Structural alterations of double-stranded DNA in complex with the adenovirus DNA-binding protein. Implications for its function in DNA replication. The Adenovirus DNA-binding protein (DBP) binds to single-stranded (ss) DNA as well as to double-stranded (ds) DNA and forms multimeric protein-DNA complexes with both. Gel retardation assays indicate rapid complex formation for both DNAs. DBP rapidly dissociates from dsDNA, indicating a dynamic equilibrium, whereas the ssDNA-DBP complex is much more stable. We investigated the complex between DBP and dsDNA in more detail. Electron microscopical analysis shows thick filament-like and beaded structures in which the length of the DNA is not significantly altered. Cryo-electron micrographs suggest the presence of interwound protein fibres around the DNA. Ligase-mediated cyclization, but not linear multimerization, of DBP-saturated DNA fragments exceeding the persistence length was severely inhibited. This suggests that DNA may be organized by DBP into a rigid structure. Under those conditions, DBP induces distinct changes in the circular dichroism spectrum of the DNA, indicative of structural DNA changes. No bending or twisting of the complex was observed. Hydroxyl radical footprinting showed that the breakdown pattern of DNA at saturating DBP concentrations is much more regular than the protein-free DNA. This suggests the removal of tertiary structures, which may be related to the effects of DBP on enhanced NFI binding and chain elongation during Adenovirus DNA replication. Using purified proteins in an in vitro replication system, we correlate the structural changes with the effects of DBP on enhancement of NFI-binding as well as on DNA replication.


Antioxidant-dependent amelioration of brain injury: role of CuZn-superoxide dismutase. Copper-zinc-superoxide dismutase (CuZn-SOD), a cytosolic antioxidant enzyme that is specific for scavenging superoxide radicals, is involved in neuroprotective mechanisms in brain injury following trauma and cerebral ischemia. Liposome-entrapped CuZn-SOD exhibit beneficial effects in vivo on cold-induced vasogenic edema and on blood-brain barrier disruption. The increased levels of edema and infarction following a focal cerebral ischemia also are decreased by the pretreatment of liposome-entrapped CuZn-SOD. The protective role of SOD on brain injury was further extended and confirmed in studies using transgenic mice overexpressing human CuZn-SOD. Our studies so far suggest that increased cerebral levels of SOD, either by means of external pharmacological application or by genetic manipulations, ameliorate brain edema and infarction induced by trauma and focal cerebral ischemia.


New CNS-specific calcium antagonists.

New CNS-specific calcium antagonists. Ischemic insults to the brain in stroke or traumatic brain injury produce excessive release of glutamate from depolarized nerve terminals. This excessive glutamate release in turn stimulates massive calcium entry into nerve cells, activating a biochemical cascade that results in cell death. A major pathway of calcium entry into depolarized nerve cells is through voltage-sensitive, high threshold calcium channels. A large fraction of this calcium entry is mediated through "R-type" calcium channels, channels resistant to blockage by dihydropyridine calcium antagonists such as nimodipine. A newly discovered compound derived from spider venom, CNS 2103, antagonizes both R-type channels and dihydropyridine-sensitive ("L-type") calcium channels. This broad spectrum of action, coupled with selectivity for calcium channels over other classes of voltage-sensitive and ligand-gated ion channels, makes CNS 2103 an interesting lead for development of drugs to treat ischemic brain injury. Activation of presynaptic ("N-type") calcium channels in nerve terminals is a primary cause of excessive neurotransmitter release in brain ischemia. Prevention of glutamate release by blockade of N-type channels in glutamatergic nerve terminals may, at an early stage in the pathophysiological cascade, abort the process leading to nerve cell death. Cambridge NeuroScience has developed a novel rapid kinetic approach for monitoring glutamate release from brain nerve terminals in vitro, and this has led to CNS 1145, a substituted guanidine that selectively blocks a kinetic component of calcium-dependent glutamate release mediated by persistent depolarization. Additional evidence suggests that CNS 1145 antagonizes presynaptic N-type calcium channels, and this may account at least in part for its ability to block glutamate release.


Receptor-mediated endocytosis and degradation of insulin-like growth factor I and II in neonatal rat astrocytes. Receptor-mediated internalization and degradation of insulin-like growth factors, IGF-I and IGF-II, were studied in primary cultures of neonatal rat astrocytes. Surface-bound IGF-II was rapidly internalized, and 80% of cell-associated radioactivity was located intracellularly after 30 min. IGF-I was internalized at a slower rate, and only 40% of cell-associated radioactivity was inside the cell after 30 min. A pulse-chase experiment demonstrated that 55% and 70% of internalized IGF-I and IGF-II, respectively, was degraded to free amino acids after a 3-hr chase. Lysosomal and protease inhibitors had different effects on the binding, internalization, and processing of IGF-I and IGF-II. Inhibition of lysosomal acidification by chloroquine increased the amounts of surface-bound IGF-II and intracellular IGF-I and reduced the degradation of IGF-I. The chelating agent phenanthroline increased the surface binding of IGF-I and IGF-II and internalization of IGF-II and reduced the degradation of IGF-I and IGF-II. Finally, receptor-bound IGF-II on the cell surface was decreased with increasing cell density, whereas IGF-I binding was unaltered. Our data suggest that cell-surface expression of IGF-I receptors and IGF-II receptors is regulated by different mechanisms and that receptor-bound IGF-I and IGF-II are trafficked and processed by different intracellular pathways in neonatal rat astrocytes.


Muscimol and N,N-dimethylmuscimol: from a GABA agonist to a glycine antagonist. By using molecular modeling methods, a molecular mechanism was identified which can explain how the incorporation of two methyl groups in place of two hydrogen atoms on the terminal nitrogen atom of muscimol can not only convert this potent agonist at GABAnergic receptors to an inactive molecule at these receptors, but also can convert this new derivative to an antagonist of glycine at glycinergic receptors. This insight into the molecular mechanism operative in the conversion of physiological function provides a basis for understanding how a single molecule may be able to act at both the GABA- and glycine-inhibitory receptors.


Peripheral nerve injury down-regulates CNTF expression in adult rat sciatic nerves. Ciliary neurotrophic factor (CNTF) is a 200-amino acid protein expressed in high concentrations by peripheral nerves and is thought to be important for the survival and regeneration of injured motoneurons (Lin et al., J Biol Chem 265:8942-8947, 1990). To better understand CNTF's role in nerve injury we have characterized the effects of crush injury on the expression of CNTF in adult rat sciatic nerves using specific antibody and RNA probes. Following a crush injury, both the protein and mRNA levels undergo pronounced decreases distal to the crush. These changes in CNTF expression were qualitatively distinct from changes in the expression of the low-affinity NGF receptor (p75NGFR), which increases following crush. Thus, the changes in CNTF levels do not reflect an overall down-regulation of mRNA during degeneration, and are inconsistent with the proposed role of CNTF in neuronal injury, since its levels are decreasing at the same time as the requirement for neurotrophic support is increasing.


Dexamethasone blocks nerve growth factor induction of nerve growth factor receptor mRNA in PC12 cells. Glucocorticoids and nerve growth factor (NGF) have been shown to have antagonistic effects on chromaffin cells in vivo. Here we determined the effect of the synthetic glucocorticoid, dexamethasone, on levels of mRNA for the nerve growth factor receptor (NGFR) in rat PC12 pheochromocytoma cells. Following administration of dexamethasone (1 microM) there is a decline in NGFR mRNA expression. More importantly, administration of dexamethasone appears to block the NGF-mediated induction of NGFR when both agents are administered simultaneously. These data support the hypothesis that glucocorticoids and NGF act in opposition in determination of the phenotype of chromaffin cells.


Schwann cell nerve growth factor receptor expression during initiation of remyelination. Initiation of remyelination is a promising therapeutic strategy to treat patients with demyelinating diseases, but specific factors that control remyelination are not clear. We first reported that expression of nerve growth factor receptor (NGFR) was increased during initiation of remyelination (Fan and Gelman, Journal of Neuropathology and Experimental Neurology 49: 312, 1990). In this study, we characterized the timing and cellular localization of NGFR expression in a model of segmental demyelination and remyelination using immunohistochemistry and monoclonal antibody 192-IgG, and compared it to an axonal neuropathy. At the onset of demyelination induced by tellurium (Te) poisoning, NGFR antigenicity was selectively expressed within and around demyelinating internodes in rat sciatic nerve. Dual fluorescence staining with myelin-specific antigen showed that NGFR colocalized with demyelinated internodal units with relative specificity; Schwann cell S-100 protein showed a concomitant down-regulation in injured internodes. Peak expression of NGFR occurred during the transition between demyelination and remyelination (day 8 of Te), then declined exponentially. NGFR expression was most prominent in the cytoplasm of daughter Schwann cells as they established contact with denuded axons, and was sharply repressed as compact myelin began to accumulate. Rare colocalization with neurofilament antigens revealed intraxonal deposits of NGFR in segmental demyelination. In the nerve crush model, Schwann cell NGFR expression was not segmentally distributed and was upregulated for a longer period of time. Our data establish that NGFR expression in the peripheral nervous system is not strictly linked to axon elongation, and that it probably functions during the initiation of myelination.


Human papillomavirus and herpes virus DNA are not detected in benign and malignant prostatic tissue using the polymerase chain reaction. Fresh prostatic tissue removed at the time of surgery was assayed for the presence of human papillomavirus (HPV) types 6, 11, 16, 18 and 33 and herpes and varicella-zoster viruses (HV) using DNA amplification followed by specific hybridization. Thirty samples representing both benign and malignant prostatic disease were assayed. Although appropriate amplimers were present for beta globulin gene indicating successful extraction of DNA, no HPV or HV amplimers could be obtained with appropriate primers. We conclude that HPV and HV are not routinely found in human prostate.


Glycerophosphorylcholine phosphocholine phosphodiesterase activity in cultured oligodendrocytes, astrocytes, and central nervous tissue of dysmyelinating rodent mutants. The levels of GPC phosphocholine phosphodiesterase, pNP phosphocholine phosphodiesterase, CNPase, and UDP galactose: ceramide galactosyltransferase activities were estimated with pure cultures of oligodendrocytes and astrocytes; mixed primary glial cells cultures; C-6 cells; and CNS tissue of the dysmyelinating md rat, the jimpy mouse, and the quaking mouse. The highest activity of GPC and pNP phosphocholine phosphodiesterases as with CNPase and C gal T was found in the pure cultured oligodendrocytes. C-6 cells had very low or undetectable activities for these two phosphodiesterases but possessed very high CNPase activity. The activity of GPC phosphocholine phosphodiesterase was significantly decreased in the CNS tissue of the md rat and the jimpy and the quaking mouse. Similar reductions were observed for the pNP phosphocholine phosphodiesterase, CNPase, and C gal T activities. The selective cellular enrichment in oligodendrocytes of the GPC phosphocholine phosphodiesterase activity and decreases of its activity in three dysmyelinating mutants in the same ratio as for CNPase and C gal T suggest that GPC phosphocholine phosphodiesterase is a myelin marker enzyme and it may reflect the quantity of myelin and oligodendrocyte present.


