Increased expression of differentiation markers can accompany laminin-induced attachment of small cell lung cancer cells. We investigated the interaction between human lung cancer cells, laminin, and several differentiating agents. When grown on laminin coated substrate eight out of 11 small cell lung cancer (SCLC) cell lines exhibited attachment to laminin and three had extensive outgrowth of long neurite-like processes. Of seven non-small cell lung cancer cell lines, selected for their in vitro anchorage-independent growth, attachment was observed in only three cell lines, and process formation was far less extensive than in SCLC cell lines. Among several differentiating agents, only dcAMP, which alone induced attachment and some process formation, increased laminin-mediated attachment and process formation of two SCLC cell lines, NCI-N417 a variant cell line, and NCI-H345, a classic cell line. The expression of several neuroendocrine and neuronal markers was investigated in these two SCLC cell lines. The expression of the light subunit of neurofilaments increased in NCI-N417 within 3 to 4 days of seeding, while NCI-H345 exhibited approximately 5 fold increase in expression of the GRP gene and a 3 fold increase expression of the beta-actin gene. The expression of a number of other neuroendocrine and neuronal markers did not change following growth on laminin. The doubling times remained unchanged independent of the presence of and attachment to laminin while topoisomerase II gene expression levels in NCI-N417 cells decreased approximately 5 fold when cells were growing on laminin.


Immunohistochemical analysis of nm23 gene product/NDP kinase expression in pulmonary adenocarcinoma: lack of prognostic value. Levels of nm23 gene product/nucleoside diphosphate kinase (NDP kinase) expression have been demonstrated to correlate inversely with metastatic potential in several tumours, indicating that this could be a useful tool as a prognostic indicator. Using an antibody to NDP kinase, levels of nm23 gene product/NDP kinase expression in pulmonary adenocarcinoma were examined immunohistochemically. Of 88 patients tested, 39 (44%; Group B) showed strong immunoreactivity for NDP kinase in most of cancer cells within the tumour tissues, while 49 (56%; Group A) contained few or no NDP kinase-positive cancer cells. Nm23 gene product/NDP kinase was expressed independently of clinicopathological factors, and unexpectedly, no correlation of survival rates between both Groups could be demonstrated. Thus, in pulmonary adenocarcinoma, levels of nm23 gene product/NDP kinase expression may lack prognostic value.


An immunohistochemical investigation of diagnostic biopsy material taken from short and long term survivors with small cell lung cancer. An immunohistochemical study has been carried out on fibre optic-biopsy specimens from patients with small cell lung cancer (SCLC) who had either died within 3 months, or who had survived more than 2 years. Long term survivors (LTS) were identified from completed clinical trials at major UK centres and were matched for age and sex within the trial with short term survivors (STS). The panel of immunohistochemical markers included those previously reported to be associated with prognosis, and reagents representative of both neuroendocrine and epithelial differentiation. A preliminary screen of 17 antibodies identified 11 as consistently reactive on paraffin-embedded material using streptavadin-biotin immunoperoxidase. Of 186 identified patients, 110 biopsy samples were retrieved. Of these, 70 gave sufficient material for analysis. All sections were scored by three observers without knowledge of the prognosis. The analysis failed to identify any antigen whose expression was correlated with prognosis. We conclude that, in fibre-optic biopsy specimens, immunohistochemical analysis does not add prognostic information in SCLC.


Serum neuron specific enolase (NSE) is a determinant of response duration in small cell lung cancer (SCLC). Seventy-two consecutive patients were eligible for a study of clinical determinants of response and response duration in small cell lung cancer (SCLC). Pretreatment values of routine laboratory parameters, and three tumour markers: neuron specific enolase (NSE), carcinoembryonic antigen (CEA), and acidic glycoprotein (AGP) were measured. Descriptive clinical variables as performance status (PS), extent of disease, age and sex were also included in the study. All variables were analysed for influence on the type and duration of response. The complete remission probability was only related to pretreatment extent of disease. In a multivariate analysis (Cox) of response duration, only NSE and type of response had significant influence. Consequently, measurements of NSE before therapy will be useful in future clinical trials on SCLC especially in situations, where responding patients are submitted to specific treatment strategies.


Comparison of lisinopril and nitrendipine on the pulsatility index in mild essential arterial hypertension. A double-blind, randomized crossover study was performed in 21 patients with essential arterial hypertension. Nitrendipine 20 mg o.d. and lisinopril 20 mg o.d. were given in a randomized order during a period of each 8 weeks. Nitrendipine and lisinopril decreased systolic and mean arterial blood pressure to a similar level without a significant increase in heart rate. The mean diastolic blood pressure was smaller with the lisinopril treatment than with the nitrendipine treatment. The blood pressure decrease was maintained in the sitting and standing position. Furthermore, only nitrendipine decreased the pulsatility index at the tibial posterior arteries, while lisinopril did not influence it significantly. This finding means that mechanisms other than the blood-pressure lowering effect are involved in the decrease of the pulsatility index.


Effects of lobenzarit on murine acute viral myocarditis. Lobenzarit (CCA) is a newly developed immunomodulating drug that has been demonstrated to enhance suppressor T-cell number and function. In this study we investigated the effect of CCA on murine myocarditis induced by the encephalomyocarditis (EMC) virus. Mice were first inoculated with the EMC virus. CCA (100 mg/kg) was administered daily for 14 days, starting on the day the mice were inoculated. The concentrations of Lyt2+ (suppressor/cytotoxic T) cells in the peripheral blood and heart were examined by laser flow cytometry and in situ staining. The concentration of Lyt2+ cells increased significantly in the peripheral blood and heart of CCA-treated mice when compared with untreated mice (blood: 23.0 +/- 2.6% vs. 18.8 +/- 2.8%, p less than 0.05; heart: 30.1 +/- 4.6% vs. 15.8 +/- 4.3%, p less than 0.05, respectively). Surprisingly, however, the severity of myocardial inflammation in CCA-treated mice was significantly greater than in untreated mice (area of inflammation: 33.1 +/- 10.5% vs. 18.8 +/- 19.5%, respectively, p less than 0.05). Thus, these findings suggest that CCA may aggravate EMC-induced acute myocarditis by enhancing the number of Lyt2+ cells.


Evaluation of a collagen/hydroxylapatite implant for orbital reconstructive surgery. A variety of autogenous and alloplastic materials have been used to correct enophthalmos. Hydroxylapatite (HA), is a calcium-phosphate-based compound that has been extensively studied as a bone replacement material. We studied the properties of a new dense particulate form of HA in a collagen matrix (PFC/HA) implanted in the subperiosteal space of ten rabbit orbits for a period of 6 months. All animals were studied with pre- and postoperative computed tomography (CT) scans, and measurements of induced proptosis and implant volume were made. The proptosis induced by the implant averaged 2.2 mm and was stable over a 6-month period. Implant volume was constant throughout the study. Three-dimensional computer-generated images of the soft tissue, skeletal, and implant surfaces confirmed the implant stability. All animals were studied histologically with fluorochrome bone markers, which revealed minimal foreign body reaction to the implant, no evidence of infection, and marked fibrovascular ingrowth. We found the PFC/HA to possess properties that make it an ideal implant material: ease of availability, ease of handling, no resorption, minimal immunogenicity, infection resistance, no observed migration, biointegration, and no risk of disease transmission. PFC/HA may make an excellent implant material to manage orbital volume.


A novel form of glycosylphosphatidylinositol-anchor converting activity with a specificity of a phospholipase D in mammalian liver membranes. It has been reported that rat liver membranes contain a glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) which may be involved in generation of phosphoinositol-glycan, a putative insulin second messenger (Saltiel, A.R. and Cuatrecasas, P. (1988) Am. J. Physiol. 255, C1-C11). Using GPI-anchored acetylcholinesterase (AChE) from bovine erythrocytes as substrate, we attempted to isolate GPI-PLC from bovine and rat liver membranes. A major part of the GPI-anchor converting activity present in liver could be washed away from the tissue by extraction with detergent-free buffer. Solubilisation of the washed membranes with 0.25% (v/v) Nonidet P-40 and ultracentrifugation resulted in a considerable amount of detergent soluble GPI-anchor converting activity in the supernatant. Anion-exchange chromatography on a Fractogel TSK-DEAE column of detergent-soluble GPI-anchor converting activity revealed two distinct peaks eluting at 50-80 mM and 120-170 mM NaCl, respectively. Using [125I]TID-labelled mf-AChE as substrate, radiolabelled diradylglycerol was obtained with both peak activities. However, when the phosphatase inhibitors NaF and sodium orthovanadate were included in the assay systems, phosphatidic acid was detected in addition to diradylglycerol. Both GPI-anchor converting activities were Ca(2+)-sensitive and inhibited by heavy metal chelating agents. These results suggested the presence of two isoenzymes of GPI-PLD and a phosphatase, rather than a GPI-PLC activity, in liver. Further, it could be shown that the activity in the second peak was identical to GPI-PLD, abundantly present in serum, while the activity contained in the first peak seems to be genuine for liver cells and, thus, apparently represents a novel form of a GPI-PLD which is membrane-associated and distinctly different from the serum enzyme.


Electron paramagnetic resonance studies on cytochrome b-558 and peroxidases of pig blood granulocytes. Low-temperature electron paramagnetic resonance (EPR) spectrometry on granulocytes prepared from pig blood was carried out with concentrated cellular and subcellular fractions to characterize EPR signals of cytochrome b-558 (cyt b-558). A thick cell suspension (approximately 2 x 10(9) cells/ml), containing mostly neutrophils, showed typical high-spin EPR signals due to myeloperoxidase (MPO) and a low spin signal at a g value of around 3.2. A similar thick granulocyte suspension containing eosinophils showed not only these signals but also low spin heme signals at g values of 2.86, 2.13, and 1.66, which have been reported to be of cyt b-558 (Ueno et al. 1991, FEBS Lett. 281, 130-132). MPO and eosinophil peroxidase (EPO) were released from the membrane fractions with 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl, and then were highly concentrated, in which no cyt b-558 was detected by absorption spectra. The signal at a g value of 2.86 was found only in the EPO fraction, suggesting that this signal is derived from a low-spin form of an EPO-complex, but neither from MPO nor cyt b-558. The O2(-)-forming NADPH oxidase associated in the membranes was solubilized with heptyl-thio-glucoside at 0 degree C and concentrated up to 45 microM cyt b-558 with no modification of the heme moiety confirmed by its O2(-)-generating activity and lack of carbon monoxide-binding capacity. Cyt b-558 showed an anisotropic signal at a g value of 3.2 +/- 0.05, which was cyanide-insensitive and reducible with reductants. The signal intensity was concentration dependent, suggesting that the g = 3.2 signal is characteristic of the low-spin heme iron in cyt b-558.


