suggesting that changes in alpha 1-adrenoceptor mechanisms are due to changes in receptor reserve. No age-related change was observed in affinity of alpha 1-adrenoceptors. 4. The tension of the dilator induced by field stimulation increased with age from 5 to 13 weeks did not change from 13 to 180 weeks. The age-related change in tension is due to change in the amount of norepinephrine released by stimulation.


Alpha-adrenoceptors involved on the cardiovascular response induced by mianserin in the pithed rat. 1. The effects of the antidepressant drug mianserin on the cardiovascular responses induced by preganglionic electrical stimulation, and i.v. infusion of the adrenergic agonists noradrenaline (NA, alpha 1 and alpha 2), phenylephrine (alpha 1) and xylazine (alpha 2) in the pithed normotensive rat were studied. 2. Mianserin inhibited in a dose-dependent manner the pressor effect caused by electrical stimulation of spinal cord (Th7-Th9) and the infusion of NA, phenylephrine and xylazine. Cocaine increased the inhibitory effect of mianserin on the pressor effect caused by electrical stimulation and NA. 3. Mianserin blocked the xylazine-induced inhibition of cardiac nerve stimulation effect. 4. These results suggest that mianserin blocks the NA uptake, and it is more effective in blocking presynaptic alpha 2- than postsynaptic alpha-adrenoceptors.


Antagonism towards endogenous adenosine and inhibition of cGI-PDE in the cardiac effects of amrinone, milrinone and related analogues. 1. The effect of amrinone, milrinone and of three milrinone analogues was tested on spontaneous chronotropic and inotropic activity of guinea-pig isolated atria, on the activity of cGMP-inhibited phosphodiesterase (cGI-PDE) from guinea-pig heart and on specific binding of N6-cyclohexyl[3H]adenosine ([3H]CHA) to Ri adenosine receptors in guinea-pig atria. 2. The Ki-values towards [3H]CHA binding to Ri receptors were linearly related to the EC50S for the increase in force of contraction but not to the EC50S for the increase in frequency of the atria. The Ki values towards cGI-PDE were linearly related to the EC50S for the positive chronotropic effect.


Effects of estrogen pretreatment of the spare alpha 1-adrenoceptors and the slow and fast components of the contractile response of the isolated female rat aorta. 1. Estrogen pretreatment increases the responsiveness to noradrenaline and to clonidine in isolated rat female aortae. 2. Determinations of the pKA and pA2 of noradrenaline and prazosin, respectively suggest that the isolated female aorta possess a homogeneous alpha 1-adrenoceptor population. 3. The fast and slow components of clonidine-induced aorta contraction were determined using nifedipine. 4. Considering that after estrogen pretreatment, an increase in spare alpha 1-adrenoceptors probably occur and only the fast component of clonidine-induced contraction was enhanced we concluded that estrogen pretreatment increases the number of alpha 1-adrenoceptors and the amount of intracellular calcium available for contraction.


The DNA polymerase-encoding gene of Bacillus subtilis bacteriophage SPO1. The bacteriophage SPO1 DNA polymerase-encoding gene, which contains a self-splicing intron, has been sequenced and its amino acid (aa) sequence has been deduced. The aa sequence of SPO1 DNA polymerase shows a high degree of similarity with that of DNA polymerase I from Escherichia coli (Po1I). Alignment with the sequences of Po1I, and the phi 29 and SPO1 DNA polymerases indicate that the aa residues that have been implicated in 3'----5' exonuclease activities are conserved.


A modified TnphoA useful for single-stranded DNA sequencing. The TnphoA transposon constructed by Manoil and Beckwith [Proc. Natl. Acad. Sci. USA 82 (1985) 8129-8133] has been modified to permit easy isolation of single-stranded (ss) DNA of target plasmids. The intergenic region (IG) of filamentous phage f1, which consists of the phage origin of replication and packaging signal, was inserted into a nonessential region of TnphoA. This modified transposon should be useful for the analysis of genes cloned in plasmids that lack a filamentous phage IG. Transposition of TnphoA-IG into a plasmid carries the IG with it; subsequently, after infection with a filamentous helper phage, ss plasmid DNA suitable for sequence analysis and useful for oligodeoxyribonucleotide-mediated mutagenesis of TnphoA-generated fusions can be isolated. The utility of TnphoA-IG was confirmed by analysis of 'blue hops' into the bla (encoding beta-lactamase) and pspE (encoding phage shock protein) genes whose products are secreted into the Escherichia coli periplasm.