Comparison between clinical and pathological staging in low stage nonseminomatous germ cell testicular tumors. Between January 1985 and December 1990, 208 consecutive patients with low stage nonseminomatous germ cell testicular tumors underwent retroperitoneal lymphadenectomy. In all of the patients the disease was staged with post-orchiectomy serum alpha-fetoprotein and beta subunit of human chorionic gonadotropin determinations, as well as chest x-rays and computerized tomography or magnetic resonance imaging of the abdomen and pelvis. Bipedal lymphangiography was performed in 139 patients. Of the 208 patients 173 (83%) had clinical stage 1 and 35 (17%) had low clinical stage 2 disease: 21 had tumors on radiographic imaging that were smaller than 2 cm. (clinical stage 2A) and 14 had tumors between 2 and 3 cm. (clinical stage 2B less than 3 cm.). Retroperitoneal metastases were found in 31 of 156 clinical stage 1 cancer patients (19.8%) with negative or normally decreasing serum tumor markers after orchiectomy, 15 of 16 (93.8%) with persistent positive markers, 8 of 14 clinical stage 2A cancer patients (57.1%) with negative or normally decreasing markers, all 7 stage 2A cancer patients with positive markers and all 14 clinical stage 2B cancer patients. Lymphangiography added little to the reliability of clinical staging. We conclude that due to the relatively low accuracy of clinical staging, retroperitoneal lymphadenectomy remains the treatment of choice for clinical stages 1 and 2A nonseminomatous germ cell testicular tumors with normal serum markers after orchiectomy.


[Platelet structure and function: immunocytochemical localizations of membrane glycoprotein and alpha-granule protein]. The platelet membrane glycoproteins (GPs) are receptors or binding sites for adhesive proteins. GPIb and GPIIb/IIIa complex are major glycoproteins and have important roles, functionally. GPIb plays an essential role in primary hemostasis as receptor for the von Willebrand factor. The GPIIb/IIIa complex acts as the binding site for adhesive proteins on activated platelets and, as such, is essential for platelet aggregation. On the other hand, four adhesive proteins (fibrinogen, fibronectin, thrombospondin and von Willebrand factor), which are present not only in plasma but also in alpha-granules, mediate or modulate the platelet adhesive response. The interaction between these adhesive proteins and platelet membrane GPs are essential for platelet adhesion and aggregation. The present report will focus on the localization of GPIb and GPIIb/IIIa on the platelet surface and that of adhesive proteins in alpha-granules in both resting and activated human platelets.


[The regulatory mechanism of free Ca2+ concentration in activated platelets]. The change in intracellular Ca2+ concentration ([Ca2+]i) following platelet stimulation results from mobilization, influx and restoration of Ca2+. To determine whether inositol 1,4,5 trisphosphate (IP3) is involved in Ca2+ influx, the relationship between IP3 formation (IP3) and Ca2+ influx ( delta [Ca2+]i) was investigated in platelets stimulated wtih various agonists (thrombin, ADP, PAF, STA2, etc). The ratio of IP3 to delta [Ca2+]i varied among the agonists, although delta [Ca2+]i was increased, depending on the amount of agonist. Furthermore, in spite of the similar delta [Ca2+]i, IP3 was smaller at 20 degrees C compared with that at 37 degrees C in thrombin-stimulated platelets. These results indicate that Ca2+ influx in platelets might be regulated by receptor-operated Ca2+ channel rather than by an IP3 mediated mechanism. As for Ca2+ restoration, calpain was demonstrated to play a role through Ca(2+)-ATPase activation by limited proteolysis.


[Endothelial cells and vascular hemostasis]. Procoagulant, anticoagulant, and fibrinolytic activities are associated with endothelial cells and involve the production, secretion, and receptor mediated binding of proteins involved in these processes. The procoagulant aspect of endothelial cells function involves the production and release of von Willebrand Factor(vWF), the production of tissue factor, and the presence of Factor IX/IXa receptors on the cell surface. Secretion of vWf will promote the initial steps in thrombus formation by supporting platelet-platelet interaction and platelet-subendothelial matrix adhesion. Tissue factor which is undetectable in resting cells appears after exposure to various cytokines and initiates factor VIIa activation of factors IX and X. Receptors of Factor IX/IXa are also present and mediate the assembly of the prothrombinase complex on the endothelial cell surface. The anticoagulant pathway involves the cell surface protein thrombomodulin, protein C and its cofactor protein S. Thrombomodulin binds thrombin which activates protein C which in the presence of protein S cleaves and inactivates Factors V and VIII. Inactivation of these two coagulation cofactors halts the coagulation. Finally, endothelial cells also play a pivotal role in the fibrinolytic system. Production and regulated secretion of tissue plasminogen activator creates a profibrinolytic state in the endothelial cell environment. In addition, receptors for plasminogen and urokinase are also present, constituting a cell surface mediated fibrinolytic pathway. Plasminogen activator inhibitor type I, the primary inhibitor of tPA, is also produced by endothelial cells. Thus endothelial cells can promote and inhibit fibrinolysis, depending on the prevailing environmental conditions.


[Effect of cyclic AMP and phorbol ester on PAI-2 synthesis in a leukemic cell line PL-21 and on u-PA secretion in a pre-B cell lymphoma cell line RC-K8]. We investigated the effect of phorbol myristate acetate (PMA), dexamethasone (Dex) and reagents which raise intracellular cyclic AMP, on the production of plasminogen activator inhibitor type-2 (PAI-2) in human promyelocytic leukemia cell line, PL-21 and on the production of urinary type plasminogen activator (u-PA) in human pre-B cell lymphoma cell line, RC-K8. Cells were cultured in fetal bovine serum free RPMI-1640 containing the test-reagents for 48 hours. PAI-2 and u-PA antigens were measured by ELISA kits. PMA, an activator of protein kinase C (PKC), markedly increased both PAI-2 and u-PA production in each cell line. On the other hand, cAMP increased PAI-2 production in PL-21 cells, but decreased u-PA synthesis in RC-K8 cells. Similar to cAMP, Dex also increased PAI-2 production but decreased u-PA production in RC-K8 cells. Moreover, PMA and cAMP synergistically increased the PAI-2 production. This was verified by Western blot, using a monoclonal antibody against the PAI-2. These two cell lines are, therefore, useful for clarifying the role of A kinase and C kinase on PAI-2 and u-PA synthesis in human hemopoietic cells.


[New oral drugs for the treatment of congestive heart failure]. The crucial issues in the management of congestive heart failure (CHF) are improvement of depressed myocardial contractility and reduction of excessive load. For this purpose, positive inotropic agents and vasodilators have been developed as new oral drugs. The former include Denopamine which possesses beta 1 stimulating effect, Xamoterol which is a unique agent acting as a beta 1-partial agonist, and Ibopamine, Docarpamine and Phosphodiesterase Inhibitors which possess both inotropic and vasodilating effects and are called "Inodilators". The latter include Angiotensin Converting Enzyme Inhibitors. In addition, new vasodilators, such as, Vasopressin Antagonist have also been developed. However, careful long-term clinical trials are required with regard to the efficacy and adverse effects before these agents are widely used with safety in the management of CHF.


[Effect of captopril on myocardial ischemia, intracardiac hemodynamics and regional left-ventricular contractility in patients with stenocardia]. Technetium-99m pertechnetate equilibrium ventriculography was used to evaluate the effects of captopril in a single dose of 50 mg on the changes in ST segment depression during the identical bicycle ergometer exercise, as well as on systemic and regional hemodynamic parameters in 10 patients (mean age 52 years) with Functional Classes II-III exercise-induced angina pectoris. During exercise performed 45 and 90 minutes after captopril, ST segment depression decreased by 30 +/- 0 (p less than 0.05), and 32 +/- 10% (p less than 0.02), respectively as compared to baseline ST segment displacement. Following 90 minutes after the drug administration, end-systolic volume reduced both at rest and during exercise, resting stroke volume increased from 71 +/- 4 to 76 +/- 4 ml (p less than 0.01), whereas exercise stroke volume rose from 69 +/- 3 to 74 +/- 3 ml (p less than 0.03); with the drug, ejection fraction showed a 5% increase (p less than 0.02) at rest and a 4% increase (p less than 0.02) on exercise. Thus, captopril had a beneficial effect on the hemodynamics and reduced myocardial ischemia in patients with exercise induced angina.


[Lectin-induced chemiluminescence of peripheral blood neutrophils in patients with ischemic heart disease before and after blood irradiation with helium-neon laser]. The blood taken from 35 patients with coronary heart disease and 30 healthy donors was irradiated with He-Ne laser, which resulted in a decrease in its count of segmented neutrophilic granulocytes. Lectins bound to various carbohydrate determinants onto the neutrophil surface were shown to affect changes occurring after luminol-depended chemiluminescence irradiation in patients and healthy persons in different ways. The patients' neutrophils contained lower levels of radiommunologically detectable leukotriene B4. Thromboxane B2 levels also dropped following the irradiation. The laser irradiation induced elimination of some less resistant cells from blood flow, i.e. "rejuvenation" of a cell population of neutrophilic granulocytes. The remaining cells differed in the composition and reactivity of surface lectin receptors and in the content of biologically active substances, which is likely to play the key role in the mechanism responsible for the therapeutical effect of He-Ne laser.


Atrial natriuretic factor: its (patho)physiological significance in humans. The first human studies using relatively high-doses of ANF revealed similar effects as observed in the preceding animal reports, including effects on systemic vasculature (blood pressure fall, decrease in intravascular volume), renal vasculature (rise in GFR, fall in renal blood flow), renal electrolyte excretion (rises in many electrolytes), and changes in release of a number of different hormones. Whether all these changes are the result of direct ANF effects or secondary to a (single) primary event of the hormone remains to be determined. Certainly, it has been proven that more physiological doses of ANF fail to induce short-term changes in many of these parameters leaving only a rise in hematocrit, natriuresis and an inhibition of the RAAS as important detectable ANF effects in humans. This leads us to hypothesize that ANF is a "natriuretic" hormone with physiological significance. The primary function in humans is to regulate sodium homeostasis in response to changes in intravascular volume (cardiac atrial stretch). Induction of excess renal sodium excretion and extracellular volume shift appear to be the effector mechanisms. The exact mechanism of the natriuresis in humans still needs to be resolved. It appears however, that possibly a small rise in GFR, a reduction in proximal and distal tubular sodium reabsorption, as well as an ensuing medullary washout, are of importance. The pathophysiological role of ANF in human disease is unclear. One may find elevated plasma irANF levels and/or decreased responses to exogenous ANF in some disease states. Whether these findings are secondary to the disease state rather than the cause of the disease remains to be resolved. Therapeutic applications for ANF, or drugs that intervene in its production or receptor-binding, seem to be multiple. Most important could be the antihypertensive effect, although areas such as congestive heart failure, renal failure, liver cirrhosis and the nephrotic syndrome cannot be excluded. Although the data that have been gathered to date allowed us to draw some careful conclusions as to the (patho)physiological role of ANF, the exact place of ANF in sodium homeostatic control must still be better defined. To achieve this, we will need more carefully designed low-dose ANF infusion, as well as ANF-breakdown inhibitor studies. Even more promising, however, is the potential area of studies open to us when ANF-receptor (ant)agonists become available for human use.