[Ocular histoenzymatic research on an experimental viral attack]. We sought to investigate enzyme response appearing subsequent to sub-conjunctival administration of the Coxsackie B3 virus. This virus stimulates oxidising enzymes, diaphorase and leucin aminopeptidase, dihydrofolate reductase, and adenosinetriphosphatase. The most typical enzyme changes are been in the chorion of the conjunctival mucose and the corneal parenchyma there by showing that the virus, triggers local immune defence mechanisms. The appearance of highly active Langerhans cells around Bowman membrane and corneal tissue proves that the virus injected greatly stimulates the mobilisation of local immune mechanisms.


Characterization of ATP receptor which mediates norepinephrine release in PC12 cells. PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.


[Diaphorase activity at the conjunctival level in an experimentally produced viral infection]. By Coxsackie virus type B3 subconjunctival administration it has been noticed the appearance of a antiviral defense mechanism, revealed by the presence in conjunctival chorion of lymphocytes, plasmocytes and active macrophages. It had also been studied by histochemical methods the responsive way at conjunctival level of NADH2-cytocrom-C-reductase (diaphorase) in experimentally obtained viral aggression. The injected virus has determined the stimulation of the activity of this enzyme at epithelial level, which play an important role in mucous conjunctival protection. The lymphocytes appearing in a great number presented an intense enzymatic reaction in chorion, which denotes a living synthetic and secretory metabolical activity.


Gel casting of resorbable polymers. 1. Processing and applications. A gel casting technique based on resorbable, synthetic, alpha-polyesters (or lactide-glycolide polymers) is described for producing medical implants such as bone graft substitutes and timed-release carriers for medication. This solution-based method enables production of thick-section solid and microporous materials, and blending of polymers and particulate fillers. Implant degradation rate, for example, may be adjusted by variation of polymer type, molecular weight range, crystallinity and morphology. Gel casting conditions are reported for solid and microporous materials and processing characteristics are interpreted in terms of established crystallization and dissolution behaviour of polymers.


[Study of the interaction of proflavin with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in aqueous solution by uni- and two-dimensional 1H-spectroscopy]. Complex formation between acridine dye proflavine and self-complementary deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in water-salt solution was studied by the method of one- and two-dimensional 1H-NMR spectroscopy (500 MHz). Two-dimensional homonuclear 1H-NMR spectroscopy (2D-COSY and 2D-NOESY) was used for complete assignments of proton signals of molecules in solution and for qualitative analysis of the nature of interactions between proflavine and tetranucleotide. Concentration dependences of proton chemical shifts of the molecules were measured at 293 K. Equilibrium reaction constants and limiting chemical shifts of dye protons in the complexes were determined using suggested schemes of complex formation. Based on the obtained data possible types of complexes were considered. Analysis of relative content of different types of complexes was made and special features of dynamic equilibrium were revealed as a function of correlation of dye and tetranucleotide concentrations. The most favourable structure of 1:2 complex of dye with tetranucleotide was constructed using the calculated values of induced chemical shifts of proflavine protons and 2D-NOESY spectra.


High and low affinity NGF receptor mRNAs in human foetal spinal cord and ganglia. The cellular localization of mRNAs encoding the low affinity NGF receptor (here referred to as LANR) and the putative high affinity receptor for NGF, trk, have been studied in the human foetal spinal and sympathetic ganglia, and spinal cord, using in situ hybridization. The receptor mRNAs were highly expressed in the spinal and sympathetic ganglia, with most but not all neurons expressing both LANR and trk mRNA. Spinal nerve rootlets distal to the spinal ganglia expressed LANR but not trk mRNA, confirming the presence of the low affinity receptor in developing Schwann cells. In the spinal cord, LANR mRNA was found throughout the medial and lateral motor columns while, trk mRNA was detected in scattered cells in the dorsal aspect of developing grey matter.


Changes in Na,K-ATPase gene expression during granulocytic differentiation of HL60 cells. During granulocytic differentiation of HL60 cells, immediate reduction of ouabain-sensitive potassium flux is observed within the first 12 hours of addition of dimethyl sulfoxide (DMSO). We show that gene expression of the alpha 3 isoform of Na+,K(+)-ATPase, which encodes an ouabain-inhibitable Na+,K(+)-ATPase activity, significantly declines during the first 24 hours of granulocytic differentiation by DMSO of HL60 cells. The more common alpha 1 isoform decreases, but more gradually over 72 hours of DMSO induction. Loss of alpha 3 and alpha 1 messenger RNA (mRNA) are due to changes in mRNA decay; their transcription is not altered. alpha 3 mRNA half-life is 3 hours in HL60 cells; upon induction by 16 hours of DMSO, it decreases to approximately 2 hours. alpha 1 transcripts are less sensitive to DMSO induction, with their half-life being 3.5 hours in HL60 cells; upon induction, their half-life decreases to 3 hours. Experiments measuring protein stability confirm that alpha 3 protein is more labile than alpha 1. In uninduced HL60 cells, alpha 3 membrane protein comprises 30% of the total alpha isoforms, and is less stable than alpha 1, with a protein half-life of only 9 hours. Upon DMSO induction, steady-state alpha 3 protein decreases markedly within 10 hours, whereas alpha 1 protein remains stable. These results show that posttranscriptional changes during induction play a major role in the differential regulation of alpha 1 and alpha 3 isoforms of Na+,K(+)-ATPase; regulation of the latter may be important for early granulocytic differentiation, or for one of the differentiated functions of mature granulocytes.


Microbial etiology of periodontal diseases. Where are we? Where are we going? Many studies indicate a strong association of Actinobacillus actinomycetemcomitans with localized juvenile periodontitis. Species associated with adult periodontitis include Bacteroides forsythus, Porphyromonas gingivalis, Prevotella intermedia, A. actinomycetemcomitans, and Wolinella recta. Capnocytophaga species may be important in pubertal gingivitis. An unnamed spirochete related to Treponema pallidum has been identified in acute necrotizing ulcerative gingivitis lesions. Species isolated from prepubertal periodontitis, peri-implantitis, pericoronitis, and human immunodeficiency virus gingivitis and periodontitis are similar to those isolated from periodontal and gingival infections. Species identification in combination with clinical characteristics facilitates periodontal diagnosis. DNA probes, immunoassays, and benzoyl-arginine naphthylamide reactivity methods can be used to indicate putative pathogens in plaque samples. Microbial identification aids in antibiotic selection and planning a treatment regimen.


Multi-step cascade of tumor cell metastasis. The most life-threatening aspects of the oncogenic process are invasion and metastasis. Even though the clinical significance of such expression of the malignant phenotype has been well appreciated, advances in understanding the molecular mechanisms involved in metastasis have been painstakingly slow. To tackle this complex, multi-step problem, investigators have separated invasion and metastasis into a series of defined, sequential steps, and focused on one step at a time. Efforts combining the disciplines of cell biology, protein biochemistry, and molecular genetics have resulted in a surge of new information, suggesting novel strategies for prognosis and therapy of metastatic cancer.


Surgical considerations for the prosthodontic patient. Surgical procedures for the prosthodontic patient have advanced greatly over the last decade. Both the aesthetics and function of the final restoration can be greatly enhanced by various surgical procedures. Some are very predictable, and others are still in the developmental stages. This review is divided into three basic categories: aesthetic root coverage; crown lengthening; and ridge augmentation procedures.


Hepatocellular neoplasia in fish, rats and man: a selected comparative review. Liver cancer is a common neoplasm of man that is especially frequent in parts of the world where hepatitis B virus is endemic and high aflatoxin ingestion is experienced. Hepatocellular carcinoma (HCC) has a very aggressive behavior, is quite resistant to radiotherapy and chemotherapy and is often inoperable, all of which lead to a five-year patient survival of less than 5 percent. Studies in lower animals (e.g. fish, rats) lend themselves to preplanned manipulations aimed at answering specific questions which are intended to elucidate the biology of HCC. Information derived from these studies can be applied to the human condition with the hope of earlier diagnosis, improved treatment and possibly prevention. This review touches on selected areas of similarity and dissimilarity in the histology, histochemistry, metastasis, etiology and molecular biology of HCC in fish, rats and man.


All-ceramic crowns.

All-ceramic crowns. Despite the good appearance and biocompatibility of dental porcelains, failures are still of considerable concern because of some limited properties common to all-ceramic crown systems. As in the years before, pertinent scientific articles published between November 1990 and December 1991 focused on strengthening mechanisms and compared fracture toughness for different ceramic systems by using various test methods. Some evaluated the clinical implications thereon for seating and loading crowns and measured wear against different ceramic surface conditions. Recently introduced with pleasing aesthetic qualities, IPS-Empress (Ivoclar, Schaan, Liechtenstein), a new European leucite-reinforced glass-ceramic, has finally drawn attention in some journals and has been reviewed with promising in vitro test results. Using a simple press-molding technique, well-fitting crowns, inlays, and veneers can be fabricated without an additional ceramming procedure. Again, only long-term clinical trials will validate achievements compared with other all-ceramic systems and with well-established metal ceramics.


Host modulation with tetracyclines and their chemically modified analogues. Recent studies have suggested the use of drugs to modulate host response as a new approach in periodontal therapy. In this regard, the tetracycline antibiotics have been found to inhibit host-derived collagenases and other matrix metalloproteinases by a mechanism independent of the antimicrobial activity of these drugs; this effect may suppress connective tissue breakdown during periodontal disease and during a variety of medical disorders including (but not limited to) noninfected corneal ulcers, serious (sometimes life-threatening) skin-blistering diseases, rheumatoid arthritis and osteoarthritis, systemically--as well as locally--induced bone loss, and perhaps even tumor-induced angiogenesis. Two therapeutic strategies based on the host-modulating properties of tetracyclines are currently being developed: 1) the use of low-dose doxycycline (the most potent anticollagenase of commercially available tetracyclines) formulations, which do not appear to result in tetracycline side effects such as the emergence of antibiotic-resistant microorganisms; and 2) the production of a family of chemically modified tetracyclines that have lost their antimicrobial activity, but have retained their anticollagenase activity. A description of several of these compounds and a discussion of their efficacy in inhibiting collagenases in vitro and reducing tissue destruction in several animal models of periodontal and medical diseases is presented.