Transformation of cucumber tissues by microprojectile bombardment: identification of plants containing functional and non-functional transferred genes. The microprojectile bombardment method was used to transfer DNA into embryogenic callus of cucumber (Cucumis sativus), and stably transformed cucumber plant lines were obtained. A total of 107 independently regenerated cucumber plants were assayed for the presence and expression of the transferred Nos-NPTII gene (encoding nopaline synthase-neomycin phosphotransferase II). Genomic blot hybridization analyses showed that a high percentage (16%) of the cucumber plants were transformed with Nos-NPTII; however, only about 25% of these transgenic plants expressed Nos-NPTII. Inactivity of Nos-NPTII in many of the transformed cucumber plants may be associated with the transfer of multiple copies of Nos-NPTII. PCR and genomic blot hybridization analyses were used to show that the transferred gene was inherited in the subsequent plant generation.


The complete sequence of genome segment 8 of bluetongue virus, serotype 1, which encodes the nonstructural protein, NS2. Bluetongue virus has a ten-segment double-stranded RNA genome, of which segment 8 encodes a nonstructural protein NS2. This protein is the only bluetongue viral protein to be phosphorylated and also has the ability to bind single-stranded RNA. At present, the function of NS2 is unknown and in order to analyse its characteristics in more detail, it was first necessary to obtain a full-length cDNA clone of the genome segment.


A mouse c-ros genomic clone: identification of a highly conserved 22-amino acid segment in the juxta-membrane domain. The mouse c-ros protooncogene genomic sequences have been cloned; an analysis of the partial genomic clone revealed a high conservation of the exons encoding the juxta-membrane (JM) and the 5' most protein tyrosine kinase domains. We have identified a segment of 22 amino acids conserved between the human and mouse JM domains; this segment may have a critical role in the function of the c-ros-encoded protein tyrosine kinase receptor.


[Analysis of the regulation of HLA class II genes by forskolin]. The expression of HLA class II molecules is mainly regulated transcriptionally and this regulation is thought to play an important role to the control of immune response. In this report, we have studied the effect of adenylate cyclase activator, forskolin, to the expression of HLA class II molecules on the cell surface of an human multiple myeloma cell line, RPMI8226. On the northern blot analysis and FACS analysis, we have revealed that forskolin upregulated the expression of mRNAs of DQB and DRB gene and their products on its surface. On the sequence analysis of upstream of HLA-DQB gene, we have identified not only Y-,X-, W-box, which were thought to regulatory region of truncated gene, but also cAMP responsible element (CRE) like regulatory region, which located upstream of W-box. On the gel retardation assay, when we used DNA probes that were specific for CRE like region and Y-box, we have found newly detectable bands, which appeared by forskolin treatment. These data suggest that forskolin upregulates HLA class II molecules by means of the interaction between CRE and cAMP responsible element binding protein (CREB).


Two-dimensional NMR studies on 1-10 fragment of adrenocorticotropic hormone. Various two-dimensional NMR techniques have been used to obtain complete resonance assignments of the protons in the 1-10 fragment of adrenocorticotropic hormone (ACTH). 1H-1H coupling constants among the backbone protons and the chemical shift values measured in aqueous and in dimethyl sulphoxide solutions indicated preference for extended but different conformations in the two solvents.


X-band electron paramagnetic resonance spectra of bovine serum albumin-copper(II) and bovine serum albumin-copper(II)-aminoacid systems. X-band electron paramagnetic resonance (epr) spectra of the binary systems, BSA-copper(II) (1:1 and 2:1), and the ternary systems, BSA-Cu(II)-aminoacid (1:1:1), are described. In the binary system, two distinct epr features have been observed. One of the features (towards the low pH), showing broad and overlapping epr signals, has been attributed to non-specific bonding of copper(II) to the albumin and other feature (towards higher pH), showing sharp intense epr signals, has been attributed to the specific bonding. The change from non-specific to specific binding is favoured by increase in pH as well as by increase in protein concentration. Specific binding of copper(II) in BSA-Cu(II) has been suggested to be similar to that in HSA-Cu(II). Spectra of BSA-Cu(II)-aminoacid (1:1:1) show simultaneous presence of binary BSA-Cu(II) and ternary BSA-Cu(II)-aminoacid.


Determination of total proton release in purple membrane suspension by umbelliferone fluorescence quenching technique. A technique for determining total proton release from purple membrane suspension under steady illumination has been described. Illuminated purple membrane is found to quench the fluorescence life-time of umbelliferone indicating the release of protons in the medium. Besides the "stoichiometric" release of protons from bacteriorhodopsin, there seems to be release of protons from sources other than protonated retinylidene Schiff base moiety also.


Tryptophan as an endogenous photosensitizer to elicit harmful effects of ultraviolet B. Owing to stratospheric ozone depletion (SOD) the natural flux of ultraviolet B (UVB) radiation (290-320 nm) is likely to increase on the earth surface. In our efforts to identify endogenous chromophores which may absorb significantly in the UVB range and subsequently induce phototoxic reactions, we have observed that tryptophan (Trp) was quite photoreactive under UVB. It enhanced considerably the oxygen-dependent photooxidation of tyrosine (Tyr) to dopachrome, a precursor of melanin. Our data suggest that UVB-sensitized Trp produces singlet oxygen (1O2) and superoxide radicals (O2-.), and these reactive forms of oxygen may contribute to membrane-, cytoplasm- and DNA-damaging effects. In the event of an increasing SOD level, other UVB chromophores may also exhibit similar phototoxic properties to lead to a definitive imbalance between cell life, injury and death.