Fifteen-S-hydroxyeicosatetraenoic acid (15-S-HETE) specifically antagonizes the chemotactic action and glomerular synthesis of leukotriene B4 in the rat. In models of experimental glomerulonephritis, there is temporal concordance between the shift in the glomerular cellular infiltrate from neutrophils (PMN) to macrophages/monocytes and the suppression of glomerular leukotriene B4 (LTB4) generation. Since macrophages are a rich source of 15-lipoxygenase (15-LO) products, we investigated whether the principal product of arachidonate 15-lipoxygenation, 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), was capable of antagonizing the proinflammatory actions of LTB4 in the rat. PMN exhibited chemotaxis to LTB4 in a dose dependent manner with an LC50 of 10(-8) M. When rat neutrophils were pre-treated with 15-S-HETE, chemotaxis to LTB4 was inhibited in a dose dependent manner (maximal at 30 microM 15-S-HETE) but, the same concentration did not inhibit chemotaxis to n-formyl-1-methionyl-1-phenylalanine (FMP). 12-S-HETE (30 microM) did not inhibit chemotaxis to LTB4. Glomeruli from rats injected with nephrotoxic serum three hours earlier generated increased levels of LTB4; prior exposure of such glomeruli to 15-S-HETE totally normalized LTB4 production. The glomerular production of 15-S-HETE and LTB4 was also determined 3 hours, 72 hours and 2 weeks after administration of nephrotoxic serum. Whereas there was an early, short-lived, burst of LTB4 followed by a return to baseline levels, the production of 15-S-HETE increased steadily over the two week period and was present in amounts fivefold greater than LTB4. Thus, these studies assign a role for locally generated 15-LO derivatives in arresting LTB4-promoted PMN infiltration and suppressing LTB4 synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)


Quantification and modulation of thrombomodulin activity in isolated rat and human glomeruli. Thrombomodulin (TM), the endothelial cell surface receptor for thrombin-mediated activation of protein C and of its anticoagulant system, is involved in maintaining vascular nonthrombogenicity, and depressed TM activity may induce intravascular fibrin formation. TM antigen was previously found by immunohistochemical methods in rabbit glomeruli. We therefore attempted to identify the corresponding TM activity in isolated detergent-solubilized rat and human glomeruli. Like purified lung TM, rat glomeruli extracts accelerated the hydrolysis by activated protein C of the chromogenic substrate S-2238 in the presence of 10 nM thrombin, as determined by spectrophotometry. One mg glomerular protein promoted the formation of 681 +/- 115 nmol activated protein C, the equivalent of the amount generated by 845 ng of purified rabbit TM. TM activity correlated with the protein content of the glomerular extracts (r = 0.94). These extracts prolonged rat plasma activated partial thromboplastin time. Incubation of glomeruli with tumor necrosis factor-alpha (TNF) or E. coli lipopolysaccharide depressed their TM-like activity in a dose and time dependent manner. Incubation with TNF suppressed their anticoagulant activity. In human glomeruli, TM activity was also found at a level which corresponded to their TM antigen content, and was determined by ELISA with mouse monoclonal antibody. These results indicate that measurement of glomerular TM activity might help to clarify the mechanisms of intraglomerular fibrin deposition in renal diseases.


Carbamylation-induced alterations in low-density lipoprotein metabolism. Low-density lipoprotein was derived from carbamyl (carbamyl-LDL) by incubating LDL in potassium cyanate (KCNO). The proportion of free amino groups in the carbamyl-LDL was negatively correlated (r = -0.95) with the time of incubation in potassium cyanate (ranged from 5 to 360 min). The carbamylation did not change the chemical composition or the flotation characteristics of the LDL particles. However, the electrophoretic mobility of carbamyl-LDL was distinctly increased with the extent of carbamylation. The carbamyl-LDL had substantially decreased binding to the LDL apoB/E receptors of the bovine adrenocortical membranes when compared to the control-LDL. The reduced binding was already observed when only 9% of the free amino groups were derived from carbamyl. A minor carbamylation of LDL (less than 20% of the free amino groups) decreased the in vivo clearance of LDL from rabbit plasma. However, when more than 20% of the free amino groups were derived the carbamyl-LDL had accelerated clearance compared to the control-LDL. LDL isolated from uremic patients was cleared in rabbits at a slower rate than LDL isolated from a control subject. Providing that carbamylation of LDL could also occur in vivo resulting in similar alterations of the LDL binding to the LDL B/E receptors, as observed in the present study, the uremia-related accelerated atherosclerosis could have one additional mechanistic explanation.


In vitro formation and expansion of cysts derived from human renal cortex epithelial cells. Acquired renal cysts derive from terminally differentiated tubular epithelium in adults as a consequence of increased epithelial cell proliferation, fluid accumulation and extracellular matrix remodelling. To understand better how human epithelial cysts may be initiated and progressively expand, cells from primary cultures of normal human adult renal cortex were dispersed in polymerized type I collagen. The transparent matrix permitted repeated observation by light microscopy of cyst formation from individual renal cells. The cyst cells reacted strongly with distal nephron histochemical markers (cytokeratin antibodies AE1/AE3, epithelial membrane antigen, and Arachis hypogaea lectin) but inconsistently or not at all to markers of proximal tubules (Tetragonolobus purpureas lectin and Phaseolus vulgaris erthroagglutinin lectin). The number of spherical, fluid-filled epithelial cysts that developed in a standardized microscope field quantified cyst initiation. Cyst progression was determined from the increase in the diameter (surface area) of cysts and represents a hyperplastic event. EGF or TGF alpha, were required in serum-free defined medium to cause cysts to develop from individual epithelial cells dispersed in the matrix; insulin was required as a co-factor. The EC50 for EGF was approximately 0.1 ng/ml, and for insulin 1 microgram/ml. Early cultures of normal cortex formed cysts more efficiently when dispersed in collagen matrix than cells passaged several times before suspension in the gel. Agonists of adenylate cyclase (PGE1, AVP, VIP, PTH, forskolin, cholera toxin), methylisobutylxanthine, and 8-Br-cAMP, though incapable of causing cyst formation alone in defined medium, enhanced cyst initiation and progression in the presence of EGF and insulin. Angiotensin II, TNF alpha, beta-estradiol, and pertussis toxin had no effect in the absence or presence of EGF and insulin. Pertussis toxin inhibited cyst initiation and expansion caused by EGF and forskolin but potentiated cyst initiation and expansion caused by EGF and PGE1. Cyst formation and expansion were inhibited by TGF beta 1 and 2-chloroadenosine. Polarized monolayers of human renal cortical cells grown on permeable membranes were used to independently quantify the effects of agonists on the net secretion of solute and water from the basolateral to the apical surface of the cells. PGE1, forskolin, and 8-Br-cAMP stimulated net fluid secretion that was sustained for several days; EGF enhanced forskolin-stimulated fluid secretion. We conclude that the formation and expansion of in vitro cysts derived from solitary human cortex cells depends on the coordinated interplay between cellular proliferation and fluid secretion.(ABSTRACT TRUNCATED AT 400 WORDS)


[Endocrine stress response in cataract operations with local anesthesia]. This study was undertaken to investigate the endocrine stress response during cataract surgery in local anaesthesia (LA) with or without additional sedation with midazolam (Dormicum). 20 patients for cataract surgery in LA were randomly allocated to the midazolam-group (before injection of LA, sedation with single doses of 1 mg midazolam until the patient was sleeping, but awakeable), and to the control-group without sedation. Premedication and LA were standardized. The investigation was performed at 7 measuring points starting at the arrival in the preparation room up to 30 min after surgery. Adrenaline in plasma was significantly lower in the midazolam-group. Intraoperatively, adrenaline increased in the control-group and decreased in the midazolam-group. In time course, noradrenaline in plasma decreased in the midazolam-group, in contrast to the control-group. With respect to ADH, ACTH and cortisol, no significant differences were found in group levels or time course, and concentrations remained within normal range. In contrast to the control-group, mean arterial pressure decreased in the midazolam-group during the course of time. There were no differences in heart rate or arterial oxygen saturation between the two groups. Local anaesthesia and premedication were sufficient to prevent psychic and surgical stressors. The slight sympathoadrenergic response in the control-group was significantly reduced by small doses of midazolam. Thus, a careful supplementation of local anaesthesia with midazolam appears advantageous for patients with cardiovascular disorders.


[Treatment of eye tumors by accelerated proton beams. 7 years experience]. The therapeutic results of 1070 cases of intraocular tumors treated with an accelerated proton beam at the University Eye Clinic of Lausanne and at the Paul Scherrer Institute of Villigen are analysed. This total is composed of 981 cases of uveal melanomas treated by proton beam as first treatment modality, 42 cases of recurrent choroidal melanomas, 16 cases of conjunctival melanomas, 8 cases of choroidal hemangiomas, 18 cases of uveal metastases and of 5 other ocular tumors. The mortality rate at 5 years for tumors treated with an accelerated proton beam as first treatment modality was 13%, and the mortality rate of recurrent melanomas was 23%. The risk factor for life expectancy analysed with the Cox model pointed out that the largest tumor diameter, the width of the tumor, the presence of an extrocular extension and the age of the patient were the most significant parameters. The conclusions of this study are compared with those found in available literature.


[Treatment of exterior extension of choroid melanomas by accelerated proton beams]. Among the 934 patients with a choroidal melanoma treated in first intention with an accelerated proton beam since 1984, 41 (4.4%) presented an extrascleral extension, of which 11 were located anteriorly and 30 posteriorly. For these 30 cases, the diagnosis was made by B ultrasonography only when the volume of the extension was important, between 20 and 2280 mm3. The Cox model multi-variant analysis was used to study the statistically significant parameters in the development of an extrascleral extension. It appears that the maximal diameter of the intraocular tumor (p = 0.0012), the localisation of the anterior margin (p = 0.0304) and the age of the patients (p = 0.0097) are statistically meaningful. The 4 years' survival, studied with the Kaplan-Meier curves, is estimated at 60% for the patients with extrascleral extension and at 85% for the patients without. These results are similar to those of literature, whatever treatment is chosen (enucleation and radiotherapy, exenteration).