[Biology of small-cell bronchogenic carcinoma: recent advances]. In the last ten years considerable progress has been made in small-cell lung carcinoma (SCLC) biology, along with the technical progress made in molecular biology. This progress now allows us to propose a model for the genesis and the development of this type of tumor. Tobacco, the principal causal factor plays a dual role. In bringing about secretion of growth factors by the bronchial epithelia, usually involved in the normal development of lungs, and by functioning autocrinally and paracrinally, it facilitates the occurrence of mitotic mutations. Without directly contributing to cellular transformation, this autocrine functioning also gives a selective advantage to cells going through transformation or immortalization. The procarcinogenic or carcinogenic agents contained in tobacco smoke, whose level of production could be genetically determined, would also contribute to the accumulation of mutations affecting both suppressor genes and oncogenes. Two tumour suppressor genes have been identified: RB1 and P53. At least one other putative tumour suppressor gene has constantly been implied. It lies on the short arm of chromosome 3. There could also be the possibility of detecting subjects susceptible to developing an SCLC, a functional hemizygote still needing evaluation. The activated oncogenes principally belongs to the myc family. Their activation could correspond with the appearance of cellular clones having aggressive behavior independent of growth factors, chemoresistant and more metastatic. SCLC may be distinguished from other malignant lung tumors by a fairly characteristic pattern consisting of the loss of suppressor genes and the activation of oncogenes. The links between the neuroendocrine properties of this type of tumor and its characteristic description are being clarified and will contribute to a better understanding of the relationship between the different types of lung tumors. From this biologic knowledge follow several therapeutic applications under investigation (blocking autocrine loop through anti-GRP antibodies), as well as potential applications (concerning the products of suppressor genes) and possible applications such as prevention oriented towards detection of high-risk subjects.


Enhanced detection of cytomegalovirus in shell vial culture following MRC-5 monolayer pretreatment with glucocorticoids. The effect of cortisol and its synthetic derivative, dexamethasone on the infectivity of cytomegalovirus (CMV) was investigated. Twenty-four to 48 h pretreatment of MCR-5 shell vials by 100 micrograms % (compared with medium supplemented with 10 or 25 micrograms %) cortisol affected the greatest increase in virus infectivity (measured in fluorescent focus units/0.2 ml inoculum). This enhancement effect was more pronounced in the CMV strain AD-169 than in a clinical CMV isolate (NCMC 7125). Attempts to reisolate CMV from 32 frozen positive original specimens in the presence or absence of cortisol demonstrated an enhanced infectivity of 8 specimens in hormone pretreated cultures compared with two specimens showing an increased activity in untreated controls. Three specimens were reisolated only in hormone pretreated shell vials. Pretreatment of shell vials maintained in maintenance medium plus 2% FBS with cortisol or dexamethasone (10(-5) M) increased CMV infectivity to ca. 300%. This hormonal mediated effect was more pronounced in 4 rather than 16 day old shell vials. Infectivity in 16 day old shell vials maintained in 10% FBS but without hormone equalled the infectivity levels achieved in 4 day old shell vials with cortisol and either a 2 or 10% FBS supplement. This study denotes the applicability of glucocorticoid pretreatment of MRC-5 monolayers but more importantly, identifies the association between FBS concentration and glucocorticoid-mediated CMV detection using shell vial technology.


Voltage-gated currents of putative GABAergic amacrine cells in primary cultures and in retinal slice preparations. To analyze the voltage-dependent ionic conductances of putative GABAergic amacrine cells developing in vitro, whole cell patch clamp recordings were carried out on identified neurons in monolayer cultures from embryonic chick retinae. These recordings were directly compared with those performed on amacrine cells in chick retinal slice preparations. Current responses to depolarizing voltage steps observed in cultured neurons could be separated into at least four different components. A small tetrodotoxin-sensitive sodium inward current was observed in approximately 50% of the cells. The considerably larger outward potassium current consisted of a transient 4-aminopyridine-sensitive component and a sustained component. The latter was reduced in the presence of both tetraethylammonium chloride and Co2+ and thus was probably composed of two conductances. In addition, a Ca(2+)-carried inward current of small amplitude could be identified. Voltage-sensitive currents measured in amacrine cells of retinal slices were very similar. Again, only about half of the cells exhibited sodium currents. Potassium currents contained the above components, but their contributions to the whole cell current seemed to be different. Together with previous findings these results suggest that immature retinal neurons in dissociated cultures undergo a differentiation process similar to that occurring in vivo.


Early cellular swelling during cerebral ischemia in vivo is mediated by excitatory amino acids released from nerve terminals. This study demonstrates ischemic cellular swelling in vivo detected as changes in the concentration of 14C-sucrose pre-perfused into the extracellular space (ECS) as an ECS marker. Microdialysis was utilized as a means of perfusion and measurement of the extracellular concentration of 14C-sucrose ([14C-sucrose]e). Concomitant with an abrupt increase in [K+]e at 1-3 min following the ischemia induction, [14C-sucrose]e was also rapidly elevated. Since sucrose is not taken up by either cells or capillaries, the absolute amount of 14C-sucrose in the ECS must be unchanged. The increase therefore appears to represent a relative decrease in water volume in the ECS resulting from a movement of water into the cells, i.e. cellular swelling. Ca(2+)-free perfusate containing Co2+, which has been shown to block excitatory amino acid release during cerebral ischemia, significantly delayed the increase in [14C-sucrose]e and [K+]e. Kynurenic acid, a broad-spectrum antagonist of excitatory amino acids, administered in situ through the dialysis probe also significantly delayed the increase in [14C-sucrose]e and [K+]e. These findings indicate that the early cellular swelling occurring during cerebral ischemia is a result of massive ionic fluxes mediated by excitatory amino acids which are released by a Ca(2+)-dependent exocytotic process from the nerve terminals.


Presence of strong glucocorticoid receptor immunoreactivity within hypothalamic and hypophyseal cells containing pro-opiomelanocortic peptides. The presence of nuclear glucocorticoid receptor immunoreactivity (GR IR) was studied in the adrenocorticotropin (ACTH), beta-Endorphin (beta-END) and alpha-melanocyte stimulating hormone (alpha-MSH) IR neuronal populations of the rat hypothalamus and hypophysis using double immunolabelling techniques. All the nuclei of the ACTH/beta-END/alpha-MSH IR neurons of the arcuate and periarcuate nuclei were strongly GR IR in the 48 h colchicine treated animal, but very few alpha-MSH-like IR perikarya located in the dorsal and lateral hypothalamus displayed nuclear GR IR. GR IR was present in the ACTH/beta-END corticotrophs and absent in the intermediate lobe of the hypophysis. The data provide morphological evidence for a glucocorticoid action through a nuclear GR in the arcuate ACTH/beta-END/alpha-MSH IR neurons and the ACTH/beta-END corticotrophs, whereas the alpha-MSH-like IR neurons of the lateral hypothalamus and the melanotropes of the intermediate lobe may not be directly affected by glucocorticoids under normal conditions.


Medial septal cholinergic lesions increase hippocampal mineralocorticoid and glucocorticoid receptor messenger RNA expression. Loss of the cholinergic innervation of the hippocampus and failure of central (presumably hippocampal) suppressive control of hypothalamic-pituitary-adrenal axis activity are important features of Alzheimer's dementia. We have examined the effects of electrolytic lesions of the medial septal cholinergic innervation on mineralocorticoid (MR) and glucocorticoid (GR) receptor mRNA expression in rat hippocampus using in situ hybridization histochemistry. Expression of both MR and GR mRNA was significantly increased in a subregions of the hippocampus, but not neocortex, with the greatest increase in the CA1 area for MR mRNA and dentate gyrus for GR mRNA. Since glucocorticoids potentiate the effects of neurotoxins in the hippocampus, the increased expression of receptors following loss of cholinergic inputs in Alzheimer's disease may increase hippocampal neuronal vulnerability.


The role of vasopressin in the nicotine-induced stimulation of ACTH and cortisol in men. Experimental evidence indicates that arginine vasopressin (AVP) contributes to the release of ACTH under certain conditions. The present study investigates the role of vasopressin as a secretagogue of ACTH during cigarette smoking or nicotine infusion with additional injection of corticotropin releasing hormone (CRH) and using the specific AVP antagonist d(CH2)5Tyr(Me)-AVP. We first tested the effect of the AVP antagonist (10 micrograms/kg body weight i.v.) on ACTH and cortisol release following cigarette smoking in 15 healthy young male smokers. Smoking led to marked increments in plasma nicotine and to a small rise in plasma ACTH and cortisol. Mean plasma ACTH and cortisol levels were at no time significantly altered by the antagonist. This might be due to a slight agonistic effect of the AVP antagonist, to high interindividual variability of the ACTH and cortisol responses after smoking or to a negligible role of AVP in smoking-induced ACTH release. In a second study we performed the following tests in six healthy male non-smokers: (1) nicotine infusion (1.0 micrograms/kg body weight per min); (2) CRH i.v. (100 micrograms); (3) AVP antagonist i.v. (5 micrograms/kg); (4) nicotine infusion plus CRH i.v.; (5) nicotine infusion plus AVP antagonist i.v.; (6) nicotine infusion plus CRH and AVP antagonist i.v.; and (7) sham infusion. Nicotine infusion led to greater increments of AVP, ACTH and cortisol than smoking without causing nausea. Peak nicotine levels after nicotine infusion were lower than after smoking. The AVP antagonist in the reduced dosage given alone had no effect on hormone levels. However, it slightly attenuated the effect of nicotine on ACTH and cortisol (P less than 0.05, ANOVA).(ABSTRACT TRUNCATED AT 250 WORDS)


Autoradiographic visualization and characteristics of [125I]bradykinin binding sites in guinea pig brain. The present study was undertaken to localize and characterize bradykinin (BK) binding sites in 10 microns serial sections of guinea pig brain by in vitro quantitative receptor autoradiography. Specific binding of [125I-Tyr8]bradykinin ([125I]BK) was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (nTS), the area postrema (AP), the dorsal motor nucleus of the vagus (X) and the caudal subnucleus of the spinal trigeminal nucleus. No significant specific [125I]BK binding was seen in other brain regions. The specific binding (85-90% of total binding) was of high affinity and saturable with a KD of 73.5 +/- 9.9 pM and a Bmax of 27.8 +/- 1.9 amol per mm2 of tissue. In competition studies, the rank order of potencies was: BK greater than Met-Lys-BK greater than Lys-BK much greater than Des-Arg9-BK. The B2 receptor antagonist D-Arg0-Hyp3-Thi5,8-D-Phe7-BK inhibited [125I]BK binding with a Ki value of 3.5 +/- 1.5 nM while Des-Arg9-[Leu8]-BK, a B1 receptor antagonist did not significantly inhibit [125I]BK binding in concentrations up to 10 microM. Our finding of specific high affinity [125I]BK binding sites in the nTS, AP and the X is important because these brain areas are known to be involved in central cardiovascular regulation. Moreover, our results suggest that the specific [125I]BK binding sites in the guinea pig medulla are of the bradykinin B2 receptor type.


High-speed chronoamperometric measurements of mesolimbic and nigrostriatal dopamine release associated with repeated daily stress. The effects of repeated, once daily exposure to either restraint or tail pinch stress on extracellular levels of dopamine in nucleus accumbens and striatum were electrochemically monitored in conscious rats using high-speed chronoamperometry. Acutely, both tail pinch and restraint increased extracellular dopamine levels in both regions. However, the effect of restraint on mesolimbic and, to some extent, also on nigrostriatal dopamine neurotransmission increased progressively with each daily exposure. While increases in extracellular dopamine elicited by tail pinch varied across test days, no reliable daily enhancement of electrochemical responses to this stress were observed in either of the regions studied. Pretreatment with dopamine autoreceptor-specific doses of apomorphine (50 and 100 micrograms/kg s.c.) potently inhibited stress-elicited responses in nucleus accumbens, indicating that dopamine was the primary electroactive species contributing to the electrochemical signal. The results of this study indicate that the magnitude of stress-elicited increases in levels of extracellular dopamine is determined by the number of previous exposures to stress and are consistent with reports of sensitization to the behavioral effects of stress with repeated testing. The study also provides pharmacological data that are consistent with electrophysiological evidence of increased mesolimbic dopamine cell firing during exposure to stress.


Ontogenesis and binding properties of high-affinity neurotensin receptors in human brain. The ontogenesis of neurotensin binding sites was studied in human brain of subjects deceased from Sudden Infant Death Syndrome. Monoiodo-Tyr3 neurotensin specifically recognized 2 distinct classes of binding sites in human brain homogenate. The high affinity sites were already present at birth and increased to a maximal level of 240 fmol/mg protein 1 month after birth. Thereafter, the density of these sites decreased to reach a value of 8 fmol/mg protein in 15-month-old brain, a value similar to that found in adult brain. The dissociation constant of the high-affinity sites (about 0.3 nM) did not vary from birth to adulthood. The high-affinity binding sites were sensitive to GTP which decreased their affinity for neurotensin by a factor of 3, indicating that these sites are functional receptors coupled to GTP-binding proteins. By contrast, the low-affinity sites were insensitive to GTP and could be partly blocked by the antihistaminic drug levocabastine. These sites were absent in human brain during the first post-natal year and could be detected only in brain homogenate of 15-month-old infants. The transient increase in high-affinity neurotensin binding sites after birth suggests that neurotensin could act as a regulatory peptide during brain development.


[Reversibility of IUdR resistance to sensitivity to an HSV1 strain in experimental keratitis in rabbits]. During 7 serial passages of Herpes Simplex Virus (HSV1) in rabbit cornea treated with idoxuridine (IUdR) (P1 to P7), the emergence of resistance had been obtained from P3. The reversion towards IUdR sensitivity has been investigated from either viral population P3 or P6 by 6 serial passages in rabbit cornea treated by Vaseline (V1 to V6). From viral population P3, the reversion to IUdR sensitivity has been obtained at V4. In contrast, from viral population P6, the IUdR resistance was conserved from V1 to V6. In vitro, on Vero cells, the effective doses 50% (ED50) and 90% (ED90), determined by dye uptake assay and plaque reduction assay, confirmed the reversibility towards IUdR sensitivity obtained from P3 and the stability of IUdR resistance from P6.


Effects of sympathetic stimulation on use dependence of lidocaine, mexiletine, and quinidine in an intact canine model. The use- or rate-dependent effects of a continuous infusion of lidocaine (n = 6, serum level 3.1 +/- 0.34 micrograms/mL), mexiletine (n = 8, serum level 7.08 +/- 0.90 micrograms/mL), and quinidine (n = 6, serum level 6.8 +/- 1.22 micrograms/mL) were studied in an open chest canine preparation. A use-dependent effect on conduction was assessed by measuring the change in the His to surface ventricular activation (HV) time at differing atrial paced rates during drug infusion. Global sympathetic activation was achieved by nondecentralized left stellate ganglion stimulation (4-10 Hz, 6-12 V, 2 ms) and use dependence at the same cycle lengths was compared. Repolarization times were measured from epicardial monophasic action potentials recorded from the anterior left ventricle throughout the study. There was no significant change in the HV time during control studies with or without left stellate stimulation. Use-dependent slowing of conduction was seen in all studies during drug infusion. This was evident at cycle lengths of 300-190 ms for quinidine and at cycle lengths less than 250 ms for lidocaine and mexiletine. Stellate stimulation attenuated use dependence in all studies. This effect was significant from cycle lengths of 300-190 ms for lidocaine and quinidine and at cycle lengths shorter than 230 ms for mexiletine (p less than 0.05). Stellate stimulation significantly reduced use-dependent prolongation of the HV interval by an average of 60%. During stellate stimulation there was a nonsignificant trend towards cycle length independent shortening of action potential duration both at baseline and in the presence of drugs.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of gonadotropin-releasing hormone (GnRH) receptors in the ovary of common carp (Cyprinus carpio). Gonadotropin-releasing hormone (GnRH) binding sites have been characterized in the fully mature common carp ovary, using an analog of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]-GnRH; sGnRH-A) as a labeled ligand. Binding of sGnRH-A to carp follicular membrane preparation was found to be time-, temperature-, and pH-dependent. Optimal binding was achieved after 40 min of incubation at 4 degrees C at pH 7.6; binding was found to be unstable at room temperature. Binding of radioligand was a function of tissue concentration, with a linear correlation over the range of 8.0-40.0 micrograms membrane protein per tube. Incubation of membrane preparations with increasing levels of [125I]sGnRH-A revealed saturable binding at radioligand concentrations greater than 400 nM. The binding of [125I]sGnRH-A to the carp ovary was also found to be reversible; addition of unlabeled sGnRH-A (10(-6) M) after reaching equilibrium resulted in complete dissociation of [125I]sGnRH-A within 30 min, and the log dissociation plot indicated the existence of a single class of binding sites. Addition of unlabeled sGnRH-A displaced the bound [125I]sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis suggested the presence of one class of high affinity GnRH binding sites. Bound [125I]sGnRH-A was also found to be displaceable by other GnRH peptides, including sGnRH ([Trp7,Leu8]-GnRH), cGnRH-II ([His5,Trp7,Tyr8]-GnRH) and a GnRH antagonist ([D-pGlu1,D-Phe2,D-PTrp3,6]-GnRH; GnRH-ANT) in a parallel fashion, indicating that these peptides bind to the same class of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)


Cytochrome composition and oxygen-dependent respiration-driven proton translocation in Wolinella curva, Wolinella recta, Bacteroides ureolyticus, and Bacteroides gracilis. The membrane fractions of the microaerobically grown type strains of Wolinella curva, Wolinella recta, Bacteroides ureolyticus, and Bacteroides gracilis contained membrane-bound cytochrome b, cytochrome c, and CO-binding cytochrome c. Soluble cytochrome c and CO-binding cytochrome c were also present. Although B. gracilis is oxidase negative, it possessed cytochrome c. With H2 or formate as the electron donor, proton efflux from anaerobic cells occurred upon addition of a pulse of oxygen. With formate as the electron donor, the H+/O ratios of W. curva, W. recta, B. ureolyticus, and B. gracilis were 0.75, 1.66, 2.06, and 2.04, respectively. With H2 as the electron donor, the H+/O ratios of W. curva, B. ureolyticus, and B. gracilis were 1.25, 1.97, and 2.36, respectively. Proton translocation was inhibited by the protonophore carbonylcyanide m-chlorophenylhydrazone. The results confirm that the organisms are not anaerobes but are microaerophiles capable of respiring with oxygen.


Benign mixed tumor of canine mammary gland showing an r(X) and trisomy 5 as the only clonal abnormalities. We report a benign mixed tumor of the canine mammary gland which showed an r(X) and trisomy 5 as the only clonal karyotypic deviations. Clonal aberrations were observed in 79 of 160 of the metaphases. Of these, 48 cells had both the r(X) and trisomy 5, whereas the remaining metaphases were characterized by the r(X) as the only clonal aberration. We conclude that formation of the ring chromosome was the first abnormality, followed by trisomy 5 during the course of karyotypic evolution.


Direct chromosome analysis of seven primary colorectal carcinomas. Chromosome studies were performed on direct preparations of seven cases of primary colorectal carcinomas. Two cases had relatively simple chromosome changes: 48,XY,+8,+21/51, XY,+8,+9,+10,+i(17q),+21, and 47,der(X)t(X;14)(q11;q11)-Y,t(6;18)(p22;q24)+7,+8,der(19)t (19;?)(q13;?). The five others had complicated deletions and translocations; 1p- was noted in five cases, and i(17q) was noted in three cases.


Invasion of Streptococcus mutans, Streptococcus intermedius and Propionibacterium acnes into the teeth of gnotobiotic rats. Germ-free rats, fed a modified cariogenic diet 2000 with a reduced sucrose content of 25%, were inoculated with a strain of Streptococcus mutans, Streptococcus intermedius or Propionibacterium acnes, respectively. After 100 days on the diet, the rats were sacrificed. Microscopic examination of stained decalcified tooth sections revealed marked destruction of the outer zones of the dentin infected with S. mutans. The destruction was closely associated with the formation of glucan-mediated large aggregates of S. mutans. The aggregates, however, were rarely formed in the deep zones of the infected dentin. Inoculation with S. intermedius and P. acnes had little effect on the microscopic outline of the decalcified tooth sections. However, the staining behavior of the sections with Mallory's method, Alcian blue-PAS and silver was like that of the tooth sections infected with S. mutans, even in the central and inner zones of the dentin. The histochemical alterations of the deep zones of the dentin induced by the three infected bacteria appeared to be due to their proteolytic rather than to their acidogenic activities.


Effects of carbon dioxide, Nd:YAG and carbon dioxide-Nd:YAG combination lasers at high energy densities on synthetic hydroxyaptite. The aim of this study was to determine the crystalline structure and chemical alterations of synthetic hydroxyapatite after irradiation with either CO2, Nd:YAG or CO2-Nd:YAG combination lasers at high energy densities of 500-3,230 J.cm2. Further, dissolution kinetics of the lased material were analysed and compared with those of unlased apatite. Electron microscopy showed that the lased material consisted of two kinds of crystals. From the micrographs their diameters varied from 600 to 1,200 A and from 3,000 to 6,000 A, respectively. The larger crystals showed 6.9-Angström periodic lattice fringes in the transmission electron microscope. alpha-Tricalcium phosphate (TCP) was identified by X-ray diffraction. Selective-area electron diffraction identified the large crystals to consist of tricalcium phosphate while the smaller crystals were probably hydroxyapatite. Assays of dissolution kinetics showed that at these high energy densities lased material dissolved more rapidly than unlased synthetic hydroxyapatite due to the higher solubility of TCP.


The pineal adrenergic----cyclic GMP response develops two weeks after the adrenergic----cyclic AMP response. Pineal metabolism is regulated primarily by noradrenergic innervation. Stimulation of the adult gland with norepinephrine elevates both cyclic AMP and cyclic GMP production, through remarkably similar mechanisms requiring activation of both beta- and alpha 1-adrenergic receptors. As described here, however, the adrenergic stimulation of cyclic GMP is first detectable about 2 weeks after the cyclic AMP response can be detected. This indicates there is a profound difference in when cyclic AMP- and cyclic GMP-regulated processes can be adrenergically regulated.


The pharmaco-ontogeny of the paraventricular alpha 2-noradrenergic receptor system mediating norepinephrine-induced feeding in the rat. The present study was undertaken to assess the functional ontogeny of alpha 2-noradrenergic receptors in the hypothalamic paraventricular nucleus (PVN) that mediate noradrenergic stimulation of feeding in the rat. Rat pups, ranging in age from 2 to 15 days, were removed from their mothers and implanted with a brain cannula directed unilaterally at the PVN or third ventricle. On the following day, each pup was implanted with an intra-oral cannula for oral infusion of milk or water that could be swallowed or rejected. Following a 1-h period of satiation, each pup received an intracerebral injection of saline, or a single dose of norepinephrine (NE, 0.01-100.0 nmol) or the alpha 2-noradrenergic receptor agonist clonidine (0.01-1.0 nmol). Milk or water intake was then assessed following a 1-h period of infusion. Injection of NE into the PVN and third ventricle significantly enhanced milk intake at 2 days of age. NE was 10-fold more potent in the PVN than in the ventricle. While paraventricular injections of NE stimulated milk and water intake equally at 2 days of age, NE produced a greater stimulation of milk than water intake at 15 days of age. Like NE, clonidine significantly enhanced milk intake at 2 days of age following injection into the PVN. Collectively, these findings suggest that alpha 2-noradrenergic receptors, mediating noradrenergic stimulation of feeding, are functionally mature very early in the postnatal development of the rat. Moreover, consistent with evidence in the adult rat, these findings indicate that alpha 2-noradrenergic receptors relevant to feeding are located in the vicinity of the PVN.


Nitrergic transmission: nitric oxide as a mediator of non-adrenergic, non-cholinergic neuro-effector transmission. 1. The possibility that transmission at some non-adrenergic, non-cholinergic (NANC) neuro-effector junctions is mediated by nitric oxide (NO) arose from the discoveries that NO mediated the effects of nitrovasodilator drugs and that endothelium-derived relaxing factor (EDRF) was NO or a NO-yielding substance. 2. NO donated by nitrovasodilator drugs or formed by endothelial cells activates soluble guanylate cyclase in smooth muscle and the consequent increase in cyclic guanosine monophosphate (cGMP) results in relaxation. The relaxations produced by stimulation of some NANC nerves are also due to a rise in cGMP. 3. The biosynthesis of NO by oxidation of a terminal guanidino nitrogen of L-arginine is inhibited by some NG-substituted analogues of L-arginine. These substances block EDRF formation by NO synthase and endothelium-dependent vasodilatation, and the blockade is overcome by L-arginine 4. NANC relaxations in some tissues are blocked by NG-substituted analogues of L-arginine and restored by L-arginine. Other agents that affect endothelium-dependent vasodilator responses produce corresponding changes in responses to stimulation of these NANC nerves. Such observations indicate that transmission is mediated by NO: we have termed this mode of transmission nitrergic. 5. There is evidence for nitrergic innervation of smooth muscle in the gastrointestinal tract, genito-urinary system, trachea and some blood vessels (penile and cerebral arteries). 6. The recognition of a mediator role for NO in neurotransmission calls for reconsideration of previously accepted generalizations about mechanisms of transmission. 7. Studies on nitrergic transmission will provide new insights into physiological control mechanisms and pathophysiological processes and may lead to new therapeutic developments.


Increased Na+/H+ exchange activity in vascular smooth muscle cells of spontaneously hypertensive rats and possible involvement of protein kinase C. 1. Na+ influx into cultured vascular smooth muscle cells (VSMC) obtained from spontaneously hypertensive rats (SHR) and from Wistar-Kyoto rats (WKY) was measured. Na+ influx via the Na+/H+ exchange system was measured as the rate of 22Na+ influx into cultured VSMC sensitive to ethylisopropylamiloride (EIPA), a specific inhibitor of the exchange system. 2. The total 22Na+ influx rate in SHR was significantly higher than in WKY (6.08 +/- 0.16 vs 4.13 +/- 0.09 nmol/min per mg protein; P less than 0.001; n = 14). The EIPA (1 X 10(-4) mol/L)-sensitive 22Na+ influx rate in SHR was significantly higher than that in WKY (4.32 +/- 0.27 vs 2.17 +/- 0.14 nmol/min per mg protein; P less than 0.001; n = 14). There was no difference in EIPA-insensitive 22Na+ influx between SHR and WKY. The EIPA-sensitive 22Na+ influx rate into VSMC was significantly decreased in SHR but not in WKY by the addition of 1 X 10(-4) mol/L 1-(5-isoquinoline-sulfonyl)-methylpiperazine (H-7), an inhibitor of protein kinase C (PK-C). 3. These results suggest that the increase in Na+ influx in SHR may be due to elevation of the Na+/H+ exchange activity, and possible involvement of PK-C in the increased Na+/H+ exchange activity in VSMC from SHR.


Increase in serum total angiotensin-converting enzyme activity with enalapril therapy in humans: a controlled trial. 1. In a controlled, randomized double-blind trial, 15 patients with essential hypertension were treated with enalapril 5-20 mg/day, or doxazosin 1-8 mg/day, during a 7 week dose titration phase. This was followed by 7 weeks of combined treatment with doxazosin and enalapril. 2. Blood was taken after a 2 week placebo run-in phase, and at 3 and 7 weeks in the single-agent and combined treatment phases, for measurement of plasma renin activity (PRA), plasma angiotensin II (AII), plasma aldosterone and serum free and total angiotensin-converting enzyme (ACE) activities. 3. Doxazosin had no effect on serum free or total ACE activities. 4. Enalapril reduced serum free ACE activity and increased serum total ACE activity, which at 7 weeks was significantly greater than in patients receiving doxazosin. 5. In those patients who received enalapril, 10 mg/day for 3 weeks and then 20 mg/day for 4 weeks (n = 12), with or without doxazosin, mean serum total ACE activity increased by 51%. PRA was also increased in this group, but there were no changes in plasma AII or aldosterone concentrations.


Evidence for presynaptic beta-adrenoceptor stimulation 24 hours after a 6 hour adrenaline infusion. 1. The effects of 6 h infusions of adrenaline (INF-A) and dextrose (INF-D) were compared in nine normal subjects. 2. A significant increase in systolic blood pressure was observed during INF-A compared with INF-D and, 24 h after infusion, diastolic blood pressure was higher after INF-A than after INF-D. 3. Heart rate (HR) was significantly higher during INF-A than during INF-D. 4. As expected, plasma ADR increased significantly during INF-A but, unexpectedly, remained elevated 60 min post infusion compared with INF-D. Levels during activity the next morning were somewhat higher after INF-A, but not significantly different from INF-D. 5. Plasma NA increased transiently during INF-A and decreased during INF-D. Urinary NA was significantly higher during INF-A than during INF-D, and insignificantly higher during overnight recumbency, consistent with enhanced noradrenergic transmission.


Effects of argininosuccinic acid on nitric oxide-mediated relaxations in rat aorta and anococcygeus muscle. 1. Argininosuccinic acid (ASA), a naturally occurring NG derivative of arginine, and the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) were compared for their ability to reduce responses to nitric oxide (NO) derived from endothelial cells (aorta) and nitrergic nerves (anococcygeus muscle). 2. In isolated rings of rat aorta, endothelium-dependent relaxation responses to acetylcholine were abolished by L-NAME (0.1 mmol/L) and were reduced by ASA (0.1 and 0.3 mmol/L). Relaxations induced by sodium nitroprusside (SNP) were not affected by L-NAME but were reduced by ASA. 3. In rat isolated anococcygeus muscles, relaxations elicited by nitrergic nerve stimulation at 1 Hz were abolished by L-NAME (0.1 mmol/L) but were only slightly reduced by ASA (1 mmol/L). The effect of ASA was not sustained. L-Arginine (1 mmol/L) prevented the effect of L-NAME but not that of ASA. Neither ASA or L-NAME inhibited SNP-induced relaxation in the anococcygeus muscle. 4. The results suggest that ASA inhibits NOS but this does not totally account for its effects in reducing NO-mediated relaxations produced by the endothelium-dependent vasodilator acetylcholine in rat aortic rings and stimulation of nitrergic nerves in the rat anococcygeus muscle.


Mesenteric vascular angiotensin-converting enzyme is increased in experimental diabetes mellitus. 1. Diabetes mellitus was induced by streptozotocin in male Wistar rats, and angiotensin-converting enzyme measured in plasma and mesenteric vessels 3 weeks later. 2. Diabetes was associated with increased mesenteric wet weight/bodyweight ratio (control 0.2 s.e.m. 0.02 mg/g, n = 21, vs diabetes 1.0 s.e.m. 0.3 mg/g, n = 27, P less than 0.01, ANOVA). 3. Plasma angiotensin-converting enzyme activity was increased in diabetic rats (98 s.e.m. 3 nmol HL/mL per min) compared with controls (64 s.e.m. 6 nmol HL/mL per min, P less than 0.01, ANOVA). 4. Mesenteric vessel angiotensin-converting enzyme was increased in diabetes mellitus estimated by radioligand binding site density (fmol/mg protein; 1407 s.e.m. 166 fmol/mg protein) compared with controls (890 s.e.m. 56 fmol/mg protein, P less than 0.05, ANOVA) and by enzyme kinetic assay (diabetes, 15.5 s.e.m. 1.5 nmol HL/mg protein per min, controls, 8.3 s.e.m. 0.7 nmol HL/mg protein per min, P less than 0.01, ANOVA). The equilibrium dissociation constant of ligand-angiotensin-converting enzyme interaction was unchanged. 5. Increased vascular angiotensin-converting enzyme concentration may contribute to vascular hypertrophy and diabetic vasculopathy by increased local synthesis of angiotensin II.


Mesenteric resistance and brain microvascular angiotensin-converting enzyme in the spontaneously hypertensive rat. 1. Angiotensin-converting enzyme (ACE) concentration was measured in mesenteric and brain microvessels from spontaneously hypertensive rats (SHR) and compared with normotensive controls using a specific radioligand binding assay. 2. Plasma angiotensin-converting enzyme activity was similar in SHR (n = 15) and normotensive controls (n = 21; 58 +/- 1 nmol HL/mL per min, vs 64 +/- 6 nmol HL/mL per min). 3. There was no significant difference between the mesenteric vascular angiotensin-converting enzyme radioligand binding site density (Bmax, fmol/mg protein) of SHR and normotensive controls (954 +/- 77 vs 890 +/- 56, P = 0.5, unpaired Student's t-test), despite significant differences in systolic blood pressure (220 +/- 8 mm Hg vs 120 +/- 6 mm Hg respectively, P less than 0.01) and increased mesenteric wet weight to bodyweight ratio in the hypertensive rats (0.28 +/- 0.02 mg/g, n = 5 vs 0.16 +/- 0.02 mg/g, n = 7, P less than 0.01). 4. Brain vascular angiotensin-converting enzyme radioligand binding site density (Bmax, fmol/mg protein) was also similar in SHR and normotensive controls (467 +/- 62, n = 5 vs 497 +/- 64, n = 5, P = 0.7, unpaired Student's t-test). 5. These results demonstrate that vascular angiotensin-converting enzyme concentration is not altered in the SHR and that vascular ACE is not increased in this form of vascular hypertrophy or regulated by the blood pressure level.


Variation in angiotensin-converting enzyme (ACE) inhibitor affinity at two binding sites on rat pulmonary ACE: influence on bradykinin hydrolysis. 1. ACE from rat lung and testis was characterized by radioligand binding studies using [125I]-Ro 31-8472, the radioiodinated hydroxy derivative of the potent ACE inhibitor cilazaprilat. 2. Analysis of the displacement of [125I]-Ro 31-8472 from ACE by ACE inhibitors of different structure by the LIGAND program was best fitted by a two binding site model for lung ACE and a one binding site model for testis ACE. 3. There was marked variation in ACE inhibitor binding affinity at the two binding sites of lung ACE across the panel of ACE inhibitors studied (equilibrium dissociation constant; Kd; pmol/L) for site one vs site two: cilazaprilat 40 +/- 3 vs 430 +/- 92*; lisinopril 25 +/- 1 vs 848 +/- 107**; and quinaprilat 4 +/- 1 vs 1869 +/- 720; *P less than 0.05; **P less than 0.005, t-test, n = 3). Reduction in binding affinity at site two of lung ACE was related to an increase in ACE inhibitor side chain length or complexity of carboxyl terminal moiety. ACE inhibitor binding affinity at the testis ACE binding site resembled site one of lung ACE. 4. Inhibition of bradykinin hydrolysis by lung ACE in the presence of increasing concentrations of cilazaprilat or quinaprilat was similar (F = 0.64; P greater than 0.05), suggesting that bradykinin cleavage predominates at ACE active site one. 5. The differences in ACE inhibitor affinity at the two ACE active sites has implications in physiological substrate selectivity, and may influence the pharmacodynamic effects of different ACE inhibitors.


Effect of neutral endopeptidase inhibitor in rats with congestive heart failure. 1. The acute hormonal, renal and haemodynamic effects of SCH 39370, a new neutral endopeptidase (NEP) inhibitor, were evaluated in rats with congestive heart failure (CHF) produced by coronary ligation. 2. Left ventricular systolic pressure and left ventricular end diastolic pressure were significantly decreased by SCH 39370 treatment. 3. SCH 39370 improved cardiac function by increasing cardiac index and decreasing total peripheral resistance. 4. SCH 39370 induced a transient but significant diuresis and natriuresis. 5. These effects were associated with significant increases in urinary atrial natriuretic peptide (ANP) and cyclic GMP excretion, but not with an increase in plasma ANP levels. 6. The results suggest that NEP inhibition may be a new therapeutic method for treating CHF possibly by potentiating the biological activities of endogenous ANP.


Interaction between vasopressin and endothelin in renal papillary tubules: uncoupling following cell isolation and culture. 1. In freshly prepared rat renal papillary tubules, endothelin-related peptides inhibited the vasopressin-stimulated accumulation of cAMP. No inhibition was observed when tubules were cultured overnight ex vivo. 2. Endothelin-1, endothelin-3 and sarafatoxin S6b had similar potencies as inhibitors of cAMP accumulation, indicating an endothelin (ETb) receptor. 3. These results demonstrate an interaction between ETb receptors and vasopressin receptors at the level of their signal transduction pathways, and show that this relationship is lost following cell culture.


Isolation of adult cardiomyocytes initiates a return of inositol trisphosphate phosphorylating activity. 1. We have previously reported that the addition of noradrenaline to [3H]-inositol-labelled adult rat atria or isolated perfused hearts caused the release of inositol-1,4,5-trisphosphate, which was metabolized by dephosphorylation to inositol-4-monophosphate. Inositol-1,3,4,5-tetrakisphosphate and its dephosphorylation products were not detected. 2. In the current study, the addition of noradrenaline to [3H]-inositol-labelled adult rat cardiomyocytes caused the release of inositol-1,4,5-trisphosphate, which was metabolized in part by phosphorylation to inositol-1,3,4,5-tetrakisphosphate. 3. These results demonstrate that the isolation and culture of rat adult cardiomyocytes initiates enhanced generation of inositol-1,3,4,5-tetrakisphosphate. This change would be expected to enhance the calcium response of the cells to stimulation of alpha 1-adrenoceptors.


Lomefloxacin and temafloxacin: two new fluoroquinolone antimicrobials. The chemistry, mechanism of action, antimicrobial spectrum, pharmacokinetics, clinical efficacy, safety, drug interactions, and dosage and administration of lomefloxacin and temafloxacin, two new antimicrobials, are presented. Lomefloxacin and temafloxacin exhibit activity comparable to that of ciprofloxacin against the Enterobacteriaceae. Lomefloxacin has only modest activity against common gram-positive organisms. Temafloxacin exhibits increased activity against the streptococci and moderate activity against many anaerobes, as compared with ciprofloxacin. Lomefloxacin and temafloxacin have only moderate antipseudomonal activity. Their elimination half-lives are 6-10 hours and they have good oral absorption, excellent penetration into many tissues and fluids, and a better drug-interaction profile than other similar agents. Lomefloxacin has demonstrated comparable efficacy to other therapies in the treatment of lower respiratory-tract infections and urinary-tract infections (UTIs) and for prophylaxis before surgical procedures in the urinary tract. Temafloxacin has been shown to be effective in the treatment of lower respiratory-tract infections, infections of the skin and associated structures, uncomplicated and complicated UTIs, bacterial prostatitis, and gonococcal and non-gonococcal urethritis and cervicitis. The most frequent adverse effects with lomefloxacin are gastrointestinal upset and headache; with temafloxacin, gastrointestinal complaints. Lomefloxacin's dosage is 400 mg p.o. daily for 10 days for treatment of acute bacterial exacerbations in chronic bronchitis or simple cystitis and for 14 days for treatment of complicated UTIs. Lomefloxacin is a new oral fluoroquinolone that is indicated for the treatment of various bacterial infections. Temafloxacin, another new fluoroquinolone, appeared to have some favourable characteristics but was withdrawn from the market.


Anti-calculus effects of dentifrice containing pyrophosphate compared with control. A 3-month, double-blind, randomized, longitudinal, parallel clinical study was conducted to compare the effects on supragingival calculus deposits of a dentifrice containing soluble pyrophosphate to a control MFP dentifrice, which did not contain pyrophosphates. During the pre-test phase, 247 teachers were examined for presence of calculus on the lingual surfaces of the 6 lower anterior teeth, using the Periodontal Probe Method of Calculus Assessment (Volpe et al. 1965). Statistical methods employed were the two-way analysis of variance as well as the paired and unpaired t tests. The level of statistical significance chosen was p less than 0.05. At the end of the 3-month pre-test phase, 88 candidates with the highest calculus index scores met the initial criteria and were stratified into two balanced groups based on their age and sex. Each group received an oral prophylaxis and participants were assigned to use either the dentifrice containing the pyrophosphate or the MFP and instructed to brush their teeth twice daily at home for the next 3 months (test phase). The results showed that the group using the pyrophosphate dentifrice experienced a 20% greater reduction in calculus formation when compared to the MFP control There was also a greater number of calculus-free subjects and tooth surface sites in the soluble pyrophosphate group. The change in the Volpe-Manhold Index revealed significant differences for both the pyrophosphate group (change = 1.7111; p = 0.0010) as well as, though less prominent, for the control group (change = 1.4186; p = 0.0313).


Calcium efflux in rat lens: Na/Ca-exchange related to cataract induced by selenite. Sodium gradient-dependent 45Ca2+ transport occurred across the lens membrane both in the direction of Ca2+ uptake by inside-out vesicles and Ca2+ efflux after Ca2+ loading of right-side-out vesicles. Using the calcium ionophore, A23187, greater than 90% of the Na+ gradient-dependent Ca2+ uptake was estimated to be free Ca2+. A normal Na+ gradient was also required to maintain calcium homeostasis in the intact lens. The Na+ gradient contributed to Ca2+ efflux from lenses pre-loaded in medium containing 15 mM CaCl2. Therefore, a Na/Ca-exchange functions to control Ca efflux in rat lens, in addition to the Ca-ATPase. In the preweanling rat mature nuclear cataracts occurred by 96 h after subcutaneous injection of sodium selenite (30 nmol/g animal wt). A 3-5 fold increase of Ca2+ accompanied cataract formation. The loss of Ca2+ homeostasis can be detected by 48 h after treatment selenite treatment. At this time the initial rate of Na+ gradient-dependent Ca2+ uptake was 30% lower in lens vesicles from selenite-treated rats compared to controls. No significant reduction of Na+,K(+)-ATPase activity was detected. Altered Na/Ca-exchange may contribute directly to the loss of Ca2+ homeostasis that leads to nuclear cataract.


Mechanisms involved in cataract development following near-ultraviolet radiation of cultured lenses. Cultured rabbit lenses were irradiated with UV (311 nm peak; 295-340 nm) for 30 to 60 min. The entire spectrum lies in the near-UV, the major component is UVB, with a minor portion (25%) of UVA, and is henceforth referred to as near-UV(B). Posterior irradiation caused no cataract and no significant ionic imbalances compared to anterior irradiation, which caused opacification and marked changes in sodium and calcium concentrations. Anterior irradiation also resulted in reduced Na/K-ATPase activity in the epithelium. ATPase activity was not immediately inhibited; rather, only after culture was enzyme activity reduced. The concentration of reduced glutathione (GSH) decreased rapidly in the epithelium and more slowly in the underlying lens fibers. Loss of GSH was more rapid and extensive when irradiation occurred in the presence of oxygen. Irradiation under anaerobic conditions resulted in opacification but was considerably less extensive than when irradiation of lenses occurred in the presence of 7% oxygen. Near-UV(B) damage following anaerobic irradiation and 20 hrs of culture resulted in an increase in sodium levels and loss of GSH; calcium levels were not significantly elevated. Since irradiation of tryptophan solutions produced small amounts of hydrogen peroxide, the possibility of hydrogen peroxide-mediated damage was investigated but no role could be substantiated. Peroxide detoxification by the epithelium of near-UV(B) cataracts was observed, as measured by its ability to eliminate hydrogen peroxide added as a bolus.


Extensive dermal metastases from primary signet-ring carcinoma of the urinary bladder. Dermal metastasis from internal carcinoma occurs infrequently. Bladder carcinoma is a rare source of skin metastasis. This report details the case of a twenty-five-year-old man with a fatal primary signet-ring adenocarcinoma of the bladder who experienced unique, extensive, infiltrative, cutaneous metastases within a few weeks of his initial presentation with an occultly carcinomatous ruptured spleen. The literature on cutaneous metastasis from carcinoma of the bladder is reviewed.


Chorion gene cis-regulatory DNA restricts tissue specificity of reporter gene expression in transformed Drosophila. P element mediated germ-line transformation was used to study the developmental specificity of Drosophila chorion gene regulatory sequences directing expression of the bacterial reporter genes for chloramphenicol acetyltransferase (CAT) and beta-galactosidase (lacZ). DNA fragments containing 5' flanking plus the entire 5' untranslated and the beginning of the coding region of either the s36 or the s15 chorion gene are able to confer on the reporter genes normal tissue as well as temporal specificity of expression, exclusively in the ovary of transformed female flies. However, if 5' untranslated and coding regions are omitted, normal ovarian expression is maintained but tissue specificity is relaxed: expression of the reporter gene is detected both in the ovary and in specific non-ovarian tissues of transformed females and males. The evidence suggests that the missing 5' untranslated and coding sequences may include negative elements that normally suppress expression in non-ovarian tissues, and that these putative elements are distinct from those that prevent premature expression in the ovarian follicles. The exact location of ectopic lacZ expression within the internal male genitalia depends on the constellation of 5' flanking chorion regulatory sequences included in the P element constructs. Ectopic expression of the CAT gene in the male genitalia under s15 promoter control can be abolished by mutating the hexamer TCACGT, a sequence previously shown to be essential for the normal expression of this chorion gene in the ovary.


The laws of cell energetics.

The laws of cell energetics. Recent progress in membrane bioenergetics studies has resulted in the important discovery that Na+ can effectively substitute for H+ as the energy coupling ion. This means that living cells can possess three convertible energy currencies, i.e. ATP, protonic and sodium potentials. Analysis of interrelations of these components in various types of living cells allows bioenergetic laws of universal applicability to be inferred.


Experimental and modelling studies on the DNA cleavage by elsamicin A. The ability of elsamicin A, an antitumour antibiotic, to cleave DNA in the presence of ferrous iron and reducing agents, has been analysed using experimental and theoretical approaches. Experimentally, the antibiotic causes DNA breakage in the presence of ferrous ions and a reducing agent. The DNA-cleaving activity appears to be partially blocked by the action of superoxide dismutase and catalase. These results indicate that the elsamicin aglycone moiety (chartarin) can be involved in the production of free radicals. We have performed a broad theoretical study based in the quantum-mechanical framework, which allow us to determine the redox properties of elsamicin that lead to the generation of radical species. Our results clearly show that elsamicin acts as a true catalyst in the production of superoxide radicals. Moreover, it is suggested that the oxidation/reduction mechanism of the aglycone moiety of elsamicin (a lactone), leading to DNA breakage, is different from the mechanism followed by other well-known anti-cancer drugs, whose chromophore is a quinone.


Ligand-independent reduction of cAMP receptors in Dictyostelium discoideum cells over-expressing a mutated ras gene. Drug-resistance selection in Dictyostelium discoideum transformants resulted in up to eight-times-higher ras protein levels. Over-production of the wild-type ras protein did not lead to an aberrant phenotype. Increased levels of the mutated [G12T]ras protein, however, were correlated with severe deficiencies in aggregation and development. This aberrant phenotype is associated with reduced cAMP binding, due to a lower number of cell-surface receptors. We show that both RNA and cAMP-receptor-protein levels are reduced. These results indicate that ras in Dictyostelium discoideum seems to be involved in regulating cAMP-receptor-gene expression.


Preferential binding of insulin-like growth factor-II (IGF-II) to a putative alpha 2 beta 2 IGF-II receptor type in C2 myoblasts. We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)


A reinvestigation of the pre-steady-state ATPase activity of the nitrogenase from Azotobacter vinelandii. The pre-steady-state ATPase activity of nitrogenase has been reinvestigated. The exceptionally high burst in the hydrolysis of MgATP by the nitrogenase from Azotobacter vinelandii communicated by Cordewener et al. (1987) [Cordewener J., ten Asbroek A., Wassink H., Eady R. R., Haaker H. & Veeger C. (1987) Eur. J. Biochem. 162, 265-270] was found to be caused by an apparatus artefact. A second possible artefact in the determination of the stoichiometry of the pre-steady-state ATPase activity of nitrogenase was observed. Acid-quenched mixtures of dithionite-reduced MoFe or Fe protein of Azotobacter vinelandii nitrogenase and MgATP contained phosphate above the background level. It is proposed that due to this reaction, quenched reaction mixtures of nitrogenase and MgATP may contain phosphate in addition to the phosphate released by the ATPase activity of the nitrogenase complex. It was feasible to monitor MgATP-dependent pre-steady-state proton production by the absorbance change at 572 nm of the pH indicator o-cresolsulfonaphthalein in a weakly buffered solution. At 5.6 degrees C, a pre-steady-state phase of H+ production was observed, with a first-order rate constant of 2.2 s-1, whereas electron transfer occurred with a first-order rate constant of 4.9 s-1. At 20.0 degrees C, MgATP-dependent H+ production and electron transfer in the pre-steady-state phase were characterized by observed rate constants of 9.4 s-1 and 104 s-1, respectively. The stopped-flow technique failed to detect a burst in the release of protons by the dye-oxidized nitrogenase complex. It is concluded that the hydrolysis rate of MgATP, as judged by proton release, is lower than the rate of electron transfer from the Fe protein to the MoFe protein.


Inhibition of the GlcNAc transferase of the glycosylphosphatidylinositol anchor biosynthesis in African trypanosomes. A wide variety of eukaryotic membrane proteins are anchored to the cell surface by a covalent linkage to glycosylphosphatidylinositol. One of the best characterised examples is the variant surface glycoprotein of the protozoan parasite, Trypanosoma brucei. The pathway for the formation of the glycosylphosphatidylinositol precursor has been previously described, with the first step being the transfer of GlcNAc, from UDP-GlcNAc to endogenous phosphatidylinositol to form N-acetyl-glucosaminylphosphatidylinositol [Doering, T. L., Masterson, W. J., Hart, G. W. & Englund, P. T. (1989) J. Biol. Chem. 264, 11,168-11,173]. Here we report that low concentrations of sulphydryl alkylating reagents irreversibly inhibit this transferase in a trypanosome-derived cell-free system. The site of inactivation by N-ethylmaleimide appears to be at, or close to, the enzyme active site, since incubation of the enzyme preparation with the donor molecule UDP-GlcNAc substantially protects the enzyme from inactivation. The protection appears to be primarily dependent on the nucleotide portion of the molecule, since UMP and UDP can mimic the protection seen with UDP-GlcNAc.


Non-linear inhibition curves for tight-binding inhibitors of dimeric ubiquinol-cytochrome c oxidoreductases. Evidence for rapid inhibitor mobility. Steady-state electron flow through and electron delivery into isolated dimeric bc1 complex (ubiquinol--cytochrome c oxidoreductase) from Neurospora crassa and beef heart mitochondria were studied in the presence of increasing concentrations of antimycin A, funiculosin and/or myxothiazol. Parabolic or linear inhibition curves were obtained, depending upon the different quinols and inhibitors that were used. Linear curves occur when the inhibitor directly affects the rate-determining step. The most reasonable explanation for the parabolic curves is given by a fast intradimeric exchange of the hydrophobic inhibitors antimycin A, funiculosin (rate less than 500 s-1) and of myxothiazol (rate greater than 1 s-1). Using mitochondria from beef heart, the shape of the inhibition curve with antimycin A is parabolic if the quinol--O2 oxidoreductase turns over at about 300 s-1, but hyperbolic if the rate is 5 times less. The hyperbolic titration curve may be the result of both intradimeric and an additional interdimeric redistribution (rate approximately 100 s-1) of inhibitors between enzymes incorporated in a continuous phospholipid membrane. This explanation is supported by experiments with chromatophores obtained from Rhodobacter capsulatus. As recently described [Fernandez-Velasco, J. & Crofts, A. R. (1992) Biophys. J. 2, A153], cytochrome b becomes fully reoxidized within 1 s after a flash at substoichiometric concentrations of antimycin A. This kinetic of the slow reoxidation can be expressed in terms of the intradimeric and interdimeric redistribution with rate constants of about 10 s-1 and 2 x 10(6) M-1 s-1, respectively. It seems that rapid inhibitor redistribution may be a widespread phenomenon for hydrophobic inhibitors of enzymes incorporated in lipid membranes.


Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome-c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron-sulfur subunit. Cytochrome-c reductase was isolated from Saccharomyces cerevisiae GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE. Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Q0 center (Q, ubiquinone) of cytochrome-c reductase, were analysed. The single amino acid substitution in the cytochrome-b subunit, N256Y in the mutant Myx-119 and G137R in the mutant Myx-118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations. The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The Km of the G137R mutant was not affected and the Vmax was 50% higher. Cytochrome-c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate-stilbene and myxothiazol by means of fluorescence-quench and red-shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDS/PAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron-sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type. This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron-sulfur protein, the second important catalytic subunit of the Q0 center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.


A soluble form of the glycolipid-anchored receptor for urokinase-type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria. The cellular urokinase-type plasminogen-activator (uPA) receptor (uPAR) is a glycolipid-anchored membrane protein thought to be involved in pericellular proteolysis during cell migration and tumor invasion. In the present study, we have identified and characterized two soluble forms of uPAR which have retained their ligand-binding capability. One variant was generated in vitro by treatment of intact normal cells with either a phosphatidylinositol-specific phospholipase C (PLC) or endoproteinase Asp-N. The other soluble uPAR variant was secreted in vivo from peripheral blood leukocytes affected by the stem-cell disorder paroxysmal nocturnal hemoglobinuria (PNH), and was found in the plasma from these PNH patients as well as in the conditioned medium from cultured PNH leukocytes. Under normal conditions, we find no evidence for any shedding or secretion of a soluble uPA-binding counterpart to human uPAR in plasma. Unlike normal leukocytes, the PNH-affected cells do not express uPAR on the cell surface, although they do contain apparently normal levels of uPAR-specific mRNA. The secreted uPAR derived from PNH cells has a mobility in SDS/PAGE that is slightly higher than that of uPAR solubilized by PtdIns-specific PLC or detergent, but resembles that of a truncated, recombinant uPAR variant, which has its C-terminus close to the proposed glycolipid-attachment site, suggesting that the secreted protein has been proteolytically processed for glycolipid attachment. The presence in plasma from PNH patients of such a secreted, hydrophilic form of uPAR lends support to the hypothesis that the lesion underlying the PNH disorder resides either in glycolipid biosynthesis or in the function of an as-yet-unidentified transamidating enzyme assumed to cleave and assemble the truncated uPAR with the preformed glycolipid moiety.


Molecular cloning of a new bombesin receptor subtype expressed in uterus during pregnancy. The homology screening approach has been used to clone a new member of the guanine-nucleotide-binding-protein-coupled receptor superfamily from guinea pig uterus. The cloned cDNA encodes a 399-amino-acid protein and shows the highest amino acid similarity to members of the bombesin receptor family; 52% and 47% similarity to the gastrin-releasing-peptide (GRP) receptor and the neuromedin-B receptor, respectively. Binding experiments with the stably transfected LLC-PK1 cell line expressing the new receptor protein confirmed the bombesin-like nature of the cloned receptor. The relative order of ligand affinity, GRP = neuromedin C much greater than neuromedin B, suggests that the cloned cDNA represents the GRP subtype rather than the neuromedin-B subtype of bombesin receptors. Northern-blot analysis of mRNA species from several guinea-pig tissues showed that the mRNA for the new bombesin receptor subtype is expressed mainly in uteri of pregnant animals.


In-vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE. Evidence that SecB prevents the precursor from aggregating by forming a functional complex. We characterised the behaviour of the purified precursor protein prePhoE upon dilution from 8 M urea by CD, fluorescence spectroscopy and gel-filtration techniques. It is demonstrated that prePhoE rapidly adopts beta structure, folds and aggregates upon dilution to urea concentrations below 3 M. These processes are paralleled by a loss of translocation competence. Furthermore the interaction of prePhoE with SecB was investigated. SecB is shown to have a very high content of beta structure, therefore we propose that precursor recognition by SecB is mediated through beta-beta interaction. It is shown that SecB has little effect on the adoption of secondary structure and tertiary folding upon dilution of the precursor from urea. However, SecB prevents the precursor from aggregating by forming a functional and stable complex.


Involvement of a labile axial histidine in coupling electron and proton transfer in Methylophilus methylotrophus cytochrome c''. Methylophilus methylotrophus cytochrome c'' is an unusual monohaem protein (15 kDa) undergoing a redox-linked spin-state transition [Santos, H. & Turner, D. L. (1988) Biochim. Biophys. Acta 954, 277-286]. The midpoint redox potential of cytochrome c" was measured over the pH range 4-10. The pH dependence of the midpoint redox potential was interpreted in terms of a model that considers the redox-state dependence of the ionization of two distinct and non-interacting protonated groups in the protein. This analysis led to the following pKa values within the pH range studied: pKa10 = 6.4, pKa1r = 5.4 and pKa2r = 8.1. Proton-NMR spectroscopy was used to assist the characterization of the two ionizing groups responsible for the observed redox-Bohr effect: the group ionizing with a lower pKar was assigned to a haem propionic acid substituent and the other to the axial histidine ligand which becomes detached upon reduction, which has a pKa0 too low to be measured. It is shown that M. methylotrophus cytochrome c" is able to couple electron and proton transfer in the physiological pH range through a mechanism involving reversible change in the haem-iron coordination. Possible implications for the physiological role of the protein are discussed.


Detection of cGMP dependent protein kinase isozymes by specific antibodies. Two isozymes of cGMP-dependent protein kinase (cGMP kinase) have been identified. Polyclonal antibodies were developed which recognize both isozymes or specifically the I alpha and I beta isoform. The specificity of these antibodies was verified by using the recombinant or purified I alpha and I beta isozymes. The antibodies cross-reacted with the purified isozymes of cGMP kinase from bovine tracheal smooth muscle. The tissue concentration of cGMP kinase was determined by ELISA. High concentrations (greater than 10 pmol/g wet tissue) were present in bovine lung, rumen, trachea, aorta, uterus and stomach. The tissue distribution of the isozymes I alpha and I beta was investigated by immunoblots using crude extracts of the different tissues. The I beta-specific antibody yielded strong signals with extracts of trachea, aorta, stomach and uterus, whereas heart, cerebellum and lung apparently contain mainly the I alpha isozyme.


Activation by ATP of a proton-conducting pathway in yeast mitochondria. The growth of Saccharomyces cerevisiae cells under aerobic conditions, in the presence of an energy-rich source, leads to production of an excess of NAD(P)H. Since the redox balance must be maintained, it has been postulated that NAD(P)H reoxidation is accelerated by the activation of energy-dissipating reactions, which would, in turn, explain the low growth efficiencies observed. It has been demonstrated already in S. cerevisiae cultures that these putative energy-dissipating reactions are stimulated both by oxygen and high cytosolic ATP levels. In this paper, we show that ATP induces a proton-permeability pathway in mitochondria at concentrations which are within the physiological range, as revealed both from the ATP stimulation of respiration and from the induction of H(+)-dependent swelling. We also demonstrate that phosphate acts as a competitive inhibitor of the nucleotide, and since activation is observed even in the presence of atractylate, we postulate that the ATP-binding site is located in the outer face of the mitochondrial inner membrane.


Structural requirements for glycosyl-phosphatidylinositol-anchor attachment in the cellular receptor for urokinase plasminogen activator. The urokinase-plasminogen-activator receptor (u-PAR) is a glycosyl-phosphatidylinositol(glycosyl-PtdIns)-anchored membrane protein. Using site-directed mutagenesis, we have studied features in the u-PAR sequence important for successful glycosyl-PtdIns attachment. Two critical sequence elements were identified. In the sequence Ser282-Gly283-Ala284, simultaneous substitution of all of these residues prevented membrane anchoring. Individual substitution of each of the residues indicated that Gly283 is the more critical residue and the likely attachment site. However, it was unexpectedly found that mutation of this residue gave rise only to a partial impairment of glycosyl-PtdIns attachment. We therefore propose that more than one residue within this sequence can be utilized as glycosyl-PtdIns-attachment site. In the last eight COOH-terminal amino acids encoded in u-PAR cDNA, deletion of this sequence (residues 306-313) completely prevented glycosyl-PtdIns attachment. However, the remaining COOH-terminal region proved still to possess a potential glycosyl-PtdIns signal activity; it could be converted to a new functional glycosyl-PtdIns signal by substitution of a single positively charged residue (Arg304). Substitution of Arg304 by Leu converted this truntaced u-PAR to a glycosyl-PtdIns-anchored protein, indistinguishable from the wild type. Substitution of Arg304 by a negatively charged residue (Glu) led to a partial acquisition of the glycosyl-PtdIns-anchoring ability. These findings show that charged amino acids placed in the COOH-terminus interfere negatively with glycosyl-PtdIns-anchoring, and, furthermore, that this effect is more pronounced for positively charged than for negatively charged amino acid residues.


Transiently and stably introduced CCAAT/enhancer-binding-protein genes are constitutively expressed in cultured cells. CCAAT/enhancer-binding protein (C/EBP) is expressed in certain cell types including hepatocytes and adipocytes. In order to understand the mechanisms that control the expression of the mouse C/EBP gene in the liver as well as in adipocytes, we have studied both the endogenous gene and transfected C/EBP gene constructs. The initiation site of transcription was identified and a strong liver-specific DNase-I hypersensitive site located at -3 kb, which does not appear to contribute functionally to the regulation of the gene in a variety of either transiently or stably transfected cells with constructs which include sequences up to 6-kb upstream of the transcription start. C/EBP gene expression during the transition from preadipocytes to adipocytes was shown to be controlled at the level of transcription. However, adipocytes stably transfected with constructs that include -3.3 kb upstream of the C/EBP gene do not express the reporter genes in a differentiation-specific manner. We detected several DNA-binding proteins that interact with the upstream sites of the C/EBP gene. Those include two labile and two heat-stable site-specific DNA-binding proteins that are present in nuclear extracts from several tissues and cultured cell lines.