Ubiquitin with a non-ATP-dependent slow intrinsic proteolytic activity: a mild and rapid purification procedure. Ubiquitin has been purified to homogeneity, through a dialysis membrane having a NMW cutoff of 12 kDa, by taking advantage of its non-dialysable nature under these conditions. The dialysate was continuously recycled through a CM-52 cation exchange column at pH 4.5. The adsorbed fraction was eluted selectively at pH 7.2. Ubiquitin (25 mg) was obtained from 500 ml of packed RBCs. On SDS PAGE, ubiquitin showed varying mobility depending on the time of boiling in SDS. With 2 min of boiling, the molecular weight seemed to be 10.5 kDa, whereas 10 min of boiling resulted in a molecular weight of 8.5 kDa. Ubiquitin showed a slow intrinsic proteolytic activity against SDS-denatured beta-galactosidase in the absence of ATP. For the first 4 hr, there was no detectable degradation, but degradation was nearly complete after 8 hr. These data are not in agreement with those of Freid et al. [Proc. Natl Acad. Sci, USA, 84 (1987), 3685] who have reported a proteolytic activity comparable to that of other proteolytic enzymes.


Effects of cadmium salt on phosphomonoesterases activity and fertilizing ability of fowl spermatozoa. Daily administration of cadmium salt for 25 days (2.5 mg per Kg body weight) in the male domestic fowl caused the end of treatment period. An increased incidences of concentration. Fertility dropped to zero at the end of the treatment period. Activity of acid and alkaline phosphomonoesterases were also drastically reduced by the end of treatment period. An increased incidences of morphological abnormalities of spermatozoa were noticed in the treated birds. After 46 days cessation of the treatment, full recovery of the above measures was found. These alterations suggest the reversible type of effect of cadmium chloride on the spermatozoa of male domestic fowl.


Polyclonal B-lymphocyte activation of sheep by Mycobacterium phlei against sheep pox virus. A trypsinized preparation of Mycobacterium phlei, non specific stimulator of immunity (NSI), and Sheep Pox Virus (SPV) were inoculated in different groups of sheep to activate B-lymphocytes and induce SPV neutralizing substance(s). NSI sensitized sheep B-lymphocytes in the presence of NSI or lymphokine elaborated SPV neutralizing substance(s). The SPV sensitized B-lymphocytes also mediated such neutralizing substance(s). Healthy control sheep B-lymphocytes failed to show any appreciable amount of viral neutralizing substance. However, a significant virus neutralizing substance(s) was detected when healthy sheep B-lymphocytes were cultured in presence of NSI antigen along with lymphokines.


Sodium channel expression in optic nerve astrocytes chronically deprived of axonal contact. Immunocytochemical and electrophysiological methods were used to examine the effect of retinal ablation on the expression of sodium channels within optic nerve astrocytes in situ and in vitro. Enucleation was performed at postnatal day 3 (P3), and electron microscopy of the enucleated optic nerves at P28-P40 revealed complete degeneration of retinal ganglion axons, resulting in optic nerves composed predominantly of astrocytes. In contrast to control (non-enucleated) optic nerve astrocytes, which exhibited distinct sodium channel immunoreactivity following immunostaining with antibody 7493, the astrocytes in enucleated optic nerves did not display sodium channel immunoreactivity in situ. Cultures obtained from enucleated optic nerves consisted principally (greater than 90%) of glial fibrillary acidic protein (GFAP)+/A2B5- ("type-1") astrocytes, as determined by indirect immunofluorescence; GFAP+/A2B5+ ("type-2") astrocytes were not present, nor were GFAP-/A2B5+ (O-2A) progenitor cells. Sodium channel immunoreactivity was not present in GFAP+/A2B5- astrocytes obtained from enucleated optic nerves; in contrast, GFAP+/A2B5- astrocytes from control optic nerves exhibited 7493 immunostaining for the first 4-6 days in culture. Sodium current expression, studied using whole-cell patch-clamp recording, was attenuated in cultured astrocytes derived from enucleated optic nerves. Whereas 39 of 50 type-1 astrocytes cultured from intact optic nerves showed measurable sodium currents at 1-7 days in vitro, sodium currents were present in only 6 of 38 astrocytes cultured from enucleated optic nerves. Mean sodium current densities in astrocytes from the enucleated optic nerves (0.66 +/- 0.3 pA/pF) were significantly smaller than in astrocytes from control optic nerves (7.15 +/- 1.1 pA/pF). The h infinity-curves of sodium currents were similar in A2B5- astrocytes from enucleated and control rat optic nerves. These results suggest that there is neuronal modulation of sodium channel expression in type-1 optic nerve astrocytes, and that, following chronic loss of axonal association in vivo, sodium channel expression is down-regulated in this population of optic nerve astrocytes.