[Evaluation of the method of DNA amplification (PCR, polymerase chain reaction) for diagnosis of superficial ocular herpes]. We evaluated the polymerase chain reaction (PCR) as a diagnostic method in superficial corneoconjunctival herpes and compared it to viral culture. A total of 38 patients were included and divided into 3 groups according to the clinical aspect. Fourteen patients that had a typical clinical aspect for herpes served as reference to evaluate the method. Nine patients had atypical lesions (herpes-suspects) and 15 patients (13 foreign corneal bodies and 2 adenovirus keratoconjunctivitis) made up the control group. All culture positive cases were also positive with the PCR method (in total 10 cases). In the group of herpes-suspects, one case had both a positive viral culture and a positive PCR; four patients that responded well to antiviral therapy had a negative culture and a positive PCR and four patients were negative with both methods. In the control group two cases of foreign bodies were false-positive. As culture was not performed for these cases it is impossible to known if it was a contamination during the PCR procedure or if concomittant viral shedding occurred. Compared to viral culture, a sensitivity of 100% and a specificity of 87.5% was calculated. PCR seems to be a very sensitive diagnostic method having an acceptable specificity for the diagnosis of superficial ocular herpes simplex disease that proved useful in atypical cases.


[Retinal and vitreal metastasis of a small cell bronchus cancer]. The rare form of retinal metastasis with infiltration of the vitreous is presented in terms of a 47-year-old female patient suffering from an oat cell bronchogenic carcinoma with remote metastasis. Clinical appearance, differential diagnosis, diagnostic evaluation, histology and evolution are discussed.


[Morphologic aspects of therapy-resistant cytomegalovirus retinitis]. Intravenous ganciclovir treatment was performed in eight male AIDS patients with primary unilateral CMV-retinitis. Three patients developed slowly progressive CMV-retinitis in the fellow eye despite adequate dose of ganciclovir. These different CMV-manifestations are shown in a sequence of fundus pictures. Three types of CMV-lesions were observed in connection with this study. Untreated central lesions showed the aspect of crumbled cheese and ketchup. Untreated lesions in the peripherie were yellowish-white, granular, "dry" and showed in most cases no haemorrhages. Lesions appearing during treatment showed initially "dry" white opaque subretinal areas, turning later on to the typical aspect of untreated lesions. The progression could not be stopped by highdose ganciclovir i.v. and thus bilateral blindness resulted after 12 to 22 months. The level of CD4-lymphocytes in the blood was diminished in all patients, but much more in patients with progressive disease.


The effect of in vitro aging on human lung fibroblast beta-adrenergic receptor density, coupling and response. Age-related changes in regulation of receptor response have been observed in several tissues and include regulation of beta-adrenergic receptor (beta-receptor) responses. The role of cellular aging in age-related changes in receptor response is not clear. We have examined the effect of aging in vitro on human fibroblast beta-receptor function. MRC-5 (embryonic lung) fibroblasts were aged by replication to produce cells of early, middle and late stages corresponding to the following cumulative population doublings: 15-20, 35-45 and greater than 50, respectively. Fibroblast membrane beta-receptor responses to isoproterenol (ISO, 0.1 mM) did not differ between the three stages. Adenylate cyclase responses to prostaglandin E1 (PGE1, 1 microM), guanosine triphosphate (GTP, 0.1 mM) and 5'-guanylimidodiphosphate (Gpp(NH)p, 0.1 mM) were also similar between the stages. Beta-receptor density (Bmax) was unaffected by in vitro aging. Beta-receptor agonist affinity, an indication of the capacity for beta-receptor coupling to the nucleotide binding protein (NS), was also unaffected by cell aging. These findings suggest that cellular aging in fibroblasts alone is not accompanied by changes in beta-receptor function.


Facial nerve regeneration through semipermeable chambers in the rabbit. Peripheral neural regeneration, over a 10-mm transectional gap, was determined in 70 rabbit buccal divisions of the facial nerve using two entubational systems (semipermeable and impermeable silicone chambers) prefilled with three natural occurring media (serum, blood, and saline) during a 5-week period. The number of myelinated axonal regenerates at the midchamber and at 2 mm in the distal transected neural stump were counted in each group and compared to pooled myelinated axonal counts in 9 normal rabbit buccal divisions of the facial nerve. Semipermeable porous chambers had an overall greater regeneration success rate (75% vs. 42.8%) and regained, on the average, a higher number of myelinated axons (51.4% vs. 26.1%) than silicone chamber regenerates. Semipermeable chambers prefilled with serum or blood had significantly higher regeneration success rates, myelinated axonal counts, and percentages of neural innervation of the distal transected neural stump. Both entubational systems produced similar axonal counts with intraluminal saline. The highest overall success rate (93.7%) and average number of myelinated axons per chamber (3072) were achieved in semipermeable chambers prefilled with serum. The greatest variability in myelinated axonal counts (0 to 3266 axons) and percentage of distal stump innervation (5.5% to 98.1%) was seen in silicone chambers filled with saline. The percentage of myelinated axons from the midchamber that innervated the distal stump was greater in semipermeable chambers with blood (73%) and serum (54%) than in silicone saline chambers (43%). On the average, the distal stumps from semipermeable chambers filled with serum (47%) and blood (33.5%) regained a higher percentage of normal myelinated axonal counts than silicone-saline chambers (12.5%). These results suggest that both the construction of entubational chamber and the intraluminal medium can have significant influence on neurite regeneration. Semipermeable chambers prefilled with serum have a strong neurite-promoting potential in peripheral neural regeneration of rabbit facial nerves.


Laser Raman spectrum of calcified human aorta. Raman spectra of highly calcified areas of human aorta were obtained using long wavelength excitation at 740 nm to minimize background fluorescence interference. Raman spectra resembling that of hydroxyapatite were obtained for segments of highly calcified aorta that were thawed frozen samples, from which the surface layer had been removed, or were dried. These are the first reported spectra obtained in air or in normal saline solution showing the hydroxyapatite peak in calcified plaque for samples which were untreated except for freezing, which is encouraging for the application of Raman spectroscopy as a method of detection of plaque during laser angioplasty. Spectra obtained from calcified aorta using excitation at 514.5 nm did not show evidence of the hydroxyapatite band because of the severe interference by background fluorescence generated at this wavelength.


Mechanism of inhibitory action of ketone bodies on the production of reactive oxygen intermediates (ROIS) by polymorphonuclear leukocytes. We determined an effect of acetoacetic acid (AcAc) and 3-hydroxybutyrate (3-OHB) on the production of reactive oxygen intermediates (ROIs) in polymorphonuclear leukocytes from healthy volunteers. Both AcAc and 3-OHB inhibited the luminol-dependent chemiluminescence (LDCL) activities assessed with initial slope and the inhibition rates were about 42%, 44% respectively by AcAc and 3-OHB when the leukocytes were preincubated with 10 mM AcAc or 3-OHB for 60 minutes. The LDCL activity was reduced by 16% and 42% following the addition of 1mM and 10 mM AcAc. The similar reduction of the LDCL activity was observed in the addition of 3-OHB. Either 3-OHB or AcAc failed to show a significant reduction of myeloperoxidase (MPO) activity. However, both 3-OHB and AcAc dose-dependently inhibited superoxide anion (O2-) production, measured by using cytochrome c. These data provided evidence that both 3-OHB and AcAc suppress neutrophil oxidative metabolism with respect with O2- production.


Delta 9-tetrahydrocannabinol suppresses concanavalin A induced increase in cytoplasmic free calcium in mouse thymocytes. It has been shown that delta-9-tetrahydrocannabinol (THC) suppresses thymocyte, lymph node, and splenic lymphocyte proliferation in response to a mitogenic stimulus. It has also been reported that increases occur in the cytosolic free calcium concentration (Ca2+) in mitogen treated lymphocytes. In an attempt to understand a portion of the molecular basis of the THC induced suppression of lymphocyte proliferation, we have examined the effects of THC on the Concanavalin A (Con A) induced cytosolic free Ca2+ mobilization in mouse thymocytes measured by fluorescent Ca2+ probes and spectrofluorometry. The results show that a 10 minute pretreatment with THC suppresses the normal rise in intracellular free Ca2+ in response to Con A. A THC concentration of 4 micrograms/ml (13 microM) was suppressive and the drug vehicle, DMSO, had no effect. In addition, we found that THC pretreatment did not inhibit the binding of FITC labeled Con A to the thymocytes suggesting that the drug did not interfere with lectin binding to the cell surface. To further define the nature of the Ca2+ response affected by THC, mouse thymocytes containing fura-2 were exposed to Con A either in the presence or absence of Ca(2+)-containing medium. It was observed that THC abrogated both intracellular release (measured in Ca(2+)-free medium) as well as extracellular Ca2+ influx. These results suggest that a portion of the proliferation defect in THC treated lymphocytes may be related to a drug induced inhibition of Ca2+ mobilization that normally occurs following mitogen treatment.


Tumoricidal activity of Kupffer cells augmented by anticancer drugs. The effect of Mitomycin-C (MMC) and Adriamycin (ADM) on the antitumor-associated function of Kupffer cells was examined. MMC and ADM enhanced the production of superoxide by Kupffer cells in cultures at low concentrations likely to occur in clinical use. The expression of interleukin-2 receptor, Ia antigen and asialoGM1 antigen, measured by flowcytometry, was increased by contact with MMC. Growth inhibition of AH130, rat ascites hepatoma, and P815, murine mastocytoma, by Kupffer cells treated with anticancer drugs was greater than that by Kupffer cells alone or anticancer agent alone. These results show that MMC and ADM activate Kupffer cells, leading to synergistic antitumor activity. The results suggest that some anticancer agents act through immunological mechanisms as well as through direct antineoplastic activity.


The application of 13C NMR to the characterization of phospholipid metabolism in cells. 31P and 13C NMR spectroscopy of lipid extracts of T47D human breast cancer spheroids and the use of 13C-labeled lipid precursors [3-13C]serine,[1,2-13C]ethanolamine, and [1,2-13C]choline enabled us to determine the rate of 13C incorporation into the major phospholipids and to show that the synthesis of phosphatidylethanolamine in T47D cells is via both the CDP-ethanolamine pathway and serine decarboxylation, with the extent of each depending on the concentration of ethanolamine in the medium. In the presence of low ethanolamine (3.4 microM), both pathways contribute in equal proportions, while in the presence of high ethanolamine, the CDP-ethanolamine pathway predominates.


A putative role for colloidal silicates in primitive evolution deduced in part from their relevance to modern pathological afflictions. Biogenic and bioactive silicas have structural subtleties that are only now beginning to be appreciated. Their ability to interact with particular biological systems is related to these structures. Some of these interactions result in pathological effects. It is suggested that such interactions mirror events occurring in a prebiotic environment.


Psoriasis: current concepts and new approaches to therapy. Psoriasis is a common disorder characterized by marked increases in keratinocyte proliferation, abnormal patterns of keratinocyte differentiation, prominent alterations in dermal capillary vasculature and the presence of dermal and epidermal T cells, monocytes/macrophages and neutrophils. It is now known that psoriasis can occur due to abnormalities in essential fatty acid metabolism, lymphokine secretion, free radical generation, lipid peroxidation and eicosanoid metabolism. It is possible to suppress almost completely psoriatic lesions by judicious use of methotrexate, cyclosporine A, and eicosapentaenoic acid. Our studies have shown that in patients with psoriasis there is an increase in the generation of free radicals with an alteration in essential fatty acid metabolism and that side-effects of anti-cancer drugs can be blocked by essential fatty acids in vivo. Thus, essential fatty acid metabolism seems to play a crucial role both in the pathogenesis and treatment of psoriasis.


[Effect of metabolic control on the renal effects of human atrial natriuretic peptide-(95-126) (urodilatin) in normotensive patients with type I diabetes mellitus]. To examine the impact of metabolic control on renal responses to human atrial natriuretic peptide (hANP) in type 1 diabetes mellitus, 13 patients with HbA1 less than 8.5%, nine patients with HbA1 greater than 8.5% and ten healthy volunteers were studied. According to a randomized, single-blind trial design, 0.5 and 2.0 micrograms/kg hANP-(95-126) (Urodilatin) (Bissendorf Peptide, Hannover) or placebo were given as iv bolus injections at 90-minute intervals. Patients with HbA1 greater than 8.5% differed from those with HbA1 less than 8.5% in longer diabetes duration, more prevalent retinopathy and neuropathy and increased somatomedin C levels and urinary albumin excretion (p less than 0.05). In response to hANP, patients with HbA1 greater than 8.5% had decreased responses of urinary volume and sodium excretion in comparison to patients with HbA1 less than 8.5% (p less than 0.05) in whom renal responses to hANP did not differ from controls. Despite similar hANP levels, hANP-stimulated urinary cGMP excretion in patients was higher than in controls (p less than 0.01). Impaired renal responses to hANP in diabetes patients with insufficient glycemic control apparently contribute to the mechanisms of diabetic sodium retention. Near-normoglycemia may prevent this phenomenon which is intimately involved into the pathogenesis of diabetic nephropathy.


[Morphometrical analysis of non-cancerous liver tissue with special reference to clinicopathological features of hepatocellular carcinoma]. This study was undertaken to investigate the relationship between morphometrical characteristics of noncancerous liver tissues and clinicopathological features of hepatocellular carcinoma (HCC) in 89 cases which underwent either hepatectomy (n = 56) or autopsy (n = 33). Using Automatic Image Analyzer (IBAS-2), we determined interstitial ratio (IR) of the non-cancerous liver tissues as a morphological parameter in all cases. In addition, mean values of area (MA), maximal diameter, and shape factor of pseudolobules were determined in the cirrhotic patients. IR in cases with main tumors smaller than 3 cm was higher than that in cases with tumors larger than 3 cm (23.5 +/- 6.7% vs 18.5 +/- 8.9% mean +/- SD: p less than 0.01), and IR in cases with histologically proven intrahepatic metastases (im positive) was also higher as compared to im negative cases (17.5 +/- 9.0% vs 21.3 +/- 8.0%; p less than 0.05). Among the other parameters determined in the cirrhotics, MA was higher in cases with tumors larger than 3 cm than in cases with smaller tumors (2.54 +/- 1.87 mm2 vs 1.78 +/- 1.01 mm2; p less than 0.05), and MA was higher in im positive cases as compared to im negatives (2.67 +/- 1.91 mm2 vs 1.67 +/- 0.96 mm2; p less than 0.01). These data indicated that non-cancerous liver tissue has a close relation not only to the carcinogenesis but to subsequent tumor growth and progression of HCC.


[Type I, type III, and type IV collagenolytic enzyme activity in lung cancer tissue]. To investigate the changes of collagen metabolism in patients with lung cancer, activities of types I, III, and IV collagenolytic enzymes in specimens obtained from lung cancer tissue and pathomorphological changes of the corresponding specimens were analyzed. And the comparison of the biochemical and morphological changes were evaluated. Nine resected pulmonary carcinomas were used and 5 samples were obtained from each carcinoma tissue. In each sample, types I, III, and IV enzyme activities were measured and its histopathological examination was done. Enzyme activities of 5 samples from the same carcinoma tissue were considerably different in all subtype of collagenolytic enzymes. The relationship between each collagenolytic enzyme activity of these samples and their histopathological findings are as follows: 1) No relationship is found between the amount of carcinoma cells in cancer tissue and enzyme activity in any subtype, 2) increase in the fibrosis in cancer tissue is accompanied by decrease in type I collagenolytic enzyme activity, 3) the substantial increase in inflammatory cells coexisting with carcinoma is accompanied by increase in types I and III collagenolytic enzyme activities. Moreover, type III collagenolytic enzyme activity elevates significantly when inflammatory cells infiltrate among carcinoma cells.


Mutant constructs of the beta-adrenergic receptor that are uncoupled from adenylyl cyclase retain functional activation of Na-H exchange. beta-Adrenergic receptor (beta AR) agonists modulate a number of intracellular effectors; for example, they stimulate adenylyl cyclase and Ca2+ channels, inhibit Na+ channels and Mg2+ efflux, and activate Na-H exchange. Regulation of adenylyl cyclase, Ca2+, Na+, and Mg2+ by the beta AR is mediated through receptor coupling to the GTP-binding protein Gs. We have previously determined, however, that beta AR stimulation of Na-H exchange occurs independently of receptor coupling to Gs. In the present study, we analyzed mutant beta ARs containing deletions of amino acid residues within the third cytoplasmic domain, to determine whether there is a structural basis for the ability of the beta AR to couple divergently to the Gs-dependent stimulation of adenylyl cyclase and the Gs-independent activation of Na-H exchange. Receptor constructs with deletions of residues 222-229 and 258-270, which were previously shown to be defective in coupling to Gs and adenylyl cyclase, retained an isoproterenol-induced activation of Na-H exchange that was similar in time course and magnitude to that observed with the wild-type beta AR. These results confirm our previous findings that the beta AR activates Na-H exchange independently of Gs, and they further suggest that distinct molecular determinants of the receptor divergently stimulate adenylyl cyclase and Na-H exchange.


Involvement of specific hydrophobic, but not hydrophilic, amino acids in the third intracellular loop of the beta-adrenergic receptor in the activation of Gs. Mutagenesis and biochemical analysis have indicated that amino acid residues at the amino terminus of the third intracellular loops of guanine nucleotide-binding protein (G protein)-coupled receptors are important in mediating the coupling of the receptors to G proteins. Because the primary sequence of this region is not conserved among all receptors that couple to the same G protein, it has been suggested that some other physicochemical property of this domain may determine G protein activation. To determine the relative contributions of charge distribution and amino acid side chain interactions within this domain of the beta-adrenergic receptor (beta AR) to the activation of the G protein Gs, point mutations were introduced into this region of the beta AR. Replacement of all four of the basic amino acid residues within this region (amino acids 222-236) with serine residues had a negligible effect on the ability of the beta AR to activate Gs. In contrast, replacement of the hydrophobic amino acids within this same region with leucine residues resulted in a mutant receptor that was poorly coupled to Gs. These results suggest that specific hydrophobic interactions within this region of the receptor may play a more significant role than ionic or hydrophilic interactions in mediating G protein activation.


Activation of protein kinase C selectively inhibits the gamma-aminobutyric acidA receptor: role of desensitization. The effects of protein kinase C (PKC) activators on gamma-aminobutyric acidA (GABAA) receptor function were studied by two-electrode voltage-clamp in Xenopus oocytes expressing brain mRNA or subunit cDNAs and in isolated mouse brain cerebellar membrane vesicles (microsacs), using 36Cl- uptake. Both oocytes and microsacs showed transient (desensitizing) and sustained (nondesensitizing) GABAA receptor responses. In oocytes expressing brain mRNA, the PKC activator phorbol myristoyl acetate (PMA), but not the inactive analog phorbol 12-monomyristate, inhibited both transient and sustained GABA-gated chloride currents. The inhibition by PMA was concentration dependent, with an EC50 of approximately 5 nM, and resulted in a decrease in the efficacy, but not the potency, of GABA. Additionally, PMA inhibited GABA-gated chloride currents in oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs. The effect of PMA on recombinant receptors was significantly antagonized by PKC inhibitory peptide (PKCI). In the microsac preparation, the PKC activators (-)-7-octylindolactam V and PMA inhibited the sustained phase of 36Cl- flux without altering the transient phase. The action of PMA was blocked by kinase inhibitors and by depletion of Mg-ATP and was mimicked by protein phosphatase inhibitors. These results demonstrate that activation of PKC inhibits GABAA receptor function, and the results from the microsac experiments suggest that PKC-dependent phosphorylation preferentially inactivates a nondesensitized form or state of the receptor.


Functionally relevant gamma-aminobutyric acidA receptors: equivalence between receptor affinity (Kd) and potency (EC50)? Many neurotransmitter receptors bind agonists with high affinity (Kd in the nanomolar range), whereas micromolar concentrations of the same agonists are required to elicit a functional effect. We have identified low affinity agonist binding sites for the gamma-amino-butyric acidA (GABAA) receptor-chloride channel under conditions normally used in 36Cl- uptake assays (a measure of receptor function). The GABAA agonist [3H]muscimol bound to a population of receptors with a Kd (2 microM) similar to its EC50 value for 36Cl- uptake. Binding was inhibited by the GABA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol and by the GABA antagonist bicuculline methiodide. A reduction in the number of [3H]muscimol binding sites (Bmax) by a thiol-modifying reagent produced a corresponding decrease in the Emax for muscimol. The benzodiazepine diazepam enhanced the potency of muscimol in ion flux experiments but did not alter the affinity of [3H]muscimol binding sites. We propose that benzodiazepines enhance GABAergic function by increasing receptor-ion channel coupling, rather than by increasing GABAA receptor affinity. These studies suggest that the study of physiologically relevant (low affinity) binding sites is necessary when examining regulation of receptors by cellular processes, drugs, and disease.


NADPH and oxidized thioredoxin mediate redox interconversion of calf-liver and Escherichia coli thioredoxin reductase. The activity of pure calf-liver and Escherichia coli thioredoxin reductases decreased drastically in the presence of NADPH or NADH, while NADP+, NAD+ and oxidized E. coli thioredoxin activated both enzymes significantly, particularly the bacterial one. The loss of activity under reducing conditions was time-dependent, thus suggesting an inactivation process: in the presence of 0.24 mM NADPH the half-lives for the E. coli and calf-liver enzymes were 13.5 and 2 min, respectively. Oxidized E. coli thioredoxin fully protected both enzymes from inactivation, and also promoted their complete reactivation after only 30 min incubation at 30 degrees C. Lower but significant protection and reactivation was also observed with NADP+ and NAD+. EDTA protected thioredoxin reductase from NADPH inactivation to a great degree, thus indicating the participation of metals in the process; EGTA did not protect the enzyme from redox inactivation. Thioredoxin reductase was extensively inactivated by NADPH under aerobic and anaerobic conditions, thus excluding the participation of O2 or oxygen active species in redox inactivation. The loss of thioredoxin reductase activity promoted by NADPH was much faster and complete in the presence of NAD+ glycohydrolase, thus suggesting that inactivation was related to full reduction of the redox-active disulfide. Those results indicate that thioredoxin reductase activity can be modulated in bacteria and mammals by the redox status of NADP(H) and thioredoxin pools, in a similar way to glutathione reductase. This would considerably expand the regulatory potential of the thioredoxin-thioredoxin reductase system with the enzyme being self-regulated by its own substrate, a regulatory protein.