A histochemical study of the activity of nonspecific cholinesterase in C6 glioma cells in culture. The activity of nonspecific cholinesterase (nChE) was revealed histochemically in a small number of C6 glioma cells under conditions of spontaneous growth in culture. The nChE-positive cells (nChE+) first appeared in 4-day-old cultures. They occurred solitarily or in small groups with a cluster- or chain-like arrangement. The incidence of nChE+ cells increased with in vitro age and/or higher population densities of cultures but it never exceeded 3% of the whole population. These results indicate the necessity of histochemical as well as biochemical data on nChE activity in glioma cells both in situ and in culture for the discovery of the heterogeneous distribution of this enzyme. db-cAMP induced a decrease in the proportion of nChE+ cells to total number of cells. These changes suggest that the original C6 glioma cell seed stock contains a small subclone of less differentiated cells exhibiting nChE activity. The greater number of nChE+ cells in older cultures is probably due to recovery and multiplication of initially scarce subclone cells. C6 glioma cells in culture form a suitable model for investigation of changes in nChE activity of glial cells provided that a histochemical method of detection is applied.


Type-1 and type-2 astrocytes are distinct targets for prostaglandins D2, E2, and F2 alpha. Accumulating evidence has revealed that astrocytes are potential targets for various neurotransmitters. Here we investigated the effects of prostaglandins (PGs) on signal transduction in purified primary cultures of rat type-1 and type-2 astrocytes. PGF2 alpha, PGD2, and 9 alpha,11 beta-PGF2, a metabolite of PGD2 and a stereoisomer of PGF2 alpha, evoked a rapid rise in the intracellular Ca2+ concentration ([Ca2+]i) in type-1, but not in type-2, astrocytes. STA2, a stable analogue of thromboxane A2, was less effective, and PGE2 showed little effect. The PG-induced rise in [Ca2+]i was not blocked by an antagonist of either PGD2 receptor or thromboxane A2 receptor. PGF2 alpha and 9 alpha,11 beta-PGF2 stimulated rapid formation of inositol trisphosphate followed by inositol bisphosphate and inositol monophosphate. On the other hand, PGE2 increased the intracellular level of cyclic AMP in type-2 astrocytes, rather than in type-1 astrocytes. The potency of PGs for cyclic AMP formation was in the following order: PGE2 greater than PGE1 greater than or equal to STA2 much greater than iloprost, a stable analogue of PGI2. PGD2 and PGF2 alpha had no effect on cyclic AMP formation. These results demonstrate that type-1 astrocytes preferentially express PGF2 alpha receptors, the activation of which leads to phosphoinositide metabolism and [Ca2+]i elevation, whereas type-2 astrocytes possess PGE receptors that are linked to cyclic AMP formation.


Viral gastrointestinal infections.

Viral gastrointestinal infections. Numerous disease-causing viruses of the gastrointestinal tract have been described. The diseases that they induce range from subclinical to fatal gastrointestinal or systemic organ infection. Recent advances in molecular biology and virus culture techniques, as well as improvements in animal models of these infections, have increased our understanding of pathologic processes associated with these viruses. Although our overall understanding of these viruses has improved to the extent that many more viruses can be identified as pathogens, we are still a long way from achieving a thorough understanding of the pathogenesis of viral infection and the mechanisms that mediate immunity.


Regulation of beta-endorphin receptor expression in mouse spleen cells with Con A and rIL-2. The expression of the beta-endorphin receptor on both activated and unstimulated mouse spleen cells was studied. Results showed that unstimulated cells have only one type of beta-endorphin receptor with a specific low affinity (Kd = 1.034 +/- 0.0237 x 10(-7) M, 25,000 sites/cell). After Con A stimulation, cells express two types of receptors, one with a low affinity (Kd = 1.034 +/- 0.024 x 10(-7) M, 320,000 sites/cell) and the other with a high affinity (Kd = 1.052 +/- 0.033 x 10(-9) M, 49,000 sites/cell). The kinetic experiments during 4 days after Con A activation indicated that the receptor of high affinity emerged from 24 to 72 h, while the low affinity one increased in number after stimulation. The receptor numbers of both high and low affinity ones reached a maximum peak at 72 h, then began to decline. The addition of exogenous rIL-2 depressed the Con A-induced increment of the receptor numbers of both the high and low affinity ones, but enhanced the proliferative response of the cells. It is suggested that the degree of the expression of the receptors does not simply depend on the mitogenic degree of the cells. In addition, our experiment demonstrated that splenocytes cultured in medium with or without Con A or Con A + rIL-2 for 96 h did not secrete any detectable amount of beta-endorphin with use of the RIA assay, which is sensitive enough to detect the much lower levels of beta-endorphin than that necessary for biological effects. We suggest that the expression of the high affinity beta-endorphin receptor on the activated T-lymphocytes may have to precede the production of IL-2 to potentiate the T-cell proliferative response. The mechanisms and modes of interaction between the neuroendocrine system and the immune system were discussed.