Three immunologically similar atrial natriuretic factor receptors. One of the atrial natriuretic factor (ANF) receptors is a 180 kDa protein (180 kDa mGC) which possesses the extraordinary characteristic of being bifunctional: it is both a receptor and a guanylate cyclase. In addition to the 180 kDa mGC, there exists another 120-130 kDa protein which is also bifunctional and a 120 kDa disulfide-linked dimeric cell surface protein that is an ANF receptor, but is not a part of guanylate cyclase. A fundamental question that needs to be resolved is: Are these three apparently biochemically distinct ANF receptors structurally similar? With the aid of affinity crosslinking techniques, a highly specific antibody to the 180 kDa mGC, and GTP-affinity techniques, we now demonstrate the presence of three immunologically similar proteins in rat adrenal gland and testes. These proteins migrate as 180 kDa, 130 kDa and 65 kDa under denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis and specifically bind ANF, raising one or more of the following possibilities about their relationships: 1) Degradation of 180 kDa to 130 kDa and 65 kDa occurs during purification; 2) 180 kDa bears a precursor-product relationship with 130 kDa and 65 kDa, suggesting the role of a protease in the processing procedure; 3) these proteins are a result of gene splicing; or 4) they are the products of three separate, but very closely related genes.


Intracellular cAMP determines the extent of degradation and not the synthesis of collagen by rat hepatocytes. Intracellular collagen degradation in normal rat hepatocytes was exponentially stimulated by db-cAMP (10-100 microM). The effect was manifested as a decrease (p less than 0.01) in net collagen production. The extent of degradation directly co-related with the intracellular cAMP levels, only up to a threshold concentration (16.2 +/- 1.3 p moles/10(6) cells) elicited by 100 microM of db-cAMP. Higher concentrations induced no further increment. Forskolin adenylate cyclase activator (10-50 microM), produced similar effects demonstrating cAMP dependence of the phenomenon. Both db-cAMP as well as Forskolin stimulated collagen degradation (p less than 0.05) in hepatocytes from rats administered CCL4. However, the extent of stimulation was significantly (p less than 0.01) less compared to that observed in normal hepatocytes. Our data demonstrates that elevated cAMP levels regulate net collagen content by signalling intracellular collagen degradation and not synthesis.


Cyclic nucleotide fluctuations during newt limb regeneration depend on injury and nerve action. Impressive cyclic GMP accumulations have been previously observed in stump tissues of amputated newt forelimbs. The aim of the present study was to measure cGMP and cAMP levels in innervated and in denervated stumps, in order to detect a possible nervous influence on the cyclic nucleotide accumulation of stump tissues. Denervation experiments were carried out in amputated newt forelimbs. Tissue sampling was made at several intervals (3, 9, 14 and 20 days) and cyclic nucleotide levels were measured by RIA. Cyclic nucleotide levels of innervated unamputated animals served as controls. Cyclic GMP and cyclic AMP contents of innervated regenerating stumps were compared with cGMP and cAMP contents of contralateral denervated stumps. Results showed clearly that cGMP accumulation was sharply reduced by denervation while cAMP levels were less modified. We suggest that dedifferentiating cells are the main source for the stump accumulated cGMP which increases under nervous control.


Voltage-dependent phosphorylation may recruit Ca2+ current facilitation in chromaffin cells. Bovine chromaffin cells have two components of whole-cell Ca2+ current: 'standard' Ca2+ currents that are activated by brief depolarizations, and 'facilitation' Ca2+ currents, which are normally quiescent but can be activated by large pre-depolarizations or by repetitive depolarizations to physiological potentials. The activation of protein kinase A can also stimulate Ca2+ current facilitation, indicating that phosphorylation can play a part in facilitation. Here we investigate the role of protein phosphorylation in the recruitment of facilitation Ca2+ currents by pre-pulses or repetitive depolarizations. We find that recruitment of facilitation by depolarization is a rapid first-order process which is suppressed by inhibitors of protein phosphorylation or by injection of phosphatase 2A into cells. Recruitment of facilitation Ca2+ current by voltage is normally reversible but phosphatase inhibitors render it irreversible. Our results indicate that recruitment of these Ca2+ currents by pre-pulses or repetitive depolarizations involves voltage-dependent phosphorylation of the facilitation Ca2+ channel or a closely associated regulatory protein. Voltage-dependent phosphorylation may therefore be a mechanism by which membrane potential can modulate ion channel activity.


Phosducin is a protein kinase A-regulated G-protein regulator. Signal transduction by G-protein-coupled receptors is regulated by various mechanisms acting at the receptor level; those studied most thoroughly are from the beta-adrenergic receptor/Gs/adenylyl cyclase system. We report here a regulatory mechanism occurring at the level of the G proteins themselves. A protein with M(r) 33,000 that inhibits Gs-GTPase activity was purified from bovine brain. This protein is very similar or identical to phosducin, a protein previously thought to be specific for retina and pineal gland. Recombinant phosducin inhibited the GTPase activity of several G proteins, and also inhibited Gs-mediated adenylyl cyclase activation. Blockade of its inhibitory effects by protein kinase A suggests that phosducin may be part of a complex regulatory network controlling G-protein-mediated signalling.


Multiple dopamine D4 receptor variants in the human population. The dopamine D4 receptor structurally and pharmacologically resembles the dopamine D2 and D3 receptors. Clozapine, an atypical antipsychotic that is relatively free of the adverse effects of drug-induced parkinsonism and tardive dyskinesia, binds to the D4 receptor with an affinity 10 times higher than to the D2 and D3 receptors. This may explain clozapine's atypical properties. Here we report the existence of at least three polymorphic variations in the coding sequence of the human D4 receptor. A 48-base-pair sequence in the putative third cytoplasmic loop of this receptor exists either as a direct-repeat sequence (D4.2), as a fourfold repeat (D4.4) or as a sevenfold repeat (D4.7). Two more variant alleles were detected in humans. Expression of the complementary DNA for the three cloned receptor variants showed different properties for the long form (D4.7) and the shorter forms (D4.2, D4.4) with respect to clozapine and spiperone binding. To our knowledge, this is the first report of a receptor in the catecholamine receptor family that displays polymorphic variation in the human population. Such variation among humans may underlie individual differences in susceptibility to neuropsychiatric disease and in responsiveness to antipsychotic medication.


Platelet-derived growth factor stimulates synthesis of PtdIns(3,4,5)P3 by activating a PtdIns(4,5)P2 3-OH kinase. Although the hormone-stimulated synthesis of 3-phosphorylated inositol lipids is known to form an intracellular signalling system, there is no consensus on the crucial receptor-regulated event in this pathway and it is still not clear which of the intermediates represent potential output signals. We show here that the key step in the synthesis of 3-phosphorylated inositol lipids in 3T3 cells stimulated by platelet-derived growth factor is the activation of a phosphatidylinositol(4,5)-bisphosphate (3)-hydroxy (PtdIns(4,5)P2 3-OH) kinase. A similar conclusion has been applied to explain the actions of formyl-Met-Leu-Phe on neutrophils, and it may be that receptors that couple through intrinsic tyrosine kinases or through G proteins stimulate the same step in 3-phosphorylated inositol lipid metabolism. The close parallel between these two mechanisms for the activation of PtdIns(4,5)P2 3-OH kinase and those described for the activation of another key signalling enzyme, phospholipase C (ref. 7), focuses attention on the product of the PtdIns(4,5)P2 3-OH kinase, PtdIns(3,4,5)P3, as a possible new second messenger.


Human cytomegalovirus UL97 open reading frame encodes a protein that phosphorylates the antiviral nucleoside analogue ganciclovir. Human cytomegalovirus (HCMV, a betaherpes virus) is the cause of serious disease in immunologically compromised individuals, including those with acquired immunodeficiency syndrome. One of the compounds used in the chemotherapy of HCMV infections is the nucleoside analogue 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (ganciclovir). The mechanism of action of this drug is dependent on the formation of the nucleoside triphosphate, which is a strong inhibitor of the viral DNA polymerase. Thymidine kinase, which is encoded by many of the herpesviruses, catalyses the initial phosphorylation of ganciclovir. But there is no evidence for the coding of this enzyme by HCMV, and DNA sequence analysis of the HCMV genome has shown that there is no open reading frame characteristic of a herpesvirus thymidine kinase. Here we present biochemical and immunological evidence that the HCMV UL97 open reading frame codes for a protein capable of phosphorylating ganciclovir. This protein seems to be responsible for the selectivity of ganciclovir and will be useful tool in the understanding and refinement of the antiviral activity of new selective anti-HCMV compounds.


A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells. Human cytomegalovirus (HCMV) is a major pathogen in immunosuppressed individuals, including patients with acquired immune deficiency syndrome. The nucleoside analogue ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)-guanine) is one of the few drugs available to treat HCMV infections, but resistant virus is a growing problem in the clinic and there is a critical need for new drugs. The study of ganciclovir-resistant mutants has indicated that the selective action of ganciclovir depends largely on virus-controlled phosphorylation in HCMV-infected cells. The enzyme(s) responsible have not been identified. Here we report that the HCMV gene UL97, whose predicted product shares regions of homology with protein kinases, guanylyl cyclase and bacterial phosphotransferases, controls phosphorylation of ganciclovir in HCMV-infected cells. A four-amino-acid deletion of UL97 in a conserved region, which in cyclic AMP-dependent protein kinase participates in substrate recognition, causes impaired ganciclovir phosphorylation. The implications of these results for antiviral drug development and drug resistance are discussed.


Structure of astacin and implications for activation of astacins and zinc-ligation of collagenases. Astacin, a digestive zinc-endopeptidase from the crayfish Astacus astacus L., is the prototype for the 'astacin family', which includes mammalian metallo-endopeptidases and developmentally regulated proteins of man, fruitfly, frog and sea urchin. Here we report the X-ray crystal structure of astacin, which reveals a deep active-site cleft, with the zinc at its bottom ligated by three histidines, a water molecule and a more remote tyrosine. The third histidine (His 102) forms part of a consensus sequence, shared not only by the members of the astacin family, but also by otherwise sequentially unrelated proteinases, such as vertebrate collagenases. It may therefore represent the elusive 'third' zinc ligand in these enzymes. The amino terminus of astacin is buried forming an internal salt-bridge with Glu 103, adjacent to His 102. Astacin pro-forms extended at the N terminus, as observed for some 'latent' mammalian astacin homologues, did not exhibit this 'active' conformation, indicating an activation mechanism reminiscent of trypsin-like serine proteinases.