Sympathetic nervous system modulates macrophage function. We have reported previously that sympathectomy augments immune responses in mice and rats. In the present study, we show that ablation of the sympathetic nervous system augments macrophage function as measured by increased TNF secretion. We also show that a factor present in the sympathetic ganglia of newborn rats, suppresses secretion of TNF by LPS-stimulated macrophages as does the beta-adrenergic agonist isoproterenol.


Upregulatory effects of cefpimizole natrium on human leukocytes. Cefpimizole natrium (CPIZ), an antibiotic belonging to the cephalosporins, was examined regarding its influence on neutrophil functions. Neutrophil superoxide (O2-) generation increased by intravenous CPIZ in patients with maxillofacial diseases. In vitro examination revealed that CPIZ directly stimulates neutrophils to generate O2- in a dose-dependent manner, though the induction ability is not as strong as phorbol myristate acetate (PMA). Protein kinase C (PKC) activity in the neutrophil plasma membrane increased after CPIZ treatment, while the activity in the cytosol fraction decreased. CPIZ cooperated with biological response modifiers (BRMs) such as sizofilan, lentinan, OK-432, rIL-2 and rIFN-gamma in neutrophil O2- generation. Non-specific cytotoxicity against K562 cells and candida cells was also enhanced by neutrophil pretreatment with both CPIZ and one of the BRMs except for sizofilan and rIL-2. From these results it can be concluded that CPIZ directly enhanced neutrophil O2- generation and that these CPIZ activations are further beneficial to protection against bacterial infections.


Radiotherapy versus radiotherapy enhanced by cisplatin in stage III non-small cell lung cancer. Between January 1987 and June 1991, 173 patients with inoperable non-small cell lung cancer, Stage III, were entered into a randomized trial comparing radiotherapy only (RT) (45 Gy/15 fractions/3 weeks) (arm A) versus RT and a daily low dose of cDDP (6 mg/m2) (arm B). An overall response rate of 58.9% was observed in arm A and 50.6% in arm B, respectively. No differences in the pattern of relapse were noted between the two treatment groups. Median time to progression was 10.6 months for arm A and 14.2 months for arm B. Median survivals were 10.3 months and 9.97 months, respectively. Toxicity was acceptable and no treatment-related death occurred in either treatment schedule. In this study no significant advantage of the combined treatment over radiation therapy only was found. The encouraging results achieved in some trials together with the intractability of the disease suggest that further efforts should be made to optimize clinical trial protocols, perhaps by reviewing the radiobiological and pharmacological basis of the combined treatment.


Combined modality therapy for stage III non-small cell lung carcinoma: results of treatment and patterns of failure. Patients with Stage III non-small cell lung carcinoma continue to pose a therapeutic problem with dismal cure rates. In an effort to improve on these results, 129 patients with biopsy-proven clinical Stage III non-small cell lung carcinoma from November 1982 through November 1987, were entered into two consecutive Phase II studies at Rush-Presbyterian-St. Luke's Medical Center. Treatment in the first study consisted of Cisplatin and 5-Fluorouracil infusion with concomitant split course radiation; in the second Etoposide was added. Radiation and chemotherapy were given simultaneously on days one through five of each cycle in a preoperative fashion for four cycles in patients considered eligible for surgery and in a definitive fashion for six cycles in patients considered ineligible for surgery. Radiation was given in 2 Gy fractions for a planned preoperative dose of 40 Gy and a definitive dose of 60 Gy. Surgical resection was attempted four to five weeks later in patients treated preoperatively. Thus, 83 patients were treated preoperatively and 46 definitively. Eighty-three patients (64%) had IIIA disease and IIIB disease was found in the remainder of the patients. Sixty-two patients (75%) in the eligible for surgery group had a thoracotomy after the combined treatment with a resectability rate of 97% and an operative mortality rate of 5%. There were 17 patients (27%) with no evidence of residual cancer in the resected specimen. Three-year survival for the eligible for surgery group at 40% was significantly better than 19% observed in the ineligible for surgery group (p = 0.003). Seventy-six percent of the patients with no residual cancer in the resected specimen are recurrence-free at three years compared to 34% of the patients with gross residual. A total of 81 patients have failed after their treatment; 49 (59%) in the eligible for surgery group and 32 (70%) in the ineligible for surgery group. Of all the patients who failed, local failure alone and as a component occurred in 21 (26%) and 36 (44%) patients, respectively. Failure in distant sites alone was noted in 56% of the overall failures. Severe toxicity was unusual. There were three treatment related deaths (2%). Radiation esophagitis and pneumonitis were only mild to moderate seen in less than 10% of the patients. Survival rates and patterns of failure according to the stage of the disease, histology, treatment group and pathologic response will be presented in detail.


Should patients with post-resection locoregional recurrence of lung cancer receive aggressive therapy? The outcome of thirty-seven patients with a post-resection locoregional recurrence of non-small cell lung cancer treated with radiation therapy alone between 1979 and 1989 was compared to that of 759 patients with unresected non-small cell lung cancer also treated with standard radiation during the same period. Each patient's locoregional recurrence was staged using the current American Joint Committee on Cancer staging system. Comparison of pretreatment characteristics between the two groups, including age, sex, extent of weight loss, performance status, stage, and histologic subtype revealed fewer patients with greater than 5% weight loss (35 vs. 47%, p = 0.04) and more cases with squamous histology (54 vs. 28%, p = 0.01) among the patients with locoregional recurrences than those with newly diagnosed lesions. Over 80% of both groups had clinical stage III lesions. The median radiation doses were 56 and 59 Gy for recurrent and newly diagnosed cases (p = NS). For the patients with locoregional recurrences, the median time from resection to recurrence was 13 months (range: 3-118 months), and the recurrences were predominantly nodal in 25 cases, chest wall/pleural in four and at the bronchial stump in eight. When measured from the date of documented recurrence, the median survival time and 2-year actuarial survival rate of the patients with recurrent lesions were 12 months and 22%, as compared to 12 months and 26% for the newly diagnosed patients (p = NS). Freedom from documented locoregional tumor progression at 2 years was 30% for both groups. Patients with bronchial stump lesions had superior survival to those with nodal or chest wall recurrences, with a median survival time of 36 versus 9 months. A therapeutic approach to selected patients with post-resection locoregional recurrence of non-small cell lung cancer equally aggressive to that for newly diagnosed lung cancer patients is justified by these results, especially for patients with bronchial stump recurrences.


Radiation therapy in the management of medically inoperable carcinoma of the lung: results and implications for future treatment strategies. Surgery is the treatment of choice for resectable non-small cell lung carcinoma. For patients who are medically unable to tolerate a surgical resection or who refuse surgery, radiation therapy is an acceptable alternative. We reviewed the records of 152 patients with medically inoperable non-small cell lung carcinoma treated at our institution between 1982 and 1990. Patients with metastatic disease, mediastinal lymph node involvement or unresectable tumors were excluded. The actuarial overall survival at 2 and 5 years was 40% and 10%, respectively. The disease-free survival at 2 and 5 years was 31% and 15%. The disease-free survival for patients with T1 tumors was 55% at 2 years, versus 20 and 25% for T2 and T3 lesions, respectively (p = .0006). Increasing tumor dose was also associated with increasing disease-free survival (p = .0143). Overall, 66% percent of the patients were considered to have failed. Of these, 70% showed a component of local failure and 45% failed distantly. Patients with T1 tumors experienced a lower probability of failing locally or distantly than did patients with T2 or T3 tumors. A reduced risk of local and distant failure was seen for patients treated to doses of greater than 65 Gray, especially for T1 tumors. We conclude that radical radiation therapy is an effective treatment for small tumors when treated to doses of 65 Gray or more. Since local failure is the prominent pattern of relapse in patients with large tumors, new therapeutic strategies should be considered for this patient group.


Adverse influence of younger age on outcome in patients with non-small cell lung carcinoma (NSCLC) treated with radiation therapy (RT) alone. Treatment outcome of 63 patients younger than 50 years of age initiated on a course of once-daily definitive radiation therapy without concurrent or preirradiation chemotherapy for clinical Stages I-III unresected non-small cell lung carcinoma from 1978 to 1988 was compared to the outcome of 695 patients over the age of 50. Follow-up ranged from 24-110 months with follow-up until death in 88% of patients. The actuarial overall survival rate for all patients was 22% at 2 years with a median survival time of 11.5 months. Patients less than 50 and greater than or equal to 50 years old were similar in male:female ratio, distribution of histologic subtype, performance status, and extent of weight loss. Poorly differentiated histologic grade was more prevalent among the younger patients (59% vs 41%, p = .005). Ninety-four percent of younger patients and 86% of older patients had clinical stage III disease (p = NS). Survival was significantly worse for patients who were younger than 50 years old (p = .05), with a median survival time of 7.8 months. Median survival time for those patients 50 years of age or older was 12.4 months. Poorer survival outcome among young patients was most pronounced among patients with unfavorable characteristics of poor performance status (greater than or equal to 2) or weight loss (greater than 5%) (p = .002). Distant failure (p = .029) and brain failure (p = .003) as initial site of relapse was more common among younger patients. Among young patients, poor histologic grade was associated with both distant failure (p = .003) and brain metastasis (p = .002). The difference in distribution of histologic grade, incidence of distant failure, particularly in the brain, and poorer survival outcome among patients less than 50 may be indicative of more aggressive tumor behavior in the younger patients. These results indicate that patients less than 50 may require alternate treatment strategies. Age should be considered a stratification variable in non-operative randomized trials of non-small cell lung carcinoma which include patients with non-favorable characteristics.