Coexisting corticotroph and lactotroph adenomas: case report with reference to the relationship of corticotropin and prolactin excess. A 57-year-old obese woman with hypertension, diabetes mellitus, osteoporosis, and a 40-year history of secondary amenorrhea was diagnosed with corticotropin-dependent Cushing's syndrome. Dynamic endocrine testing and radiological evaluation did not reveal definitively the source of the excess corticotropin. Bilateral adrenalectomy was performed with resolution of the signs and symptoms of hypercortisolism. Four years later, the patient was noted to have rising serum corticotropin levels and an enlarging pituitary mass; hyperprolactinemia also was documented. A diagnosis of Nelson-Salassa syndrome was made, and she underwent a transsphenoidal adenomectomy. A histological examination of the specimen revealed two distinct, albeit contiguous, adenomas: a corticotroph adenoma and a lactotroph adenoma. Postoperatively, the serum prolactin and corticotropin levels decreased significantly. Although the stalk section effect resulting from compression by a pituitary adenoma can raise serum prolactin levels, a concurrent lactotroph adenoma should be considered in patients with nonfunctional or functional pituitary adenomas of other types associated with significantly elevated prolactin levels. The mechanisms underlying simultaneous adrenocorticotropic hormone and prolactin excess are discussed.


Surgeon's foot: a report of sural nerve palsy. Although compression neuropathies are encountered frequently in neurosurgical practice, involvement of the sural nerve is described rarely. We report a case of bilateral compression neuropathy of the sural nerve with an unusual mechanism of injury. The case is discussed, and the pertinent literature is reviewed.


DNA comparisons of Trypanosoma evansi (Indonesia) and Trypanosoma brucei spp. Two clones from separate isolates of Trypanosoma evansi in Indonesia were found by polymerase chain reaction (PCR) analyses to contain 3 different repeated nuclear DNA sequences of Trypanosoma brucei spp: the consensus sequence for a highly repetitive 177 base pairs and the gene repeats encoding procyclin and the spliced leader. In addition, the 994 bp minicircle sequence of one of the clones was determined, and PCR amplification primers specific for minicircles of T. evansi were identified that do not amplify minicircle sequences in the T. brucei spp. clones tested.


Bronchoalveolar lavage fluid cytology reflects airway inflammation in beagle puppies with acute bronchiolitis. Beagle puppies infected with both canine parainfluenza virus type 2 (CPI2) and Bordetella bronchiseptica (Bb) develop more severe acute bronchiolitis and airways hyperresponsiveness than do those infected with CPI2 or Bb alone. The aim of our study was to characterize the inflammatory response associated with airway hyperresponsiveness, and to determine whether the inflammatory cell response of bronchoalveolar lavage fluid (BALF) reflected changes in the bronchioles in this model. We investigated 25 beagle puppies (ages 76 +/- 5 days, mean +/- SEM) in four groups: controls (n = 6), or puppies inoculated with both CPI2 and Bb (CPI2-Bb) (n = 11), with only CPI2 (n = 4), or only Bb (n = 4). The puppies were killed 3-4 days after inoculation, the lungs excised, the intermediate lobe lavaged, and BALF and the bronchiolar wall tissue examined for neutrophils and other inflammatory cells. Control puppies had no evidence of inflammation. However, the CPI2-Bb puppies had developed cough and rhinitis, positive cultures for CPI2 and Bb, and a neutrophilic cellular response in both the bronchioles and the BALF. Puppies inoculated with only CPI2 or Bb had milder illnesses and no significant bronchiolar and BALF neutrophilic response. For all groups, the severity of bronchiolar wall inflammation correlated with the total number of BALF inflammatory cells, and bronchiolar wall neutrophil counts correlated with the percentage of neutrophils in the BALF. The illness and the airway hyperresponsiveness observed in the CPI2-Bb group were associated with airway neutrophilia. Our studies support the hypothesis that neutrophils are associated with airway dysfunction in this model, and the use of BALF to study the process.


[Severe forms of herpes simplex in newborn infants]. Generalized herpetic infection in the newborn runs its course in association with cerebral lesions as well as with injuries to the lungs, heart and liver. The infection diagnosis should take into account the mother's epidemiological anamnesis, the clinical manifestations in the child and the results of the simultaneous use of several methods (fluorescent antibody test, IEA, virological test). Of paramount importance is the simultaneous examination of the mother. Of no less importance is echoencephalography, namely the detection of cysts in white matter of the frontal lobes.


[Clinical picture of congenital Cytomegalovirus infection in infants in their first year of life]. A study was made of the clinical picture of congenital cytomegaloviral infection in 40 children of the first year of life. The diagnosis was established on the basis of demonstration in all the patients of IgM antibodies to cytomegalovirus (IgM anti-CMV) by EIA with the aid of the commercial test systems. Besides, 81% of the children were found to have cytomegalic-specific cells in urine, 17.6% in saliva, and 72.5% demonstrated IgG anti-CMV (in accordance with EIA). The majority of the patients (70%) were born premature. The most typical clinical signs of congenital cytomegaloviral infection were adynamia, jaundice, liver and CNS injury, prenatal hypotrophy; 45% of the children had developmental abnormalities and stigmas of dysembryogenesis. In 97.5% of the mothers, the pregnancy ran a pathological course. The disease history often pointed to miscarriages (10%) and death of the children due to intrauterine infection in the early times after birth (15%).


Basolateral membrane conductance in A6 cells: effect of high sodium transport rate. Conductance of apical and basolateral membranes in short-circuited cultured renal distal cells (A6) was determined using microelectrodes. Epithelia were pre-incubated with 0.1 mumol/l dexamethasone in the presence of 4 mumol/l amiloride to prevent increase in apical Na+ entry. Omission of amiloride increased the Isc from 5.7 to 27.6 microA/cm2 due to the rise in apical membrane conductance from 21 to 595 microS/cm2. Apical fractional resistance decreased from 0.89 to 0.40 and cells depolarized from -52 to -4 mV. Basolateral membrane conductance, which was 320 microS/cm2 at partially inhibited transport, was not significantly altered during the first 2 min following establishment of high transport activity; it started to increase thereafter reaching a more than threefold higher value of 1324 microS/cm2 within 12 min. The gain cannot be explained by increase in partial K+ conductance. Disappearance of the conductance after reduction of basolateral Cl- or in the presence of the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate indicates a Cl- conductance, which appears to be activated by depolarization.


Interleukin-2 inhibits sodium currents in human muscle cells. The effect of the T cell growth factor interleukin-2 (IL-2) on muscular Na+ channels was studied in myoballs produced from primary human muscle cultures. The transient Na+ inward currents of the myoballs, elicited by repetitive stimulation at 1 Hz and recorded in the whole-cell mode, were inhibited by IL-2 applied to the external solution, the half maximum effect occurring at 300 U/ml. The effect was complete within 5 s and was totally reversible, the on and off effects having identical time courses. The h infinity curve was shifted in negative direction indicating that the mechanism of IL-2 action is a conversion of the Na+ channels into a state of inactivation. The reaction of the IL-2 solution with an anti IL-2 antibody neutralized the inhibitory effect on the Na+ currents, indicating a specific effect of the peptide growth factor interleukin-2 on muscular Na+ channels. The connection of IL-2 and Na+ channels may be important in inflammatory processes of muscle and nerve.


Cloning and characterisation of the S. pombe rad15 gene, a homologue to the S. cerevisiae RAD3 and human ERCC2 genes. The RAD3 gene of Saccharomyces cerevisiae encodes an ATP-dependent 5'-3' DNA helicase, which is involved in excision repair of ultraviolet radiation damage. By hybridisation of a Schizosaccharomyces pombe genomic library with a RAD3 gene probe we have isolated the S. pombe homologue of RAD3. We have also cloned the rad15 gene of S. pombe by complementation of radiation-sensitive phenotype of the rad15 mutant. Comparison of the restriction map and DNA sequence, shows that the S. pombe rad15 gene is identical to the gene homologous to S. cerevisiae RAD3, identified by hybridisation. The S. pombe rad15.P mutant is highly sensitive to UV radiation, but only slightly sensitive to ionising radiation, as expected for a mutant defective in excision repair. DNA sequence analysis of the rad15 gene indicates an open reading frame of 772 amino acids, and this is consistent with a transcript size of 2.6 kb as detected by Northern analysis. The predicted rad15 protein has 65% identity to RAD3 and 55% identity to the human homologue ERCC2. This homology is particularly striking in the regions identified as being conserved in a group of DNA helicases. Gene deletion experiments indicate that, like the S. cerevisiae RAD3 gene, the S. pombe rad15 gene is essential for viability, suggesting that the protein product has a role in cell proliferation and not solely in DNA repair.


Both yeast W double-stranded RNA and its single-stranded form 20S RNA are linear. Most yeast strains carry a cytoplasmic double-stranded RNA (dsRNA) molecule called W, of 2.5 kb in size. We have cloned and sequenced most of W genome (1), and we proposed that W (+) strands were identical to 20S RNA, a single-stranded RNA (ssRNA) species, whose copy number is highly induced under stress conditions. Recently it was proposed that 20S RNA was circular (2). In this paper, however, we demonstrate that both W dsRNA and 20S RNA are linear. Linearity of W dsRNA is shown by the stoichiometric labelling of both strands of W with 32P-pCp and T4 RNA ligase. The last 3' end nucleotide of both strands is about 70 to 80% C and 20 to 30% A. Linearity of 20S RNA is directly demonstrated by a site-specific cleavage of 20S RNA with RNase H, using an oligodeoxynucleotide complementary to an internal site of 20S RNA. The cleavage produced not one but two RNA fragments expected from the linearity of 20S RNA.


A ribosomal RNA gene promoter at the telomere of a mini-chromosome in Trypanosoma brucei. The parasitic protozoan Trypanosoma brucei has some hundred mini-chromosomes of 50-150 kb, which mainly consist of telomeric repeats, sub-telomeric repeats and internal 177-bp repeats. Their primary function seems to be to expand the repertoire of non-transcribed sub-telomeric variant surface glycoprotein (VSG) genes. Here we report that two of the smaller mini-chromosomes (55 and 60 kb) contain sequences homologous to the ribosomal RNA gene promoter region. We have targeted by homologous recombination the neomycin phosphotransferase (neo(r)) gene behind the promoter on the 55 kb chromosome and show that this promoter mediates the efficient synthesis of properly trans-spliced and polyadenylated neo mRNA. The resulting high resistance to G418 (a neo analogue) is stable in the absence of drug showing that mitotic segregation of this mini-chromosome is precise. Downstream of the transcription start the wild-type version of the ribosomal promoter is flanked by telomeric repeats. The absence of the sub-telomeric repeats found in other T.brucei chromosome ends suggests that the rDNA-telomeric junction has been formed by de novo addition of telomeric repeats to a broken chromosome end (healing). Our results provide a plausible explanation for the alpha-amanitin-resistant transcription of telomeric repeats in T.brucei reported by Rudenko and Van der Ploeg and they show that trypanosomes can efficiently use RNA polymerase I for the expression of sub-telomeric genes, supporting the notion that the alpha-amanitin-resistant transcription of sub-telomeric VSG genes may also be catalyzed by this enzyme.