A reappraisal of the role of prophylactic cranial irradiation in limited small cell lung cancer. The use of prophylactic cranial irradiation in limited stage small cell lung cancer remains controversial. Prospective trials have demonstrated that PCI can reduce central nervous system relapse rates, but the impact on survival remains questionable except for the possible evidence of a beneficial effect for long term survivors. With higher rates of thoracic control now obtainable with hyperfractionated radiation and concomitant chemotherapy, it becomes important to analyze the benefit of PCI in that setting. Before 1982, we included PCI in the management of all patients with limited stage small cell lung cancer; thereafter, we discontinued its use. This report compares the outcome of the two treatment approaches and addresses the role of PCI among patients who achieve durable local control. There were 36 limited stage small cell lung cancer patients treated with PCI from 1979-1982 and 26 patients treated without PCI from 1985-1989. Induction chemotherapy was followed in both groups by thoracic irradiation (45 Gy). The PCI patients received 30 Gy to the whole brain in 10 fractions. Both groups received maintenance chemotherapy. Of complete responders, brain failure was the first failure in 18% (4/22) of PCI (+) versus 45% (10/22) of PCI (-) (p = .04). Survival at 2 years was 42% for PCI (+) versus 13% for PCI (-) (p less than .05). When the analysis was limited to those patients permanently controlled in the thorax; there were 25% (4/16) brain failures PCI (+) versus 70% (7/10) PCI (-) (p = .03). For this same subset the 2-year survival was 56% PCI (+) versus 14% PCI (-) (p less than .05). There were no 5 year survivors without PCI compared to 38% (6/16) with PCI. These data suggest that PCI appears to be effective in enhancing survival of patients who achieve durable thoracic control. Prospective trials are necessary to evaluate the use of PCI combined with therapeutic regimens with a documented ability to achieve high rates of sustained control of thoracic disease.


Extramammary Paget's disease of the perineal skin: role of radiotherapy. We have reviewed our treatment results in 65 patients with extramammary Paget's disease arising in the vulva, perianal area, or scrotum. In 30 patients with primary disease, positive surgical margins were found in 53%, and there was an actuarial local recurrence rate of 40% within 5 years. The median follow-up period for primary extramammary Paget's disease patients treated with surgery alone was 198 months, and none died of this disease. Three patients treated with definitive radiotherapy were without recurrence at 12, 21, and 60 months after 56 Gy of supervoltage x-rays. In 22 patients with extramammary Paget's disease and associated adnexal or rectal adenocarcinoma, nine treated with surgery alone had a 75% local control rate. Three patients treated with surgery and adjuvant radiotherapy all had local control; of two patients treated with radiotherapy alone, one had persistent adenocarcinoma. The median survival for all patients with extramammary Paget's disease and adenocarcinoma was 22 months. We conclude that patients with extramammary Paget's disease have excellent survival but that local recurrence and morbidity from surgery, especially in the elderly, can be high. Radiotherapy greater than 50 Gy as primary treatment for extramammary Paget's disease in those medically unfit for surgery, or as an alternative to further surgery for recurrence after surgery and for anyone wishing to avoid mutilating surgery, is indicated. For those with adenocarcinoma and extramammary Paget's disease, the use of adjuvant postoperative radiotherapy in doses greater than 55 Gy is indicated because of the high risk of local recurrence after surgery alone.


A Coxiella burnetti repeated DNA element resembling a bacterial insertion sequence. A DNA fragment located on the 3' side of the Coxiella burnetii htpAB operon was determined by Southern blotting to exist in approximately 19 copies in the Nine Mile I genome. The DNA sequences of this htpAB-associated repetitive element and two other independent copies were analyzed to determine the size and nature of the element. The three copies of the element were 1,450, 1,452, and 1,458 bp long, with less than 2% divergence among the three sequences. Several features characteristic of bacterial insertion sequences were discovered. These included a single significant open reading frame that would encode a 367-amino-acid polypeptide which was predicted to be highly basic, to have a DNA-binding helix-turn-helix motif, to have a leucine zipper motif, and to have homology to polypeptides found in several other bacterial insertion sequences. Identical 7-bp inverted repeats were found at the ends of all three copies of the element. However, duplications generated by many bacterial mobile elements in the recipient DNA during insertion events did not flank the inverted repeats of any of the three C. burnetii elements examined. A second pair of inverted repeats that flanked the open reading frame was also found in all three copies of the element. Most of the divergence among the three copies of the element occurred in the region between the two inverted repeat sequences in the 3' end of the element. Despite the sequence changes, all three copies of the element have retained significant dyad symmetry in this region.