Mechanisms of intermolecular homologous recombination in plants as studied with single- and double-stranded DNA molecules. To elucidate the mechanism for intermolecular homologous recombination in plants we cotransformed Nicotiana tabacum cv Petit Havana SR1 protoplasts with constructs carrying different defective derivatives of the NPTII gene. The resulting kanamycin resistant clones were screened for possible recombination products by PCR, which proved to be a valuable technique for this analysis. Our results show that the double-stranded circular DNA molecules used in this study recombine predominantly via a pathway consistent with the single-strand annealing (SSA) model as proposed for extrachromosomal recombination in mammalian cells. In the remaining cases recombination occurred via a single reciprocal recombination, gene conversion and possibly double reciprocal recombination. Since single-stranded DNA is considered to be an important intermediate in homologous recombination we also established the recombination ability of single-stranded DNA in intermolecular recombination. We found that single-stranded DNA enters in recombination processes more efficiently than the corresponding double-stranded DNA. This was also reflected in the recombination mechanisms that generated the functional NPTII gene. Recombination between a single-stranded DNA and the complementing DNA duplex occurred at similar rates via a single reciprocal recombination and the SSA pathway.


A negative transcriptional control region of a developmentally-regulated gene co-localizes with the origin of replication of an endogenous plasmid in Dictyostelium. The endogenous nuclear plasmid Ddp1 from the wild-type Dictyostelium discoideum strain NC4 has been cloned, its origin of replication has been localized, and plasmid-encoded genes have been mapped that are preferentially expressed during growth or development. Here we present an analysis of the regulation of the Ddp1-encoded gene d5, which, in wild-type cells, is expressed only during the multicellular stages of development. In this study, we show that sequences 3' to the d5 coding region are required to suppress constitutive expression of d5 from aberrant transcriptional start sites and that this regulatory region acts at a distance and in an orientation-independent manner. The cis-acting negative regulatory element(s) necessary for repression of aberrant d5 expression is either very tightly linked or identical to sequences required for extrachromosomal replication, such that all 3' deletions that cause constitutive d5 expression result in the integration of the plasmid into the D. discoideum genome. Placing d5 (without the 3' regions containing the Ddp1 origin) on an extrachromosomal vector based on another endogenous plasmid (Ddp2) did not restore proper transcriptional regulation, suggesting that an extrachromosomal environment alone is not sufficient to confer proper transcriptional regulation to d5.


Bladder cancer, 1992.

Bladder cancer, 1992. Although the incidence of bladder cancer has not changed in the last 40 years, treatment has greatly decreased morbidity. Invasive disease, however, remains fatal. Bladder cancer is not common in the general population, and current laboratory tests of urine are not adequate for screening the population at large. However, bladder cancer is not uncommon in certain high-risk groups, and urine studies in these patients can be helpful. Hematuria and/or an irritative voiding pattern in an elderly man or a patient otherwise at high risk for bladder cancer should be evaluated carefully. Fortunately, most bladder cancers are superficial when discovered and can be treated with local therapy with or without intravesicular chemotherapy. Cystectomy is still the treatment of choice for invasive disease, but preliminary reports of new methods of radiation therapy and chemotherapy show promising results.


The new macrolide antibiotics. Azithromycin and clarithromycin. Azithromycin (Zithromax) and clarithromycin (Biaxin Filmtabs) are new macrolide antibiotics with several advantages over erythromycin. Azithromycin has an expanded spectrum against gram-negative bacilli. Clarithromycin is more active than erythromycin against gram-positive cocci; combination with its 14-hydroxy metabolite enhances its antimicrobial activity. These new agents penetrate well into tissues and concentrate in macrophages and polymorphonuclear leukocytes. They offer improved bioavailability and an extended half-life. The high tissue-to-serum level and extended elimination half-life of azithromycin allow for once-daily dosing and short-course therapy. Clarithromycin and 14-hydroxyclarithromycin maintain high serum levels and tissue-to-serum concentrations. Both of these new agents have been effective in streptococcal pharyngitis, acute sinusitis, acute lower respiratory tract infections, skin and soft-tissue infections, and sexually transmitted diseases. A single dose of azithromycin is effective for genital chlamydial infections. Adverse reactions to these agents have usually been mild and have not included serious organ toxicity. In clinical trials, the rate of premature discontinuation of therapy has been less than observed with erythromycin. Azithromycin and clarithromycin should be used according to the current guidelines of the Food and Drug Administration; their future role will be determined by ongoing laboratory and clinical evaluations.


Effect of dietary sodium zeolite A and graded levels of calcium and phosphorus on growth, plasma, and tibia characteristics of chicks. Sodium zeolite A (SZA), a synthetic sodium aluminosilicate having a high ion exchange capacity, has been shown to influence Ca and P utilization in chickens. A 3 x 2 x 2 factorial arrangement of treatments was used to investigate the effect of dietary P (.41, .55, and .69% total P), Ca (.6 and 1%), and SZA (0 and .75%) on growth, plasma, and tibia characteristics of chicks from 5 to 15 days of age. Growth, feed intake, gain:feed ratio, and tibia characteristics were influenced by dietary Ca and P in a manner consistent with dietary recommendations for these macro minerals. The addition of Ca, SZA, or both exacerbated the adverse effects of feeding low-P diets, yet alleviated the adverse effects of feeding a low-Ca, high-P diet. Dietary SZA had no effect (P greater than .5) on plasma Ca or alkaline phosphatase; however, SZA reduced (P less than .01) plasma P. Dietary SZA increased (P less than .02) tibia Mn, Zn, Cu, and Al. The SZA-induced increase in tibia Al was most evident in chicks fed low levels of P (SZA by P interaction, P less than .02). The overall response to dietary SZA addition paralleled the response observed from Ca supplementation, indicating that SZA increased Ca utilization, reduced P utilization, or contributed to both of these effects. These data demonstrate that the effects of SZA are influenced by the dietary concentration of Ca and P and that the addition of SZA to diets low in P results in bone Al accumulation.


Luteinizing hormone-releasing hormone receptor bindings of the hen pituitary: difference between laying and nonlaying hens, effects of ovarian steroid hormones in vivo, and changes during an ovulatory cycle. The equilibrium dissociation constant (Kd) of the pituitary luteinizing hormone-releasing hormone (LHRH) receptors and the maximum binding capacity (Bmax) per milligram of membrane protein were greater in laying hens than in nonlaying hens. A single i.m. injection of progesterone, estradiol-17 beta, or 5 alpha-dihydrotestosterone into nonlaying hens caused an increase in Kd and Bmax values. During an ovulatory cycle of the laying hen, Kd and Bmax values decreased from 16 to 14 h and 8 to 6 h before ovulation. A decrease in the Bmax value was also found 24 to 21 h before ovulation. A single i.v. injection of chicken LHRH-I caused a decrease in the Kd and Bmax values within 2 min after the injection. The results suggest that the hen pituitary LHRH receptor bindings are affected by ovarian steroid hormones and that their changes during the ovulatory cycle may relate to the release of a gonadotropin from the pituitary.


Immunomodulatory ionophore copolymers, T150R1 and T130R2, induce corticotropin from anterior pituitary cells. T150R1 is a synthetic copolymer with Na+ ionophore activity. We demonstrated previously that T150R1, when injected into mice, produces rapid thymic involution with depletion of cortical thymocytes. Elevated serum ACTH and corticosterone levels, as well as abrogation of the effects of T150R1 on the thymus by adrenalectomy and hypophysectomy, suggested a pituitary-mediated mechanism. In this work, we investigated the ability of T150R1, and of the related ionophore copolymer T130R2, to stimulate ACTH in vitro from the mouse anterior pituitary cell line AtT-20. Copolymer-induced ACTH release was dose-, time-, and temperature-dependent. Hormone induction peaked at 30 degrees C for T150R1 and 37 degrees C for T130R2. The temperature dependence of ACTH release paralleled that of ionophore activity measured in red blood cells, providing evidence that the ability to induce ACTH is related to the ionophore property of the copolymers. Peak ionophore activity and hormonal release occurred at the temperatures when the copolymers form partially soluble complexes which interact optimally with cell membranes. Cotreatment with exogenous phospholipase C inhibited the effects of T150R1, which suggests that the enzyme either blocks the insertion of T150R1 into the cell membrane or that the phospholipase C-induced increase in intracellular calcium inhibits the ionophore activity of T150R1. These data support an ionophore mechanism for copolymer-induced ACTH release in which changes in the physicochemical structure of the copolymers may affect their interaction with cell membranes. The data also suggest that direct stimulation of pituitary ACTH accounts for at least some of the in vivo immunomodulatory effects of T150R1.


Possible involvement of endogenous beta-endorphin in the pathophysiological mechanisms of Pichinde virus-infected guinea pigs. Previously, we demonstrated that naloxone, an opiate antagonist, prolonged survival of strain 13 guinea pigs infected with Pichinde virus. Thus, endogenous opiates may be involved in the pathogenesis of this viral disease. To determine whether endogenous opiate levels were affected by Pichinde viral infection, beta-endorphin concentrations in plasma and cerebrospinal fluid (CSF) of normal and infected strain 13 guinea pigs were measured by radioimmunoassay. Cerebrospinal fluid beta-endorphin concentrations were 78.0 +/- 13.2 pg/ml on postinoculation day (PID) 7, 59.0 +/- 5.6 pg/ml on PID 12, and 58.8 +/- 5.4 pg/ml on PID 14. These values were significantly higher than baseline levels of CSF beta-endorphin: 30.8 +/- 1.9 pg/ml. Plasma beta-endorphin concentrations of infected animals increased significantly to 202.1 +/- 17.9 pg/ml on PID 7 and to 154.2 +/- 21.4 pg/ml on PID 12 from a mean baseline value of 84.2 +/- 13.1 pg/ml. After a primer intravenous injection of beta-endorphin (10, 15, or 30 micrograms/kg), followed by constant infusion of beta-endorphin (15, 45, or 90 micrograms/kg.hr) to control noninfected guinea pigs, heart rate (except with the lowest dose) and mean blood pressure decreased markedly. Under these experimental conditions, concentrations of plasma and CSF beta-endorphin increased simultaneously with different magnitude. Because both Pichinde viral infection and beta-endorphin administration produced a similar trend of cardiovascular disturbances, leading to hypotension and bradycardia, increased concentrations of plasma and CSF beta-endorphin may play a partial role in the pathophysiological mechanisms of Pichinde virus infection.