Energy transduction in the methanogen Methanococcus voltae is based on a sodium current. We provide experimental support for the proposal that ATP production in Methanococcus voltae, a methanogenic member of the archaea, is based on an energetic system in which sodium ions, not protons, are the coupling ions. We show that when grown at a pH of 6.0, 7.1, or 8.2, M. voltae cells maintain a membrane potential of approximately -150 mV. The cells maintain a transmembrane pH gradient (pH(in) - pH(out)) of -0.1, -0.2, and -0.2, respectively, values not favorable to the inward movement of protons. The cells maintain a transmembrane sodium concentration gradient (sodium(out)/sodium(in)) of 1.2, 3.4, and 11.6, respectively. While the protonophore 3,3',4',5-tetrachlorosalicylanilide inhibits ATP formation in cells grown at pH 6.5, neither ATP formation nor growth is inhibited in cells grown in medium at pH 8.2. We show that when grown at pH 8.2, cells synthesize ATP in the absence of a favorably oriented proton motive force. Whether grown at pH 6.5 or pH 8.2, M. voltae extrudes Na+ via a primary pump whose activity does not depend on a proton motive force. The addition of protons to the cells leads to a harmaline-sensitive efflux of Na+ and vice versa, indicating the presence of Na+/H+ antiporter activity and, thus, a second mechanism for the translocation of Na+ across the cell membrane. M. voltae contains a membrane component that is immunologically related to the H(+)-translocating ATP synthase of the archaeabacterium Sulfolobus acidocaldarius. Since we demonstrated that ATP production can be driven by an artificially imposed membrane potential only in the presence of sodium ions, we propose that ATP production in M. voltae is mediated by an Na+-translocating ATP synthase whose function is coupled to a sodium motive force that is generated through a primary Na+ pump.


A temperature-regulated, retrotransposon-like element from Candida albicans. A repetitive element was isolated from the genome of Candida albicans. This repetitive element, which we designated alpha, was localized to a 500-bp fragment of genomic DNA. The alpha element was dispersed in the genome and varied in copy number and genomic location in the strains examined. Analyses of various loci containing the alpha element identified a locus containing a composite element. This composite element consisted of two direct repeats of the alpha element separated by approximately 5.5 kb of DNA, a structural arrangement similar to that of retrovirus-like transposable elements. The flanking alpha elements of the composite structure were 388 bp in length and were identical in sequence. They were bounded by the nucleotides 5'-TG. ... CA-3', which were part of a delimiting inverted repeat, a feature conserved in the long terminal repeats of retroviruses and retrovirus-like elements. As in retrovirus-like elements, the entire composite element, including the alpha elements, was transcribed into an approximately unit-length mRNA. The expression of this transcript was greatly increased when cells were grown at 25 versus 37 degrees C. As has been found in many retrotransposons, the composite element was flanked by a 5-bp duplication and varied in both copy number and genomic location in various strains. We conclude that the composite element is a retrotransposon-like element, and we have designated this element Tca1. We suggest that Tca1 may be relevant to the genomic evolution of C. albicans and the pathogenic potential of the organism.


New thermosensitive plasmid for gram-positive bacteria. We isolated a replication-thermosensitive mutant of the broad-host-range replicon pWV01. The mutant pVE6002 is fully thermosensitive above 35 degrees C in both gram-negative and gram-positive bacteria. Four clustered mutations were identified in the gene encoding the replication protein of pVE6002. The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position. Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed. The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range. Using pVE6002 as the delivery vector for a transposon Tn10 derivative in Bacillus subtilis, we observed transposition frequencies of about 1%.


dcd (dCTP deaminase) gene of Escherichia coli: mapping, cloning, sequencing, and identification as a locus of suppressors of lethal dut (dUTPase) mutations. In Escherichia coli, most of the dUMP that is used as a substrate for thymidylate synthetase is generated from dCTP through the sequential action of dCTP deaminase and dUTPase. Some mutations of the dut (dUTPase) gene are lethal even when the cells are grown in the presence of thymidine, but their lethality can be suppressed by extragenic mutations that can be produced by transposon insertion. Six suppressor mutations were tested, and all were found to belong to the same complementation group. The affected gene was cloned, it was mapped by hybridization with a library of recombinant DNA, and its nucleotide sequence was determined. The gene is at 2,149 kb on the physical map. Its product, a 21.2-kDa polypeptide, was overproduced 1,000-fold via an expression vector and identified as dCTP deaminase, the enzyme affected in previously described dcd mutants. Null mutations in dcd probably suppress the lethality of dut mutations by reducing the accumulation of dUTP, which would otherwise lead to the excessive incorporation of uracil into DNA.


Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe. CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed.