Local potentiation of bradykinin-induced vasodilation by converting-enzyme inhibition in isolated coronary arteries. The interaction of angiotensin-converting enzyme (ACE) inhibitors and bradykinin was investigated in isolated bovine and human coronary arteries. Rings with and without endothelium were mounted in organ chambers for measurement of isometric force. The effects of the ACE inhibitors lisinopril, enalaprilat, fosinoprilat, ramiprilat, and captopril were determined during submaximal stimulation with bradykinin or other vasodilators. Lisinopril and captopril alone did not affect vascular tone; however, in rings with endothelium partially relaxed with bradykinin (> or = 10(-10) M), all ACE inhibitors caused further relaxations. Lisinopril did not affect bradykinin concentrations in the incubation medium. Mechanical removal of the endothelium or incubation with nitro-L-arginine or the bradykinin2-receptor antagonist Hoe 140 prevented the relaxations to bradykinin and lisinopril. Other vasodilators including acetylcholine, adenosine diphosphate, substance P, or SIN-1 did not prime the rings to respond to ACE inhibitors. Endothelium-dependent relaxations to lisinopril were also observed in human coronary arteries treated with bradykinin (> or = 10(-7) M). Thus, ACE inhibitors potentiate endothelium-dependent relaxations to submaximal concentrations of bradykinin in bovine and human coronary arteries. This local mechanism occurs regardless of elevated bradykinin concentrations in the blood and reduced angiotensin II generation.


Role of prostaglandins in the cardiovascular effects of bradykinin and angiotensin-converting enzyme inhibitors. The cardiovascular effects of bradykinin require additional vasoactive mediators for a fully balanced response. This includes arachidonic acid (eicosatetraenoic acid) and its metabolites, the eicosanoids (prostaglandins, leukotrienes, thromboxanes, and others). Eicosanoid generation by bradykinin is started by binding of the peptide to specific B2 receptors at the plasma membrane. This initiates G-protein coupled stimulation of phospholipase C, IP3-induced increases in cytosolic Ca2+, and stimulation of protein kinase C. Arachidonic acid is liberated from membrane phospholipids primarily via Ca(2+)-induced stimulation of phospholipase A2 and converted into tissue-specific eicosanoids by enzymes in the vicinity. In vascular tissue, most of the available arachidonic acid is converted into vasodilator prostaglandins, i.e., prostacyclin (PGI2) and prostaglandin E2 (PGE2). These prostaglandins are involved in vasodilator actions of the kinins. There is also some evidence for generation of vasoconstrictor eicosanoids, such as thromboxane A2, under certain conditions. The biological significance of kinin-related prostaglandin formation becomes apparent after inhibition of kinin breakdown by ACE inhibitors. These compounds prevent generation of vasoconstrictor angiotensin II and stimulate endothelial eicosanoid formation via local kinin accumulation. There is evidence suggesting that kinin-induced prostaglandin generation contributes to anti-ischemic, inotropic, and blood pressure-lowering effects of the compounds. This also includes inhibition of polymorphonuclear leukocyte (PMN) accumulation in injured myocardial tissue, which is antagonized by PGI2-related pathways, stimulated by ACE inhibition and/or bradykinin.


Heterogeneity of endothelium-dependent vasodilator effects of angiotensin-converting enzyme inhibitors: role of bradykinin generation during ACE inhibition. Endothelium-derived mediators are released in response to shear stress and a variety of endogenous substances including bradykinin and angiotensins. They may contribute to the regulation of the renin-angiotensin system in the vascular wall and in the kidney. Bradykinin is a powerful agonist at endothelial cells, and the actions of this peptide, which is generated by components of the vascular wall, during angiotensin-converting enzyme (ACE) inhibition may determine some of the vascular effects of ACE inhibitors. In vitro studies demonstrate that the relaxations to bradykinin are mostly endothelium dependent and are mediated by nitric oxide, endothelium-derived hyperpolarizing factor, and/or vasodilator prostaglandins; however, these endothelium-derived relaxing factors do not always contribute simultaneously to the relaxations in every artery. The contribution of ACE in the termination of bradykinin action, relative to the other inactivation processes (including carboxypeptidases and internalization) also may determine the ability of ACE inhibitors to augment the effects of the kinin. Furthermore, it appears that the level of ACE activity and the potency of bradykinin, respectively, are not uniform in all preparations. In arteries in which bradykinin is very efficacious and in which ACE activity may be relatively low, ACE inhibitors may prolong but not amplify the responses to the peptide. The pharmacologic characteristics of the responses of the different vascular beds to bradykinin, together with the modulation of endothelium-dependent responses to other agonists (including purines), may be of importance in the heterogeneity of the vasodilator actions of ACE inhibitors.


Role of bradykinin in the cardiac effects of angiotensin-converting enzyme inhibitors. Angiotensin-converting enzyme (ACE) inhibitors exert their beneficial effects not only via endocrine mechanisms, but most probably also via interference with autocrine-paracrine actions involving local renin-angiotensin and kallikrein-kinin systems with subsequent autacoid release. Inhibition of ACE (kininase II) results in the reduction of angiotensin II generation and kinin degradation, leading to beneficial cardiovascular effects. Bradykinin and prostacyclin release from isolated rat hearts was increased by local ACE inhibitions with ramiprilat. In different models the bradykinin-mediated effects of ACE inhibition were abolished with the specific B2 kinin-receptor antagonist Hoe 140: The cardioprotective effects of ramiprilat or ramipril such as reduction of postischemic reperfusion injuries in isolated rat hearts or the reduction in infarct size in dogs and rabbits were abolished by coadministration of Hoe 140. Furthermore, left ventricular hypertrophy in rats with aortic banding could be prevented or regression was induced when the ACE inhibitor was given in a non-blood pressure-lowering dose. These beneficial effects were also abolished by Hoe 140. In conclusion, in different experimental models, ACE inhibitors exert cardioprotective effects. An enhancement of endothelial autacoid formation (nitric oxide and prostacyclin) by inhibiting degradation of bradykinin may contribute to these effects.


Endothelium-dependent relaxations are augmented in rats chronically treated with the angiotensin-converting enzyme inhibitor enalapril. The study was designed to evaluate the effects of chronic inhibition of angiotensin-converting enzyme (ACE) on the reactivity of the endothelium and the smooth muscle to vasoconstrictor and vasodilator stimuli in normal rats. Male rats were treated orally for 6 weeks with enalapril (10 mg/kg/day, n = 10) or with placebo (n = 10). Endothelium-dependent relaxations to acetylcholine and adenosine diphosphate were augmented in aortic rings from rats treated with enalapril compared with controls, whereas the response to the endothelium-independent vasodilator SIN-1 were similar. Contractions induced by phenylephrine and angiotensin II remained unchanged. Thus, the results suggest that chronic ACE inhibition enhances the release of relaxing factors from the endothelium in normotensive rats.


Role of bradykinin in chronic antihypertensive actions of ramipril in different hypertension models. We investigated the chronic effect of bradykinin B2-receptor blockade on the antihypertensive actions of the angiotensin-converting enzyme (ACE) inhibitor ramipril in three different hypertensive rat models, the two-kidney/one-clip (2K1C) hypertensive Wistar rat, the kinin-deficient 2K1C hypertensive Brown Norway Katholieke (BN-K) rat, and the spontaneously hypertensive rat (SHR). Chronic blockade of bradykinin B2 receptors by subcutaneous infusion of the new bradykinin antagonist HOE 140 (500 micrograms/kg/day) attenuated the antihypertensive effect of ramipril only in 2K1C hypertensive Wistar rats, but not in 2K1C BN-K rats and SHR. Our data demonstrate for the first time that potentiation of endogenous kinins contributes to chronic antihypertensive actions of ACE inhibitors in experimental renal hypertension. Whether this holds also true for other forms of hypertension remains to be answered.


[Economic evaluation of prophylaxis with recombinant granulopoiesis stimulating factor]. The aim of the study was to economically evaluate the prophylactic use of the recombinant factor of the stimulation of granulocyte colonies (rG-CSF) for the prevention of episodes of febrile neutropenia (EFN) following antineoplasic chemotherapy. The methods of economic evaluation used were those of cost analysis and cost-effectivity analysis. The probability of occurence of EFN was estimated by the application of the Delphi method and the costs were calculated. The costs of EFN for the patients with solid tumors is situated between 236,000-377,000 pesetas according to hospitalary centers. For patients with acute leukemia the value was estimated as between 391,000-667,000 pesetas. The results of the cost analysis indicate that the net cost per patient prophylactically treated is between 4,000-64,000 pesetas. The results of the cost-effectivity analysis indicate that the cost per case of no incidence of EFN is between 20,000-320,000 pesetas. Episodes of febrile neutropenia carry expansive hospital and antibiotic clinical treatment. The prophylactic use of new medication for patients between the first and second cycle of chemotherapy, in agreement with the estimates calculated, does not save health care costs but may improve the quality of life in these patients and permit the continuation of a therapeutic schedule without interruption which may improve the life expectancy of the patient.


[Somatostatin in preoperative therapy and postoperative diagnosis of a patient with Verner Morrison syndrome]. We report the case of a patient with Verner-Morrison syndrome due to a malignant MEN I-associated vipoma. Marked tumor-associated hypercalcemia could be treated successfully with somatostatin analogues prior to surgical therapy of the pancreatic tumor. Sixteen months after extirpation of the primary tumor recurrent tumor growth was diagnosed; at this time the patient was clinically asymptomatic and had no abnormal laboratory test results. Liver metastases and local metastases were identified using somatostatin receptor scintigraphy. We report and discuss the use of somatostatin in the treatment of tumor-associated symptoms in endocrine tumors and the possibility of identifying endocrine tumors by means of somatostatin receptor scintigraphy.


The gal locus from Haemophilus influenzae: cloning, sequencing and the use of gal mutants to study lipopolysaccharide. The gal locus from Haemophilus influenzae was cloned and sequenced. Four genes were identified by amino acid homology: galT, galK, galM and galR. The coding direction of galT, galK and galM is divergent from that of galR. There are non-coding intergenic regions between galR and galT, galT nd galK, and galK and galM. Deletion-insertion mutations constructed in galK and galE, which is in lic3, were moved into the H. influenzae chromosome generating each of the single mutants as well as the double gal mutant. Even when grown on complex media, the double mutant failed to react with an anti-lipopolysaccharide monoclonal antibody known to react with a digalactoside epitope. Both the galE single and the galE galK double mutants were serum-sensitive and relatively avirulent in infant rats, indicating a critical role for galactose metabolism, and providing evidence to support a central role for lipopolysaccharide, in H. influenzae virulence.


Comparative studies on binding of vitronectin and fibronectin to groups A and C streptococci. Binding of 125I-labelled fibronectin and vitronectin to streptococci of group A (S. pyogenes), group B (S. agalactiae) and group C (S. dysgalactiae and S. zooepidemicus) isolated from various human infections and bovine mastitis, and S. uberis bovine isolates, was studied. Binding of vitronectin and fibronectin was common among both human groups A and C, and bovine group C streptococci. S. agalactiae strains of human and bovine origin as well as S. uberis bovine isolates bound low levels of both proteins. The binding of radiolabelled fibronectin and vitronectin to selected groups A and C streptococcal strains was specific, time-dependent and occurred with both live and heat-killed (80 degrees C for 15 min) cells. Binding declined rapidly after treatment of cells with trypsin or proteinase K, while pepsin digestion at pH 5.5 affected vitronectin but not fibronectin binding.


Detection and purification of a potential precursor protein or a prohaemolysin produced by Staphylococcus haemolyticus. The haemolytic substance H7 produced by Staphylococcus haemolyticus is composed of three peptides out of 44 amino acid residues each having a structure resembling a signal peptide. The cytoplasmic fraction for the presence of a protein precursor containing this signal sequence was investigated. Specific rabbit IgGs to substance H7 were isolated by affinity chromatography on Sepharose-4B-H7. These anti-H7 IgGs recognized mainly a 51 kD protein in the cytoplasmic fraction of S. haemolyticus from 2, 4, 6, 8, 10 and 23 h cultures. These results support the idea that the 51 kD protein could be either a prohaemolysin or else the precursor of a protein of unknown function with a signal sequence showing homology with the haemolytic peptides. After affinity chromatography on Sepharose-4B-anti-H7, the 51 kD protein was shown associated with an RNA-protein complex composed of four or five proteins and an RNA estimated at 300 nt. This complex could be associated with the machinery of protein secretion. The 51 kD protein was finally purified to homogeneity by HPLC on a Protein Pak DEAE-5PW column in the presence of 5 M urea.


Epidermal growth factor receptor at endothelial cell and pericyte interdigitation in human granulation tissue. Angiogenic immature capillaries in human granulation tissue possess many cytoplasmic interdigitations between endothelial cells and pericytes (CIDEP). Epidermal growth factor (EGF) is a potent mitogenic polypeptide which accelerates angiogenesis in vivo and in vitro. We have recently demonstrated in immature capillaries that EGF is present at the CIDEP and proposed that the CIDEP may be involved in a signaling pathway for EGF (Wakui et al., 1990c. Microvasc. Res. 40, 285-291). This study follows that previous report. In the present study, I have investigated the ultrastructural localization of the EGF receptor (EGF-r) at CIDEP. Immunoreactivity for the EGF-r in immature capillaries was located strictly at the CIDEP in large numbers, at the coated pits and vesicles of endothelial cells, and at some lysosome-like structures of the endothelial cells and the pericytes. On the other hand, immunoreactivity for EGF-r was absent in mature capillaries. At the CIDEP, EGF-r immunoreactivity was present at the cell membrane and a few cytoplasmic elements at the tip of pericyte cytoplasmic projections, but it was completely absent at the corresponding endothelial membrane indentation. The present results support our proposed hypothesis that the CIDEP in immature capillaries act as a pathway for EGF transportation from the endothelial cell to the pericyte by a receptor-mediated process.


Possible mechanism(s) for permeability recovery of venules during histamine application. Histamine is known to cause a substantial increase in the permeability of venules to both water and proteins. However, this increase is transient, i.e., the initially elevated permeability returns toward control levels, or "recovers," even during continuous histamine stimulation. In this investigation, we attempted to identify the possible chemical signal(s) initiating the permeability recovery process in single venules of rat mesentery. Specifically, we tested whether histamine's binding to H2 receptors and/or the production of prostacyclin by endothelial cells was involved in this process. To achieve this aim, the time course of endothelial cells was involved in this process. To achieve this aim, the time course of histamine-induced changes in permeability to alpha-lactalbumin was measured in the presence of H2 receptor antagonist (cimetidine) or of prostacyclin synthetase inhibitor (tranylcypromine), respectively. Permeability of individually perfused microvessels was measured using fluorescence microscopy. The results demonstrated that permeability recovery was not affected by the H2 receptor antagonist but was suppressed or even abolished by the prostacyclin synthesis inhibitor. Therefore, these results suggest that the production of prostacyclin by endothelial cells might serve as one chemical signal to initiate the permeability recovery process, whereas histamine's binding to H2 receptors is not involved in this phenomenon.


Mechanisms of augmented resistance of cyclosporin A-treated mice to influenza virus infection by trehalose-6,6'-dimycolate. Cyclosporin A (CsA), which is an immunosuppressive drug of helper T lymphocytes, diminished a resistance of mice to influenza virus infection. Mice inoculated intravenously with trehalose-6,6'-dimycolate (TDM, a glycolipid component of the cell wall of Mycobacterium) in an oil-in-water emulsion (TDM emulsion) recovered the resistance to influenza virus infection impaired by CsA. Number of antibody-producing cells was markedly reduced in CsA- and/or TDM-treated mice. Interferon production in lung of TDM-treated mice was augmented; however, it was extremely reduced not only in CsA-treated mice, but also in CsA- and TDM-treated mice. Activities of natural killer cells of CsA- and/or TDM-treated mice were not different from that of control mice. Numbers of lymphocytes in lung of TDM-treated mice and CsA- and TDM-treated mice were more predominantly increased than that of control mice. Analysis of lung lymphocytes by flow cytometry revealed no difference between the populations of L3T4+ T lymphocytes and Lyt-2.2+ T lymphocytes in CsA- and/or TDM-treated mice and the populations in control mice. However, the population of gamma delta T cell receptor positive (gamma delta TCR+) lymphocytes increased markedly in lung of TDM-treated mice and also CsA- and TDM-treated mice. In vitro experiments showed that macrophage cultures treated with TDM emulsion released a mediator(s) which activates T lymphocytes, but not B lymphocytes. These and our earlier results suggest that the recovered anti-influenza virus resistance of CsA-treated mice by treatment with TDM emulsion was caused by elicitation of macrophages with TDM, then activation of T lymphocytes, especially gamma delta TCR+ lymphocytes.


Demonstration of low molecular weight polypeptides associated with small, round-structured viruses by western immunoblot analysis. Small, round-structured viruses (SRSV) were detected in 14 of 300 fecal specimens obtained from patients with acute gastroenteritis by electron microscopy. These SRSV strains were morphologically indistinguishable from one another. While 11 of these strains had a single usual major structural protein with molecular weight of 63,000 (63K) daltons (p63), interestingly, three strains possessed a single major structural protein with molecular weight of 33K daltons (p33). Treatments of p63-SRSV with proteolytic enzymes or denaturating reagents did not affect the molecular weight of p63, and the p33 was not detectable by Western immunoblot in the ultracentrifugal supernatant of the p63-SRSV suspension. These results suggest that the p33 is neither a definitive subunit of p63 nor disintegrated component derived from the p63-SRSV but a novel polypeptide of SRSV. Immune electron microscopy and Western immunoblot analyses indicated that p63- and p33-SRSVs may share an antigenic determinant(s).


Outcome of paediatric bacterial meningitis 1979-1989. To determine the outcome of all cases of paediatric bacterial meningitis over the 11-year period 1979-1989 and discuss the role of adjunctive therapy with dexamethasone. The study was performed by retrospective case review at Flinders Medical Centre, a general teaching hospital. There were 80 episodes in 79 patients. The age range was 2 days to 15 years (mean, 1.7 years). Haemophilus influenzae type b was the commonest organism (60 of 80 cases; 75%). There were five deaths (6.3%). Sensorineural hearing loss was found in six of 71 children (8.5%) and was bilateral and severe in four (5.6%). Other problems included learning difficulties (12.7%), motor problems (7%), speech delay (7%), hyperactivity (4.2%), blindness (2.8%), obstructive hydrocephalus (2.8%) and recurrent seizures (2.8%). Bacterial meningitis remains a disease with significant morbidity and mortality. Adjunctive therapy with dexamethasone should be considered, and vaccination against Haemophilus influenzae type b should be routine.


High dose chemotherapy and autologous bone marrow transplantation in advanced Hodgkin's disease. To present the use of high dose chemotherapy with autologous bone marrow transplantation as salvage therapy for advanced Hodgkin's disease in Australia. A prospective open study for patients whose disease was resistant to conventional treatment. The bone marrow transplantation units of four Australian tertiary hospitals. Seventeen patients (median age 30 years) entered and completed the study. The stage of the disease at initial diagnosis was I or II (seven patients), III (seven patients) and IV (three patients). Histological types were lymphocyte predominant (one), nodular sclerosis (12), mixed cellularity (three) and unknown (one). Therapy before consideration for transplantation included radiotherapy (13), mustine, vincristine, procarbazine and prednisone (MOPP--17 patients) or doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD--13 patients) and other chemotherapy regimens (five). The median interval from diagnosis to transplantation was 29 months (range, 9-178 months). The patient's disease was classified as sensitive (nine) or resistant (eight) to treatment, depending on the response to the most recent course of chemotherapy. Morphologically normal autologous bone marrow was harvested and cryopreserved. The conditioning regimen given was cyclophosphamide, carmustine and etoposide (14) or busulphan and cyclophosphamide (three). The marrow was then infused. Remission (complete or partial), disease-free survival and overall survival. Over all, 10 of 17 patients (59%) entered or remained in complete remission and four of 17 (24%) achieved partial remission. The overall actuarial survival at 30 months was 70%. Eight of the nine patients with treatment-sensitive disease (89%) remain disease-free at a median of 22 months (range, 18-29 months) after transplantation. Two of the eight patients with resistant disease (25%) are disease-free at 20 and 28 months. There was one procedure-related death from haemorrhage and four disease-related deaths at six, seven, eight and 13 months after transplantation. Autologous bone marrow transplantation may provide an effective salvage therapy in advanced Hodgkin's disease, particularly for patients with treatment-sensitive disease and a low tumour burden.


Signal transduction via the fibronectin receptor: do integrins regulate matrix remodeling? An appropriate balance of matrix synthesis and degradation is required for normal morphogenesis and maintenance of tissue architecture. Extracellular matrix molecules and their receptors, as well as proteinases and their inhibitors, are all involved in matrix remodeling. This report examines the idea that extracellular matrix receptors can regulate matrix remodeling. Rabbit synovial fibroblasts and human embryonic lung fibroblasts (MRC-5) were cultured under two sets of conditions. First, they were plated in serum and allowed to establish an extracellular matrix over a 48 h period. Rat monoclonal antibody to the alpha 5/beta 1 integrin fibronectin receptor or normal rat IgG was added to the medium and the expression of the metalloproteinases was examined. Cells treated with anti-alpha 5/beta 1 expressed procollagenase and prostromelysin, whereas the control cells did not. In both cases the cells were well spread and maintained a well-organized cytoskeleton. In the second condition, cells were plated in serum-free medium on intact fibronectin, anti-alpha 5/beta 1, or fragments of fibronectin that contained the cell-binding domain. Cells attached and spread on all these substrates in a fibronectin receptor-dependent manner. They expressed collagenase and stromelysin on anti-alpha 5/beta 1 and on several fibronectin fragments, but not on intact fibronectin. These data support the hypothesis that the fibronectin receptor can exist in more than one functional state and that these functional states provide information that influences gene expression. Adhesion and spreading are supported by all states, whereas only a subset permits collagenase and stromelysin expression.


Localization of cleavage sites for human fibroblast collagenase in the bait region of five mammalian alpha-macroglobulins. The interaction between human fibroblast collagenase and five mammalian alpha-macroglobulins (human alpha 2-macroglobulin and pregnancy zone protein, rat alpha 1- and alpha 2-macroglobulin and rat alpha 1-inhibitor 3) is discussed. Complex formation involves specific limited proteolysis in the alpha-macroglobulin bait regions, activation of the internal beta-cysteinyl-gamma-glutamyl thiol esters, conformational changes and covalent complex formation. For human alpha 2-macroglobulin, and rat alpha 1-macroglobulin and alpha 2-macroglobulin it is estimated that the overall rate constant of complex formation is greater than 1.10(6) M-1s-1, while it is much lower for human pregnancy zone protein and rat alpha 1-inhibitor 3. More than 95% of the complexed collagenase is covalently bound. The identification of the sites of specific limited proteolysis in the bait regions of the five alpha-macroglobulins shows that cleavage may take place in sequences that are not related to those identified earlier in the collagens. These results greatly expand the repertoire of sequences known to be cleaved by fibroblast collagenase, and suggest that this proteinase has a primary substrate specificity resembling that of the microbial proteinase thermolysin as it preferentially cleaves at the NH2-terminal side of large hydrophobic residues. In addition, the results highlight the unique structure of the flexible alpha-macroglobulin bait region in that it can accommodate a conformation required by the highly restrictive fibroblasts collagenase. It is suggested that alpha-macroglobulins may play an important role in locally controlling the activity of collagenases and perhaps other proteinases of the extracellular matrix.


Cytochromes P450 NMa, NMb (2G1), and LM4 (1A2) are differentially expressed during development in rabbit olfactory mucosa and liver. Mammalian olfactory mucosa has a high concentration of cytochrome P450 monooxygenases (P450). The major olfactory P450 isoforms in adult rabbits include P450 NMa, which is found in both olfactory and respiratory mucosa, as well as in liver at a low level, P450 NMb (2G1), which is olfactory specific, and P450 form 4 (1A2), which is found only in liver and olfactory mucosa. In the present study, we have found that the developmental expression of olfactory P450 in rabbits is not coordinated with the ontogenesis of hepatic P450. These three P450 isoforms were detected immunochemically and found to be at a relatively high level in olfactory but not hepatic microsomes in the first 2 weeks after birth. In the liver, NMb is not detectable at any age and NMa is not detectable until the fourth week. P450 1A2 is not detectable until the third week, but its level increases rapidly in the fourth week. These P450 isoforms are also detectable in prenatal olfactory tissue at 2 days before birth, indicating that direct exposure to air is not a prerequisite for their early expression in this tissue and that the early appearance of these enzymes may be controlled by both endogenous and environmental factors. In addition, the developmental expression of 2E1, a minor olfactory P450 isoform, also occurs earlier in olfactory mucosa than in liver, and the same conclusion can be made about the expression of NADPH-P450 reductase, which is detectable in olfactory microsomes but not in hepatic microsomes from prenatal rabbits. Thus, the regulatory mechanisms that control basal prenatal expression in the olfactory tissue may be common for multiple P450 isoforms and perhaps also for other biotransformation enzymes. The tissue-specific early onset of expression of multiple forms of P450 in olfactory tissue suggests that these enzymes may play an important role in the neonatal period, when olfactory ability is vital for the survival of the newborn. The presence of relatively high levels of biotransformation enzymes in the olfactory mucosa may also have important implications for neonatal inhalation toxicology.


Binding kinetics of gamma-aminobutyric acidA receptor noncompetitive antagonists: trioxabicyclooctane, dithiane, and cyclodiene insecticide-induced slow transition to blocked chloride channel conformation. Binding kinetics and affinities are determined for 25 antagonists interacting with the noncompetitive blocker site of the gamma-aminobutyric acidA receptor complex present in bovine brain membranes. Four radiolabeled noncompetitive antagonists are 4-tert-butylbicyclophosphoro[35S]thionate ([35S]TBPS), 4-tert-butylbicycloortho[3',4'-3H2]benzoate, 4'-cyano-4-sec-[3,4-3H2]butylbicycloorthobenzoate, and the new 4'-ethynyl-4-n-[2,3-3H2]propylbicycloorthobenzoate. The other 21 antagonists are unlabeled inhibitors of three chemical classes (other trioxabicyclooctane, dithiane, and cyclodiene insecticides). The radioligands bind to a single noninteracting site in the membranes, based on linear Scatchard plots and monophasic association and dissociation kinetics. The kinetics of unlabeled ligands are estimated by their effect on the [35S]TBPS association curve, using the theoretical model of Motulsky and Mahan [Mol. Pharmacol. 25:1-9 (1984)]. The receptor affinities of trioxabicyclooctanes and dithianes correlate with their association rates, whereas those of cyclodienes correlate with their dissociation rates. The low association rate constants for all ligands (< or = 3 x 10(7) M-1 min-1 at 25 degrees) are consistent with a slow transition to a blocked receptor conformation upon binding of these channel blockers. The association rate-controlled affinity for the trioxabicyclooctanes and dithianes is suggestive of an induced-fit model in which binding of the ligand initiates a conformational change in the receptor complex to the blocked state.


Mechanism of anti-human immunodeficiency virus action of polyoxometalates, a class of broad-spectrum antiviral agents. Various polyoxometalates proved inhibitory to the replication of a number of enveloped DNA and RNA viruses, i.e., herpesviruses (herpes simplex and cytomegalo), togaviruses (Sindbis), paramyxoviruses (respiratory syncytial), rhabdoviruses (vesicular stomatitis), arenaviruses (Junin and Tacaribe), and retroviruses [human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), simian immunodeficiency virus, and murine sarcoma virus]. The most potent compounds, i.e., JM1590 [K13[Ce(SiW11O39)2]. 26H2O] and JM2766 [K6[BGa(H2O)W11O39]. 15H2O], inhibited HIV-1 and simian immunodeficiency virus at concentrations as low as 0.008-0.8 microM. The polyoxometalates also inhibited giant cell formation in co-cultures of HIV-infected HUT-78 cells and uninfected MOLT-4 cells. Studies designed to unravel the mechanism of action of these compounds revealed that they inhibit the reverse transcriptase activity associated with HIV. The polyoxometalates also proved inhibitory to the binding of HIV-1 virions to the cells. From "time of addition" experiments, whereby the polyoxometalates were added at different times after virus infection, their mechanism of anti-HIV action could be attributed to inhibition of virus-cell binding. There was a good correlation (r = 0.84) between the inhibitory effects of the compounds on HIV-1-induced cytopathicity and their inhibitory effects on syncytium formation and a close correlation (r = 0.902) between their inhibitory effects on syncytium formation and their interaction with gp120, whereas there was no correlation between their anti-HIV-1 activity and their inhibitory effects on HIV-1 reverse transcriptase. In flow cytometric studies, the compounds did not interfere with the binding of OKT4A/Leu-3a monoclonal antibody to the CD4 receptor of uninfected cells, but they inhibited binding of anti-gp120 monoclonal antibody to HIV-1-infected cells. Thus, the binding of the polyoxometalates to the viral envelope glycoprotein gp120 is responsible for their anti-HIV activity.


Effects of a benz[e]indene on gamma-aminobutyric acid-gated chloride currents in cultured postnatal rat hippocampal neurons. Benz[e]indenes (BIs) are tricyclic molecules that can be envisioned as steroids without an A-ring. Because certain steroids are known to alter gamma-aminobutyric acid (GABA) responses in central neurons, we examined the effects of a substituted BI resembling 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP) on GABA-gated chloride currents in cultured postnatal rat hippocampal neurons. The compound, BI-1, reversibly potentiated GABA currents at concentrations of > 10 nM, with an EC50 value of 0.2 microM. BI-1 increased the apparent affinity of GABA for its receptor, decreasing the GABA EC50 from 9 microM to 3 microM. BI-1 had no effect on the shape of the GABA current-voltage relationship and did not alter the GABA reversal potential. The effects of BI-1 were not altered by benzodiazepine or picrotoxin site antagonists. At concentrations up to 10 microM, where maximal effects on GABA currents were seen, BI-1 did not directly activate a membrane current. This contrasts with the effects of 3 alpha-OH-DHP, which activated chloride currents at concentrations that were subsaturating for GABA potentiation. These results suggest that the BIs may be useful for determining the mechanisms by which steroids potentiate GABA responses and directly gate chloride channels.


Myocardial fatty acid oxidation during ischemia and reperfusion. Inhibition of fatty acid oxidation is an early event in myocardial ischemia that most likely contributes to tissue injury by the accumulation of potentially toxic intermediates such as acylCoA and acylcarnitine. After reperfusion both myocardial oxygen consumption and fatty acid oxidation may rapidly recover to preischemic levels, even when contractile function remains depressed. The mechanisms underlying the apparent dissociation between contractile function and oxidative metabolism early during reperfusion are still controversial. In isolated rat hearts subjected to 60 min of no-flow ischemia myocardial oxygen consumption and oxidation of palmitate were lowered during reperfusion by 3 mM of NiCl2 and by 6 microM of ruthenium red. The results provide indirect evidence for the hypothesis that intracellular calcium transport may be involved in the mechanisms responsible for the high oxidative metabolic rate early after reperfusion.


Effects of palmitoyl CoA and palmitoyl carnitine on the membrane potential and Mg2+ content of rat heart mitochondria. Palmitoyl CoA and palmitoyl carnitine added to rat heart mitochondria in amounts above 20 and 50 nmoles/mg protein, respectively, induced a fall in transmembrane potential and loss of endogenous Mg2+. The dissipation of membrane potential by low concentrations of palmitoyl CoA in the presence of Ca2+, but not that of high concentrations of palmitoyl CoA alone, was prevented by either ruthenium red, Cyclosporin A or Mg2+, but reversed only by Mg2+. The fall of membrane potential induced by palmitoyl carnitine was not prevented by any of these factors. It is suggested that the action of both palmitoyl CoA and palmitoyl carnitine at high concentrations is due to a non specific disruption of membrane architecture, while that of low concentrations of palmitoyl CoA in the presence of Ca2+ is associated specifically with energy dissipation due to Ca2+ cycling.


Identification of a gonadotropin-releasing hormone-responsive region in the glycoprotein hormone alpha-subunit promoter. Transcriptional regulation of the glycoprotein hormone alpha-subunit gene has been studied extensively in placental cells, but much less is known about its regulation in the pituitary gland. In this study, transcriptional control of the human alpha-subunit gene by GnRH was analyzed using transient expression assays in primary cultures of rat pituitary cells using alpha promoter constructs linked to a luciferase reporter gene. Deletion mutants between -846 and -156 basepairs (bp) had little effect on basal expression, but further deletion to -132 bp reduced basal activity by approximately 50%. Deletion of a cAMP response element between -132 and -99 bp caused a marked loss of basal activity, reducing expression to that of background luciferase activity. The same constructs were analyzed for cAMP responsiveness in primary pituitary cells. The degree of stimulation with 1 mM 8-bromo-cAMP (3.6- to 6.0-fold) was relatively unaffected by deletions from -846 to -132 bp, whereas cAMP stimulation was decreased by further deletion to -99 bp, consistent with the presence of previously defined cAMP response elements in this region of the alpha promoter. GnRH stimulation of alpha promoter activity was highly dependent upon the time of hormone addition after transfection, being most effective when added soon after transfection. Under optimal conditions, GnRH stimulated -846 alpha LUC expression by 20-fold. GnRH responsiveness was retained with deletion to -346 bp, but it was decreased by 55% after deletion to -280 bp and by 79% with deletion to -244 bp.(ABSTRACT TRUNCATED AT 250 WORDS)


Posttranscriptional regulation of albumin and alpha-fetoprotein messenger RNA by transforming growth factor-beta 1 requires de novo RNA and protein synthesis. Transforming growth factor-beta (TGF beta) has been implicated in the regulation of hepatocyte function. We have examined TGF beta 1 regulation of albumin and alpha-fetoprotein (AFP) mRNA levels in a well differentiated mouse hepatoma cell line (BWTG3). TGF beta 1 reversibly decreased steady state mRNA levels of both albumin and AFP. By nuclear run-on assays, we found that TGF beta 1 caused no significant change in transcription rates for albumin or AFP. Pretreatment with actinomycin-D prevented the TGF beta 1-induced decrease in albumin and AFP mRNA levels. Also, if cells were treated with actinomycin-D after a 12-h exposure to TGF beta 1, actinomycin-D abrogated the further decrease in albumin and AFP mRNA levels that occurred after treatment with TGF beta 1 alone. Cycloheximide pretreatment blocked the TGF beta 1-induced decrease in albumin and AFP mRNA levels. TGF beta 1 altered neither the rate of BWTG3 cell growth nor the levels of mRNA for the growth-associated protooncogene c-myc. These data suggest that TGF beta 1 has regulatory effects on specific hepatocyte functions that are independent of growth regulatory effects. The decrease in albumin and AFP mRNAs caused by TGF beta 1 is posttranscriptional and dependent upon de novo RNA and protein synthesis.


Endothelial cells express insulin-like growth factor-binding proteins 2 to 6. Cultured endothelial cells have been shown to produce insulin-like growth factor-binding proteins (IGFBPs); however, the identity of these BPs has not been defined. We now demonstrate that cultured bovine endothelial cells produce IGFBP2, IGFBP3, and IGFBP4 and have mRNA specific for IGFBP2, -3, -4, -5 and -6. DNA probes for bovine IGFBP2-6 were obtained by polymerase chain reaction (PCR) amplification of cDNA from bovine large vessel pulmonary artery and aortic endothelial cells as well as omental and periaortic fat microvessel cells, using oligonucleotide primers whose sequences were based on the reported cDNA sequences of IGFBP2-6. The PCR-derived probes were labeled with 32P and used for Northern blot analysis of RNAs obtained from the four bovine endothelial cell types. Transcripts corresponding to IGFBP2-6 were found in RNA from large vessel endothelial cells (bovine pulmonary artery and bovine aorta) and microvessel cells (periaortic and omental fat). The PCR-derived probe for IGFBP4 was used to screen a bovine pulmonary artery cDNA library for a full-length bovine IGFBP4 cDNA clone. One positive clone, containing a single EcoRI insert of approximately 2.0 kilobases, was selected for further characterization by DNA sequence analysis. This clone contained an open reading frame encoding a 258-amino acid protein that was 97% identical to human IGFBP4, 268 basepairs of 5'-untranslated region, and a longer 1044 basepairs of 3'-untranslated region. IGFBP4 protein was purified from bovine pulmonary artery-conditioned medium, shown to have N-terminal amino acid sequence DEAIHCPPCSEEKLARCR (identical to human IGFBP4) and to be secreted in glycosylated and nonglycosylated forms. Immunoblots further demonstrated that microvessel cells, at early passage, secrete predominantly IGFBP2 and IGFBP3, while large vessel cells, at early and late passages, secrete IGFBP3 and IGFBP4. Thus, cultured bovine endothelial cells synthesize and secrete IGFBP2, IGFBP3, and IGFBP4 and have mRNA encoding IGFBP2-6. The production of specific IGFBPs by endothelial cells raises the interesting possibility that the vascular endothelium contributes to circulating and tissue levels of specific IGFBPs in vivo.


Cholera toxin-sensitive 3',5'-cyclic adenosine monophosphate and calcium signals of the human dopamine-D1 receptor: selective potentiation by protein kinase A. The signal transduction pathways of the dopamine-D1 receptor were investigated in two cell types stably transfected with the human D1 receptor cDNA, rat pituitary GH4C1 cells (GH4-hD1), and mouse Ltk-fibroblast cells (L-hD1). In both GH4-hD1 and L-hD1 cell lines, stimulation of the dopamine-D1 receptor induced a marked increase in cAMP accumulation. In addition, dopamine potentiated activation of L-type voltage-dependent calcium channels in a cAMP-dependent manner in GH4-hD1 cells. However, in L-hD1 cells, dopamine increased cytosolic free calcium concentrations ([Ca++]i) by mobilization of intracellular calcium rather than by calcium influx. This effect was correlated with a dopamine-induced enhancement of phospholipase C activity in L-hD1 cells. Pretreatment (24 h) with cholera toxin (CTX) was used to maximally activate the GTP-binding protein (G protein) Gs, causing a maximal elevation of cAMP levels and uncoupling the D1 receptor from Gs. The described actions of dopamine in both cell lines were abolished by pretreatment with CTX, indicating that CTX substrates (e.g. Gs) may mediate these actions. The blockade by CTX was not due to CTX-induced elevation of cAMP, since pretreatment with forskolin or 8-bromo-cAMP to activate cAMP-dependent protein kinase did not inhibit dopamine actions nor alter basal [Ca++]i. Pretreatment (1-3 h) of L-hD1 cells with forskolin (10 microM) or 8-bromo-cAMP (5 mM) altered neither the basal activity of phospholipase C nor basal [Ca++]i in L-hD1 cells but greatly enhanced the dopamine-induced increase of phosphatidyl inositol turnover and [Ca++]i. From these results we conclude that: 1) the dopamine-D1 receptor induces multiple and cell-specific signals, including elevation of cAMP levels in both GH and L cells, cAMP-dependent activation and potentiation of opening of L-type voltage-dependent calcium channel in GH cells, and a novel phosphatidyl inositol-linked mobilization of cellular calcium in L cells; 2) coupling of the D1 receptor to these responses involves CTX-sensitive proteins, possibly Gs; and 3) acute preactivation of cAMP-dependent protein kinase can markedly enhance, rather than attenuate, certain pathways of dopamine-D1 transmembrane signaling.


Beta 1 isoform-specific regulation of a triiodothyronine-induced gene during cerebellar development. Although tissue-specific expression of the alpha 1 and beta 1 thyroid hormone receptors (TR-alpha 1 and TR-beta 1) suggests isoform-specific function, transfection studies to date have failed to show consistent differences in their ability to regulate gene expression. We here provide evidence that TR-beta 1 but not TR-alpha 1 regulates the expression of the gene coding for PCP-2 in cerebellar Purkinje cells during neonatal rat development and that such regulation appears to be both T3 dependent and T3 independent. Examination of neonatal rats revealed that the levels of three mRNAs expressed in cerebellar Purkinje cells (myoinositol-1,4,5-triphosphate receptor, calbindin, and PCP-2) rise from neonatal day 1 to day 15. This rise is preceded by the previously documented surge in brain T3 and TR-beta 1. Methimazole-induced hypothyroidism sharply reduces, but does not abolish, the rise in these mRNAs. Concomitant T3 administration normalizes the process. In order to establish more directly the role of TR-beta 1 and T3, cotransfection experiments were performed in CHO cells with PCP-2-lacZ construct and TR isoforms. These studies showed that TR-beta 1, even in the absence of T3, regulated the expression of the transfected PCP-2 construct. T3 augments the response to TR-beta 1 alone by 40% (P < .01). TR-alpha 1 had no effect on PCP-2-lacZ expression either in the presence or absence of T3.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of growth hormone on levels of differentially processed insulin-like growth factor I mRNAs in total and polysomal mRNA populations. As a result of multiple transcription initiation sites and differential splicing involving the first exons and alternate exon use, many insulin-like growth factor I (IGF-I) mRNA species are produced. In the present study, we have assessed the effect of GH on transcription start site usage and splicing and have determined the apparent in vivo translatability of IGF-I mRNAs with different 5'-untranslated region (UTR) sequences by comparing their abundance in total and polysomal RNA fractions from control, hypophysectomized, and GH-treated hypophysectomized rats. Hypophysectomy decreased the level of all IGF-I mRNA species, but those initiated at start site 3 in exon 1 and the major start site in exon 2 were preferentially reduced. These same variants were preferentially increased by GH treatment. Under all conditions, exon 1 mRNAs with shorter 5'-UTR sequences were enriched in polysomal RNA at the expense of IGF-I mRNAs with long 5'-UTR sequences, in accordance with the scanning model of translation initiation. Exon 2-derived mRNAs, on the other hand, which have short 5'-UTR sequences, were not enriched on polysomes, suggesting that some aspect of the exon 2-derived 5'-UTR other than length influences translation in vivo. These results demonstrate that transcription within exon 1 and between exons 1 and 2 is differentially regulated by GH status and that the variant IGF-I mRNA species resulting from the complex patterns of transcription initiation and splicing in these leader exons are differentially translated in vivo.


Role of nitric oxide in antagonistic effects of transforming growth factor-beta and interleukin-1 beta on the beating rate of cultured cardiac myocytes. We have recently shown that transforming growth factor-beta (TGF beta) acts in an autocrine manner to maintain the beating rate of neonatal rat cardiac myocytes cultured in serum-free medium on cardiac fibroblast matrix. Interleukin-1 beta (IL-1 beta) suppresses the myocyte-beating rate, and TGF beta antagonizes this effect. We now show that TGF beta and IL-1 beta also have antagonistic effects on the secretion of nitric oxide (NO) by these myocytes, and that NO secretion, the activity of NO synthase (NOS), and expression of the inducible form of NOS correlate inversely with the effects of these two agents on the beating rate. Western blot analysis shows that treatment of myocytes with TGF beta antagonizes the induction of NOS after treatment with IL-1 beta. Release of NO, induced by IL-1 beta, is dependent upon the availability of the substrate, L-arginine, and is suppressed by a competitive inhibitor, NG-monomethyl-L-arginine. L-Arginine (> 0.25 mM) also suppresses, and NG-monomethyl-L-arginine (> 0.5 mM) enhances the myocyte-beating rate. Treatment with IL-1 beta, but not TGF beta, increases cellular cGMP, presumably by activation of guanylate cyclase by NO. Methylene blue, an inhibitor of guanylate cyclase, reverses the suppression of beating caused by IL-1 beta. Bacterial lipopolysaccharide, present in the serum-free medium, is a coinducer of NO secretion. The suppressive effects of NO on the beating rate can be overcome by altering either the set of cytokines employed to induce NO or the matrix on which the myocytes are cultured, demonstrating that additional parameters are also involved in regulation of the beating rate.


Growth inhibition of glioma cells by liposome-mediated cell transfection with tumor necrosis factor-alpha gene--its enhancement by prior gamma-interferon treatment. A new approach to the treatment of malignant glioma is cytokine gene therapy to produce growth inhibitors in cells. Previously, we showed that human glioma cells selectively transfected with the gene of interferon (IFN)-beta and/or IFN-gamma by our novel liposomes tagged with monoclonal antibody against a glioma-associated antigen achieved a remarkable growth inhibition effect. In the present experiment, we demonstrated the effectiveness of gene therapy against glioma cells using liposomes bearing a plasmid containing the gene for tumor necrosis factor (TNF)-alpha. We also found that the effect of endogenous TNF-alpha was enhanced by treatment of IFN-gamma prior to the transfection with the TNF-alpha gene.


Appearance of collagen fibers in the cerebral vascular wall following subarachnoid hemorrhage. Ultrastructural examinations of the cerebral vascular wall following subarachnoid hemorrhage (SAH) demonstrated that collagen fibrils developed in the muscle layer near the adventitia in the early stage of SAH and increased in number and volume with time. These findings suggest that accelerating factors of collagen synthesis in the muscle layer may be released from the adventitial side, and collagen synthesis is induced by extravascular factors. The changes of collagen volume with time suggest collagen to be an important phenomenon of persistent vasospasm after SAH. Histoimmunological studies showed that increased collagen fibers in the tunica media were mainly type III collagen, implying that cerebral vasospasm may be related to chemical inflammation.


Worldwide distribution of moyamoya disease. Many cases of spontaneous occlusion of the circle of Willis (moyamoya disease) have been reported in Japan, but the worldwide distribution is unclear. The total number of cases in each country reported in the literature and complications were evaluated. A total of 1063 cases have occurred worldwide excluding Japan. Cases were observed most frequently in Asia and in people of non-Caucasian origin.


Nontraumatic intradiploic arachnoid cysts--report of five cases. Five cases of nontraumatic intradiploic arachnoid cysts in elderly patients are reported. All cysts were located in the occipital bone and appeared as well-demarcated radiolucent lesions. The cysts were multiple in three cases. Presenting symptoms included headache or dizziness, but most lesions were asymptomatic and found incidentally. In the most recent three cases, magnetic resonance (MR) imaging revealed intradiploic cysts containing cerebrospinal fluid (CSF) with cerebellar herniation. Operation found the cysts filled with CSF and dural defects through which cerebellar tissue was herniating. In two patients, CSF leakage from the outer table occurred. Intradiploic arachnoid cyst seems to be congenital in origin but commonly found in the elderly. MR imaging is the most useful diagnostic method for differential diagnosis from other osteolytic skull lesions.


Recurrent brain abscess associated with hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber disease)--case report. A 53-year-old mentally handicapped female with hereditary hemorrhagic telangiectasia complicated by pulmonary arteriovenous fistula developed a recurrent brain abscess in the left frontal region. She responded well to surgical drainage and antibiotic therapy. Previously, she had two brain abscesses of the left parieto-occipital and right parietal regions removed on separate occasions at 38 and 40 years of age. Although pulmonary arteriovenous fistula was eventually confirmed by angiography at age 41 years, surgical removal of the pulmonary lesion was not indicated due to multiplicity and large vascular shunting. Early recognition of pulmonary arteriovenous fistula in patients with hereditary hemorrhagic telangiectasia is emphasized for diagnosis of brain abscess and prevention of recurrence.


Traumatic internal carotid artery occlusion--case report. A 20-year-old male was admitted comatose immediately after a motorcycle accident. Initial computed tomography demonstrated traumatic subarachnoid hemorrhage, and the diagnosis of traumatic internal carotid artery occlusion was established by angiography. Conservative management improved his symptoms, but eventually he died from delayed traumatic apoplexy. Traumatic internal carotid artery occlusion is relatively rare, but is serious and requires early diagnosis and treatment. For patients with severe head trauma and vascular occlusion, anticoagulants are contraindicated, and frequent follow-up angiography is recommended.


Moyamoya disease progressing from unilateral to bilateral involvement--case report. A 2-year-old boy with unilateral moyamoya disease demonstrated progression to the bilateral condition 1 year later. Previous reports of progression from unilateral to bilateral moyamoya disease showed that all patients were children or relatively young, and developed ischemic symptoms as the initial manifestation. These findings suggest that a considerable proportion of unilateral pediatric cases are in the initial stage of moyamoya disease and will progress to the contralateral side in the future.


Intracranial mycotic aneurysm caused by Aspergillus--case report. A 75-year-old female with chronic renal failure diagnosed as Wegener's granulomatosis was receiving steroids and immunosuppressive agents when subarachnoid hemorrhage developed. Cerebral angiography showed a fusiform aneurysm arising from an angular branch of the left middle cerebral artery. Hemorrhage occurred and the aneurysm was excised by emergency surgery. Microscopic examination of the aneurysm revealed dense infiltration of hyphae identified as Aspergillus. She died of subsequent hemorrhage. Autopsy showed numerous Aspergillus hyphae in the lung. Fungal mycotic aneurysm should be considered in the differential diagnosis of an immunocompromised patient with subarachnoid hemorrhage.


Skull base metastasis from gastric cancer--case report. A 48-year-old male presented with a metastatic skull base tumor located on the right of the clivus and the sphenoid sinus, which originated from adenocarcinoma of the stomach. The initial symptom was abducens nerve paralysis and there were no symptoms of upper gastrointestinal tract throughout the course. This gastric cancer, which rarely metastasizes to the central nervous system or osseous system, caused multiple bone metastases which produced the neurological symptoms.


Anaplastic astrocytoma of an oncocytic type occurring in the cerebellar vermis in Pierre Robin syndrome--case report. A 44-year-old male with Pierre Robin syndrome and funnel chest was diagnosed with a tumor of the cerebellar vermis and spontaneous pneumothorax. He received tube thoracostomy for pneumothorax and ventriculoperitoneal shunt for hydrocephalus, followed by radiological examination, subtotal removal of the tumor, pneumonorrhaphy, and chemoradiotherapy. Light microscopy of the tumor sample revealed marked pleomorphism of the tumor cells and numerous giant cells, without mitotic figures. Microcystic changes due to vasogenic edema were also evident throughout the tumor. Electron microscopy showed more than 50% of all tumor cells to be oncocytes, with numerous mitochondria in the cytoplasm. The tumor was diagnosed as anaplastic astrocytoma of an oncocytic type.


Innervation of the feline eyelids.

Innervation of the feline eyelids. The innervation of the eyelids is incompletely understood. This is a particular problem for those who wish to develop animal models of eyelid dysfunction in humans. Blepharospasm, for example, is a disease of uncontrolled eyelid spasm that is difficult to manage clinically because the aetiology is not understood. The anatomical literature on eyelid innervation is sparse and even conflicting. We attempted to study eyelid innervation, both sensory and motor, with injection of horseradish peroxidase (HRP) into the superior eyelid, inferior eyelid, and bulbar conjunctiva. We used 13 anesthetized weanling cats. Shape and structure of the facial nucleus varied along its rostrocaudal extent, but there was a clear demarcation of lateral and medial division. HRP-filled facial nucleus cells were ipsilateral to the injection site, and label appeared throughout the rostrocaudal length. All injection sites, including bulbar conjunctiva, labelled facial nucleus neurons located with overlapping distribution, predominantly in the dorsal part of the lateral division. Likewise, heavy labelling occurred throughout the entire ipsilateral cranial cervical ganglion and the trigeminal ganglion in all kittens. Injection of upper or lower eyelids caused some labelling in the second through the fourth cervical spinal ganglia.


Isolation of complementary DNAs for heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 genes and the expressions in post-ischaemic gerbil brain. A complementary DNA (cDNA) library was constructed with a plasmid vector from cerebral cortices of gerbils at 8 h of reperfusion after 10 min of bilateral common carotid ligation. After the 3rd screening of this cDNA library with a human genomic DNA probe for HSP70 (pH2.3), 4 cDNA clones were isolated (named pGA3' pGB1' pGD3 and pGE4' respectively). Southern and Northern blot analysis, and partial nucleotide sequence analysis indicated that pGA3 and pGE4 were the HSP70 cDNA clones, and that pGB1 and pGD3 were the HSC70 cDNA clones, which selectively recognized HSP70 or HSC70 mRNA, respectively. HSP70 mRNA is present in a very small amount in normal controls, and is greatly induced after the transient ischaemia. HSC70 mRNA is constitutively expressed in a normal gerbil brain, but is still inducible. In situ hybridization study demonstrated that the HSP70 mRNA was present in a very small amount in the hippocampal pyramidal and dentate granule cells in the sham control, and that the mRNA was greatly induced in the cells of hippocampus, dentate gyrus, medial habenula, ventral thalamic nuclei, caudate putamen, ventromedial and arcuate hypothalamic nuclei, amygdaloid nuclei and cerebral cortex after 8 h of reperfusion. HSC70 mRNA was present in almost the same areas of sham control, and was slightly induced after 8 h of reperfusion. Our results indicate that HSP70 and HSC70 cDNA clones were first isolated from post-ischaemic gerbil brain, and selectively recognize HSP70 or HSC70 mRNA, respectively. A regional difference in the induction of the HSP70 and HSC70 mRNA in post-ischaemic gerbil brain was observed by in situ hybridization.


Increased opening of blood-tumour barrier by leukotriene C4 is dependent on size of molecules. Intracarotid injections of leukotriene C4 (LTC4) will increase blood-tumour barrier (BTB) permeability for 14C aminoisobutyric acid (AIB) in RG-2 tumours in rats. In this study, rats with RG-2 tumours were given 15 minute intracarotid infusions of LTC4 (5 micrograms total dose). Blood-tumour and BBB permeability were determined by quantitative autoradiography using different sized 14C tracers; AIB (103.1 daltons, radius = 2.8 A), Sucrose (342 daltons, radius = 5 A), Inulin (5000 daltons, radius = 15 A) and Dextran (70,000 daltons, radius = 60 A), respectively (n = 7 in each group). Permeability (Ki) within tumours for AIB, Sucrose and Inulin increased significantly after LTC4 infusion (92.7 +/- 15.0 vs 39.4 +/- 6.5, 47.8 +/- 5.2 vs 29.1 +/- 8.8, and 46.4 +/- 8.4 vs 26.4 +/- 4.8 microliters/gm/min, respectively, p < 0.01) (mean +/- SD). No significant change in Ki for 14C Dextran was observed. Increased permeability within tumours after LTC4 infusion is dependent on molecular size. This suggests that LTC4 opens tight junctions rather than increases vesicular transport through endothelial cells.


Polymerase chain reaction assay of mRNA using 28S rRNA as internal standard. A method is developed for PCR assay of mRNA using 28S rRNA as internal standard and its applicability is reported for 4 types of mRNA. It consists of reverse transcription of total RNA using primers selective for 28S rRNA and the message of interest. Routinely the 28S reaction mix is diluted 20,000-fold and the dilution error is minimized by adding 10 microCi of 3H2O before the dilution and determining the level of 3H before and after the dilution. Aliquots from the diluted 28S mix and the undiluted reverse transcripts are co-amplified by PCR in the presence of 32P-dATP. The samples are analyzed by electrophoresis, bands detected by autoradiography and in each lane the ratio is determined between cpm in the band of interest and cpm in the 28S band. The ratio is a measure of the level of mRNA in the sample and can be used to monitor changes in mRNA during treatments, development and pathogenesis.


Immunoblot detection of antibodies to myelin basic protein in Alzheimer's disease patients. Based on a suspected pathological relationship of autoimmunity in Alzheimer's disease (AD), antibodies to myelin basic protein (MBP) were investigated in the sera of AD patients, healthy controls and disease controls. As detected by the protein-immunoblotting technique at a screening serum dilution of 1:400, sera from 16 of 18 (89%) AD patients were positive for antibodies to MBP. In contrast, sera from only 7 of 90 controls (healthy adults and elderlies, Parkinson's disease patients, mentally-retarded children and Down's syndrome patients) showed a positive reaction. This approximately 11-times higher incidence of antibodies to MBP in AD patients was statistically significant (P < or = 0.0001) and may indicate an autoimmune process in the pathophysiology of the disease.


Origin and distribution of NADPH-diaphorase-positive neurons and fibers innervating the urinary bladder of the rat. Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry was utilized to localize nitric oxide synthase (NOS), and thus sites where nitric oxide (NO) can be synthesized, within peripheral nervous system perikarya and fibers. Recent studies suggest that NO relaxes vascular and non-vascular smooth muscle. In this study, the origin and distribution of NADPH-diaphorase perikarya and fibers in the rat urinary bladder were examined. Results suggest that a small number of NADPH-diaphorase-positive perikarya are present within the bladder wall and within adjacent small ganglia. In addition, NADPH-diaphorase-positive nerve fibers were observed in the adventitial and muscular layers, subjacent to the urothelium and as perivascular fibers. After injection of the retrograde tracer fluorogold (FG) into the bladder wall, numerous FG-labeled perikarya in the major pelvic ganglia and the T13-L2, L6 and S1 dorsal root ganglia were NADPH-diaphorase positive. However, none of the FG-labeled perikarya in the inferior mesenteric ganglia were NADPH-diaphorase positive. The prevalence of NADPH-diaphorase-positive perikarya and fibers suggests that NO may serve a role in bladder function.


Insect glial cells show differential expression of a glycolipid-derived, glucuronic acid-containing epitope throughout neurogenesis: detection during postembryogenesis and regeneration in the central nervous system of Tenebrio molitor L. The monoclonal antibody CAF-1 recognises a glucuronic acid-containing epitope present on insect acidic glycolipids. Immunohistochemical analysis of the CAF-1 epitope has revealed its differential, temporal and spatial expression during postembryogenesis of the midbrain of Tenebrio molitor. Electron microscopic resolution demonstrated, that the CAF-1 epitope is expressed on glial cells that ensheath the glomeruli of the central body. Concomitantly, a differential pattern of expression was observed in the ventral nerve cord, exhibiting a serially homologous display on glial cells that ensheath neuronal somata in the cell body layer of the thoracic ganglia and ventral associative neuropil. Prominent, topologically restricted CAF-1 immunoreactivity was monitored in termination areas of sensory neurons in the ventral associative neuropil and corresponding nerves 6-48 h after extirpation of the respective sensory neuron somata. CAF-1 expression is correlated with structural reorganisation in postembryonic nervous tissue of T. molitor.


Nitric oxide synthase in rat brain is predominantly located at neuronal endoplasmic reticulum: an electron microscopic demonstration of NADPH-diaphorase activity. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) of the rat brain, apparently identical with nitric oxide (NO) synthase, was demonstrated at the electron microscopic level by means of the tetrazolium salt 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)tetrazolium chloride (BSPT). BSPT is a non-osmiophilic compound that yields an insoluble, osmiophilic, and lipophobic formazan on reduction. The reaction product was deposited sharply on membranes of the endoplasmic reticulum including the nuclear envelope. Other membrane structures were, as a rule, free of reaction product, likewise mitochondria. Occasionally, however, the outer membrane of mitochondria was labeled, and their contents displayed a homogeneous, medium electron density. The findings suggest that NADPH-d, i.e. neuronal NO synthase, is a predominantly membrane-bound enzyme, which is ubiquitously distributed in cells of brain tissue, but highly concentrated in nerve cells described as 'NADPH-d-positive' at the light microscopic level.


cAMP-mediated second messenger mechanisms are involved in spinule formation in teleost cone horizontal cells. A number of light adaptive changes of teleost horizontal cells are mediated by dopamine D1 receptors coupled positively with the cAMP second messenger system. Spinules, finger-like extensions from horizontal cell dendrites directed towards the cone pedicle cytoplasm, are formed in response to a stimulation of D1 receptors. We studied the second messenger mechanism associated with this process using isolated dark-adapted cyprinid retinae. Increasing intracellular cAMP concentrations by adding a membrane permeable analogue, or by stimulating the adenylate cyclase and simultaneously blocking the degradation of cAMP, resulted in a significant increase of spinule numbers in spite of the absence of light. In contradistinction to using isolated retinae for pharmacological experiments, injection of drugs into the vitreous had inconsistent or negative results.


Phakomatous choristoma of the eyelid. Immunohistochemical and electron microscopic observations. A 13-month-old Hispanic boy underwent excision of a congenital inferonasal orbital mass arising from the right lower lid. Results of histopathologic examination of the tumor showed a phakomatous choristoma of the eyelid. An immunohistochemical and electron microscopic study of this rare, benign, congenital tumor of lenticular anlage was performed. Immunohistochemistry was performed on 4-microns thick sections from paraffin-embedded tissue. Electron microscopy was performed on thin sections stained with uranyl acetate and lead citrate. The cuboidal epithelial cells that comprise this choristoma showed strongly positive cytoplasmic staining with S-100 protein and vimentin and focally positive staining with a keratin cocktail (AE1/AE3). Electron microscopy showed the presence of numerous 10-nm whorled cytoplasmic microfilaments within degenerating epithelial cells. The immunoreactivity of this tumor to keratin and vimentin are newly described in this detailed clinicopathologic report and, together with its S-100 positivity, support the proposal that this tumor is of lenticular anlage. The authors hypothesize that the intracytoplasmic 10-nm intermediate filaments observed with electron microscopic examination within the epithelial cells that comprise this choristoma represent vimentin as detected by immunohistochemistry.


Quantitative determination of morphometric indices of the total and perfused capillary network of the newborn pig brain. The purpose of this study was to determine if the newborn pig brain had a reserve of unperfused capillaries during normoxia. To accomplish this, a method was developed to determine the volume fraction, surface area, and number of both total and perfused capillaries in the newborn pig brain. Newborn pigs of either sex, 2-7 d old, were used. FITC-dextran, molecular weight 147,000, was used as a plasma marker to visualize the perfused capillaries. Alkaline phosphatase staining was used to stain the total capillary bed of the brain. Our results showed that FITC-dextran stayed within the vascular compartment, as it was not seen in areas that were not subsequently visualized with alkaline phosphatase staining. Eighty-four to 86% of the alkaline phosphatase-stained capillaries could be visualized with perfusion markers (india ink or FITC-dextran) in different brain regions. Similar results were obtained in two animals using a basement membrane stain, silver methenamine. The total volume fraction of capillaries (mm3/mm3) was cortex 0.055 +/- 0.012, cerebellum 0.062 +/- 0.011, and medulla 0.039 +/- 0.012. Capillary surface area (mm2/mm3) of different brain regions averaged cortex 23.2 +/- 1.8, cerebellum 24.8 +/- 2.5, and medulla 15.8 +/- 2.9. The total number of capillaries (per mm2) was cortex 375 +/- 37, cerebellum 329 +/- 37, and medulla 216 +/- 32. The time course of filling of the capillaries indicated that approximately 50% were perfused at 6 s, which increased to over 80% at 12 s and remained unchanged thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)


[Influence of the inflammatory reaction on the distribution of alpha1-macroglobulin and pregnancy-associated alpha1-glycoprotein in the rat]. The authors studied the distribution of proteinase inhibitors in inflammation induced by intramuscular turpentine injection. It was found that it reduces the concentration of the studied proteins in the tissues, which is attended by an increase of their plasma levels. The store of pregnancy-associated alpha 1-glycoprotein in the tissues is rapidly depleted, while the concentration of alpha 1-microglobulin reduces to a relatively stable level. The role of the both proteins in the inflammatory process is discussed.


[Content of trace elements and steroid hormones in blood and tissues of internal organs in experimental pulmonary fibrosis induced by intratracheal administration of bleomycetin]. Pulmonary fibrosis was produced in rats by intratracheal administration of bleomycetin. The development of the fibrous process in the lungs was attended by marked reduction of the zinc content in the tissues of the viscera, plasma, and formed elements of the blood, as well as of corticosterone and testosterone in blood plasma. Respiratory insufficiency is a probable cause of the developing zinc-deficiency which leads to disturbed synthesis of steroid hormones. Injection of exogenous testosterone normalizes partly the level of steroid hormones in the blood, improves the general condition of the animals, and facilitates an increase of body weight.


In vivo study of phytanic acid alpha-oxidation in classic Refsum's disease and chondrodysplasia punctata. A series of in vivo experiments is described in which [1-13C]phytanic acid was given as an oral substrate to a healthy subject and two patients showing an impairment in phytanic acid degradation, one with Refsum's disease and one with chondrodysplasia punctata. After intake of the substrate by the control in a dose of 20 mg/kg body weight, the production of 13CO2 was measured in exhaled breath air and the concomitant formation of labeled 2-hydroxyphytanic acid and of pristanic acid was demonstrated by plasma analysis. After application of a substrate dose of 1 mg/kg body weight to the control, no substantial amounts of 13CO2 were measured, whereas time-dependent analysis of labeled 2-hydroxyphytanic acid in plasma yielded a concentration curve superimposed upon the baseline value (0.2 mumol/L) of the unlabeled substance. Phytanic acid accumulated in plasma from the Refsum's disease patient [649 mumol/L, controls > 1 y (n = 100): < 10 mumol/L], whereas the pristanic acid concentration was within the control range [1.4 mumol/L, controls > 1 y (n = 100): < 3 mumol/L]. Low amounts of 2-hydroxyphytanic acid were found normally present [0.04 mumol/L, controls > 1 y (n = 11): < 0.2 mumol/L], and formation of labeled 2-hydroxyphytanic acid could not be demonstrated after ingestion of [1-13C]phytanic acid in a dose of 1 mg/kg body weight. In addition to phytanic acid accumulation (232 mumol/L), the chondrodysplasia punctata patient showed an elevated 2-hydroxyphytanic acid plasma concentration (0.4 mumol/L), whereas the plasma pristanic acid level was in the control range (0.7 mumol/L).(ABSTRACT TRUNCATED AT 250 WORDS)


hnRNP A2/B1 binds specifically to single stranded vertebrate telomeric repeat TTAGGGn. We have previously isolated a protein from mouse liver nuclei that specifically binds to single stranded (TTAGGG)n repeats. TTAGGG is the telomeric repeats of mammals and we therefore named the new protein single stranded telomere binding protein (sTBP). Further studies now identify sTBP as heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 on the basis of amino acid sequence determination and antibody reactivity. A2 and B1 form a major part of the protein component of hnRNP particles and are abundant nuclear proteins. Unexpectedly, A2/B1 has a high specificity for binding to the RNA equivalent of TTAGGG, UUAGGG, but under the same conditions does not appear to have a strong affinity for a number of other RNA species.


Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type-1 rev protein. The Human Immunodeficiency Virus type-1 rev protein binds with high affinity to a bubble structure located within the rev-response element (RRE) RNA in stemloop II. After this initial interaction, additional rev molecules bind to the RRE RNA in an ordered assembly process which requires a functional bubble structure, since mutations in the bubble sequence that reduce rev affinity block multiple complex formation. We have used synthetic chemistry to characterize the interaction between rev protein and its high affinity binding site. A minimal synthetic duplex RNA (RBC6) carrying the bubble and 12 flanking base pairs is able to bind rev with 1 to 1 stoichiometry and with high affinity. When the bubble structure is inserted into synthetic RNA molecules carrying longer stretches of flanking double-stranded RNA, rev forms additional complexes resembling the multimers observed with the RRE RNA. The ability of rev to bind to RBC6 analogues containing functional group modifications on base and sugar moieties of nucleoside residues was also examined. The results provide strong evidence that the bubble structure contains specific configurations of non-Watson--Crick G:G and G:A base pairs and suggest that high affinity recognition of RRE RNA by rev requires hydrogen bonding to functional groups in the major groove of a distorted RNA structure.


Generation of viroid conformational isomers that are stable to incubation with magnesium ions and in a nuclear extract from tomato plants. We identified conditions for heating and quick cooling viroid RNAs in the presence of salt which lead to the production of conformational isomers stable to incubation for at least 45 minutes at 30 degrees in the presence of magnesium ions. Elution in 0.3 M NaCl allowed the purification of an electrophoretically slow form of an in vitro transcript carrying a complete copy of the potato spindle tuber viroid RNA sequence. Slow forms of this transcript and of kinase-labeled linear viroid RNA persisted for longer than 20 minutes when incubated with a protein-rich extract prepared from the nuclei of uninfected tomato plants, although both were slowly cleaved by a nuclease present in this extract. The fast form of the transcript was highly resistant to this tomato ribonuclease. The slow form of the transcript was much more susceptible to cleavage by RNase T1 than the fast form of this RNA, suggesting that the reduced gel mobility of the slow forms results from their relatively open structure. The ability to purify viroid conformational isomers from polyacrylamide gels will facilitate biochemical studies aimed at identifying host components interacting with RNAs of the viroid replication complex, which may not all be present in the most thermodynamically favored rodlike structure of mature viroids.


Large scale chemical synthesis, purification and crystallization of RNA-DNA chimeras. RNA-DNA chimeras, in which both DNA and RNA monomers are site-specifically substituted in the same strand, may be prepared only by chemical synthesis. Biochemical studies have revealed a number of surprising and subtle effects resulting from the insertion of either a ribonucleotide into a DNA strand or a deoxyribonucleotide into an RNA strand. The availability of large quantities of these chimeras allows for their crystallization and subsequent x-ray structure determination. We describe a flexible and efficient method for the large-scale preparation of these compounds, their purification, and their crystallization. The methodology is based on a combination of existing DNA phosphoramidite synthons and those recently introduced for the preparation of biochemically active RNA1. We demonstrate that these two different synthons are compatible, produce large quantities of nucleic acid needed for physical studies, and that high resolution diffraction quality crystals may be grown from these chimeras. Of the duplex chimeras synthesized and crystallized, [r(G)d(CGTATACGC)]2, [d(GCGT)r(A)d(TACGC)]2 and [r(GCG)d(TATACCC) + d(GGGTATACGC)] form A-helices and d(CG)r(CG)d(CG)]2 forms a left-handed Z-helix.


The effect of loop size in antisense and target RNAs on the efficiency of antisense RNA control. Most natural antisense RNAs display a high degree of secondary structure with stem-loops as their most prominent feature. Mutations affecting the inhibitory activity of these RNAs most often map in or close to loop regions in both the antisense and target RNAs. The primary recognition loops often contain 5-7 unpaired nucleotides. Nucleotide changes in the loops affect the binding rate and, hence, the inhibitory effect on the activity of the target RNA. Here we address the question whether loop sizes affect binding rates between antisense and target RNAs, using the replication control system of plasmid R1 as a model system. By creating a series of loop size mutants we show that loop size alterations have strong effects on the binding rates between the two reactant RNAs in vitro, and that most of the mutations analyzed display corresponding effects on antisense RNA control in vivo. Our data suggest that the three-dimensional structures of antisense and target RNA stem-loops are crucial for determining binding rates. The implications of these results for the design of efficient artificial antisense RNA control systems are discussed.


Effects of lordosis-relevant neuropeptides on midbrain periaqueductal gray neuronal activity in vitro. Certain neuropeptides can facilitate lordosis by acting on midbrain periaqueductal gray (PAG) in estrogen-primed female rats. Here, we investigated responses of individual PAG neurons in vitro, to five neuropeptides: substance P (SP), luteinizing hormone-releasing hormone (LHRH), prolactin (PRL), oxytocin (OT), and thyrotropin-releasing hormone (TRH). Substance P, OT, and TRH excited spontaneous activity of PAG neurons through neurotransmitter-like actions in a dose-dependent manner, whereas LHRH and PRL virtually never affected PAG neurons this way. Oxytocin acted through oxytocin receptors located on the recorded PAG neurons, since excitatory actions of OT were 1) not abolished by synaptic blockade, 2) mimicked by the OT-specific agonist [Thr4, Gly7]OT but not by arginine vasopressin, and 3) blocked by the OT-specific antagonist [d(CH2)5,Tyr(Me)2,Orn8]vasotocin. Although LHRH had no neurotransmitter-like action on spontaneous activity of PAG neurons, it, as well as SP, could modulate responses of some dorsal PAG neurons to GABAA and GABAB agonists or norepinephrine. Neuromodulatory actions of LHRH and SP could help facilitate lordosis through PAG neurons.


[Palliative methods of the treatment of prostatic adenoma]. Semi-conservative therapies in prostatic adenoma are discussed. They may be used in patients with non-operational prostatic adenoma. Besides permanent catheterization, there are the following possibilities: extension of the prostatic tube with a balloon, application of the urologic coil, commissurotomy, therapy with cryosurgery or hyperthermia. ++Palliative therapy markedly prolongs and improves the life of patients with systemic diseases, which make radical surgery impossible.


[Preliminary clinical evaluation of systemic chemotherapy of invasive cancer of the bladder by the CMV protocol]. The authors discuss the results of preliminary clinical trials with chemotherapy with CMV (cisplatin, methotrexate, vinblastine) in the group of 17 patients with the infiltrating cancer of the bladder. Chemotherapy was used as an element of the radical treatment (in 7 patients, i.e. 41.2%) or as ++palliative treatment (in 10 patients, i.e. 58.8%). Objective response to the treatment was noted in 73.3% of the treated patients, including 13.3% of the complete recovery, and 60.0% of partial improvement. In the remaining 26.7% of patients (including all patients with distant metastases) no response to therapy was achieved. Complications of therapy are discussed. Systemic chemotherapy with CMV regimen proved efficient in the infiltrating cancer of the bladder.


[Evaluation of the results of combined treatment of patients with testicular seminoma in the II stage of clinical advancement]. An attempt to introduce combined therapy for patients with testicular seminoma in the II degree of clinical advancement was undertaken at the Centre of Oncology. Combined therapy consisted of 3 courses of PVB in the following daily doses: DDP 100 mg/m2 i.v. on the first day; VLB 10 mg i.v. on the first and second day; bleomycin 30 mg i.v. on the second, ninth and sixteenth day every 21 days, and 60Co on lymphatic glands area in which metastases were diagnosed prior to chemotherapy. Twenty three patients were treated that way between January 1985 and June 1989. Mean follow-up period after the treatment was 14 months. One patient died for the tumor, metastases to the lungs were diagnosed in one patient 9 months after completion of the treatment which ameliorated after "second" chemotherapy, and 22 patients (96%) are still free from the symptoms of active disease.


[Methotrexate-induced skin detachment].

[Methotrexate-induced skin detachment]. The undesirable effects of high-dose methotrexate on integument and mucosa are well-known, but lesions of the skin proper, characterized by distal erythema with desquamation, are rare. We report the case of a male patient presenting with extensive skin erosions after intravenous infusion of a 5 gram total dose of methotrexate to treat high-grade lymphoma. The skin disease was accompanied by renal, hepatic and mucosal lesions, as well as bone marrow aplasia. The mechanism of cutaneous necrosis is uncertain, but it might be due to direct toxicity of methotrexate to the epidermis. The patient was also treated with G-CSF. He recovered from aplasia within 6 days, without infectious complications. Haematopoietic growth factors might have reduced the risk of infection arising from the skin, by shortening the duration of cytopenia.


[Evaluation of the effectiveness of prazosin in conservative treatment of benign prostatic hypertrophy]. Prazosin--a selective blocker of alpha 1-adrenergic receptors--was administered to 30 patients with benign prostatic hypertrophy. Twenty four patients (80%) reported an improvement in voiding and observed more potent urinary stream after the treatment. Average and maximum flow rates increased in 18 patients (60%). Therapy had to be discontinued in 2 patients because of the adverse reactions (hypotension and syncope in one and exacerbation of the coronary disease symptoms in another patient).


[Monoclonal antibodies in the treatment of rheumatoid arthritis]. Monoclonal antibodies (MoAb's) make it possible to treat rheumatoid arthritis with selective immunotherapy. These antibodies may be directed against various targets, such as lymphocyte activation antigens, cytokines or subpopulations of lymphocytes (notably TCD4 +), involved in the pathogenesis of the disease. Recent open studies have demonstrated the feasibility and safety of this therapeutic method, but the number of patients who entered the trials is still low, and the clinical, biological and immunological results vary considerably in importance and duration, without remission. No response predictive factor could be elicited from these studies. The murine origin of these MoAb's exposes to the frequent risk of immunization which may interfere with the effectiveness and safety of a second treatment. Some possibilities can already be envisaged, including potentiation of the MoAb by coupling with a cytotoxic agent (anti-CD5 + ricin) and "humanization" of murine MoAb's (chimeric anti-CD4) reducing the risk of immunization. Further (controlled) trials therefore are indispensable to evaluate the true rank occupied by this therapeutic method in rheumatoid arthritis.


Search for the stable state of a short chain in a molecular field. A general approach is developed to search for stable structures of short chain fragments (e.g. of loops or bound oligopeptides) in a given molecular field. This molecular field is produced by the remaining part of a globule or by any other surface with a defined spatial structure. The fragment must be short enough to have no pronounced long-range interactions within itself. The method is illustrated by calculation of the 3-D structures of two loops of bovine pancreatic trypsin inhibitor (BPTI). Computations are based on a lattice model of conformational space and on strict and fast algorithms of 1-D statistical mechanics and dynamic programming (which are very similar in essence). This makes a search of oligopeptide structures only several times (and not several orders of magnitude) longer than that of a dipeptide.


A new lease of life. Nursing care of elderly surgical patients. Advances in surgical techniques have meant more elderly people can now be operated on. Older people are, however, more prone to postoperative complications and require greater preoperative preparation. Forward planning and setting high standards for nursing care are prerequisites in this area of care.


The molecular biology of human hereditary central diabetes insipidus. Molecular biology techniques have begun to shed light on the genetic basis of autosomal dominant central DI, but several very basic questions remain to be answered. The disorder was initially presumed to have a developmental, degenerative, or autoimmune basis based on the autopsy findings in the hypothalamus of a limited number of patients. The molecular cloning of the AVP-NP II gene and the clue from the Brattleboro rat that at least this one form of hereditary DI involved an AVP-NP II gene mutation allowed us to focus on this gene in our study of human hereditary DI. Our initial experiments did not show this gene to have a major structural alteration such as a deletion, insertion, or rearrangement, but the approach was not capable of detecting more suitable defects. The linkage studies provided substantial evidence that one particular OT-NP I haplotype was linked to the disease phenotype in each family, and thus, a mutation in the AVP/OT region of chromosome 20 is responsible for this disease. Ito et al. (1991) then identified a single base change in the AVP-NP II gene in affected members of one Japanese family. This change was not detected in unrelated, unaffected persons and thus is a good candidate for the mutation causing the disease in this family. However, there appears to be diversity in the molecular basis of autosomal dominant central DI as affected members of one of our families did not have this particular base change in either AVP-NP II allele and recently another distinct AVP-NP II gene base change has been associated with this disorder. One interesting question still to be addressed is how a mutation in the NP-II coding region of this gene prevents AVP release from the posterior pituitary in the rat or the human disease. Does the disrupted AVP-NP II coding sequence prevent normal processing of the mRNA so that it can not be properly translated into protein? Does the mutated AVP-NP II glycoprotein precursor protein interfere with normal post-translational processing to prevent release of AVP? Is an altered NP II protein not able to protect the AVP from proteolysis within the magnocellular neuron? An even more puzzling question is how a mutation in the gene encoding a hormone is inherited in an autosomal dominant pattern. The Brattleboro rat model follows the a priori expectation of autosomal recessive inheritance: the animal only exhibits a defect in hormone function if both genes encoding the hormone are defective.(ABSTRACT TRUNCATED AT 400 WORDS)


Effect of bradykinin on the cytosolic free calcium activity and phosphoinositol turnover in human glomerular epithelial cells. The effect of bradykinin (BK) on the intracellular free calcium activity [Ca2+]i and phosphoinositide (PI) turnover was investigated in human glomerular epithelial cells (GEC) in culture. Human GEC exhibited a baseline [Ca2+]i of 114 +/- 3 nmol (n = 81). BK (ED50 10(-9) mol/l) caused a rapid and transient increase in [Ca2+]i, which could also be observed in the absence of extracellular calcium. The effect of BK (10(-8) mol/l) on the [Ca2+]i was inhibited by the BK2 antagonist Hoe 140 (IC50 10(-8) mol/l). BK also induced PI turnover in a time- and dose-dependent manner. A transient increase in (1,4,5)-inositol-triphosphate (InsP3) formation from 1,445 +/- 119 to 4,629 +/- 323 cpm occurred after 5 s. Stimulation of protein kinase C (PKC) by short-term preincubation (15 min) of human GEC with phorbol-12-myristate-13-acetate (PMA) induced a dose-dependent inhibition of the BK-stimulated (10(-7) mol/l) inositol-phosphate formation. Downregulation of PKC by preincubation of human GEC with PMA (24 h, 10(-6) mol/l) or inhibition of PKC by pretreatment with staurosporin (1 h, 10(-6) mol/l) resulted in a slight but significant augmentation of the BK-induced InsP3 stimulation. The data indicate that BK induces stimulation of [Ca2+]i and PI turnover via a BK2 receptor in human GEC. PKC might exert a negative feedback function for the BK-induced PI turnover.


Inhibition of sodium-dependent transport systems in LLC-PK1 cells by metabolites of ifosfamide. Ifosfamide (IF) is an alkylating cytostatic derived from nitrogen mustard. In addition to its well-known urotoxic effects (hemorrhagic cystitis), several cases of Fanconi syndrome following IF therapy have been reported. No information is available concerning the pathomechanisms of this tubulotoxicity. We used the permanent renal epithelial cell line LLC-PK1 in order to investigate whether major metabolites of IF (i.e. 4-OH-IF, acrolein and chloracetyldehyde) induced the transport defects most frequently detected after IF therapy in vivo. LLC-PK1 cells of passages 162-177, grown in plastic culture dishes, were used in a confluent state. Sodium-dependent and independent fluxes of l-[3H]alanine and of D-[3H]glucose were determined by standard techniques. Activities of marker enzymes of apical and basolateral membranes, of mitochondria and of endoplasmic reticulum were determined in cell homogenates. IF itself has no detectable effect on fluxes of l-alanine and D-glucose in LLC-PK1 cells. The IF metabolite 4-OOH-IF induces a clear inhibition of sodium-dependent fluxes of both substrates after a 24-hour exposure of cells to 100 mumol/l of 4-OOH-IF. Chloracetaldehyde induces a biphasic response of sodium-dependent fluxes of l-alanine with increased uptake rates at low concentrations (< 200 mumol/l) and with a short incubation time, while higher concentrations and long exposure of the cells leads to a reduction in sodium coupled transport. Glucose transport is affected in a comparable way, however, in contrast to alanine transport, chloracetaldehyde also stimulates sodium-independent fluxes of glucose. Acrolein is the most toxic substance tested. It severely damages cell monolayers at concentrations beyond 75 mumol/l. Sodium-coupled glucose and alanine transport is inhibited by acrolein at concentrations higher than 50 mumol/l. Sodium-coupled glucose transport is more sensitive to all metabolites tested than alanine transport. While acrolein strongly affects both transport systems, marker enzymes of the apical plasma membrane, i.e. alkaline phosphatase and leucine amino-peptidase, are not significantly inhibited, suggesting a specificity of the toxic effect for the transport proteins. We conclude that LLC-PK1 cells represent a good model for further investigation of the pathogenesis of Fanconi syndrome after IF therapy. Sodium-dependent transport systems are more sensitive to acrolein than other cell surface proteins.


Tumor necrosis factor-alpha- and interleukin-1-stimulated intercellular adhesion molecule-1 expression by murine renal tubular epithelial cells is transcriptionally regulated and involves protein kinase C. The regulation of intercellular adhesion molecule-1 (ICAM-1) expression by renal tubular epithelial cells (TEC) has been studied in vitro. ICAM-1 expression in TEC is stimulated with phorbol 12-myristate 13-acetate (PMA), but not with forskolin, suggesting a role for protein kinase C (PKC) but not for protein kinase A (PKA). The tumor necrosis factor-alpha- (TNF-alpha) and interleukin-1 (IL-1)-stimulated ICAM-1 expression in TEC is blocked with the PKC/PKA inhibitor staurosporine, and also with the PKC-selective inhibitor calphostin C. The TNF-alpha-stimulated ICAM-1 expression is resistant however to downregulation of PKC with PMA. The TNF-alpha- and IL-1-stimulated ICAM-1 expression is also inhibited with the transcriptional inhibitor actinomycin D and with the protein synthesis inhibitor cycloheximide. Thus, ICAM-1 expression by TEC may involve a downregulation-resistant PKC which induces ICAM-1 expression at a transcriptional level.


Transmembrane signalling of cholinergic-stimulated contraction in isolated rat glomeruli. Exposure of isolated rat glomeruli to carbachol (Carb) 1 mM induces glomerular (mesangial cell) contraction which is inhibited by pirenzepine. To identify the cellular signalling mechanism, we analyzed Carb-activated phosphoinositide hydrolysis in myo-[2-3H] inositol preloaded cells of isolated rat glomeruli. Carb evoked a rapid (5 s, 30.4 +/- 8.3% of basal) and sustained (60 s, 157.2 +/- 1.2% of basal) inositol trisphosphate (IP3) response. Acetylcholine 100 microM (Ach) in the presence of eserine also evoked IP3 formation. Mobilization of Ca2+ was monitored by preloading glomerular cells with 45Ca2+ (200 microCi/ml) and analyzing the cellular efflux using a constant perifusion system. Both Carb and Ach induced transient 45Ca2+ efflux, with peak values above baseline of 69.0 +/- 19.9 and 71.5 +/- 7.9%, respectively. Incubation with pirenzepine or prior release of Ca2+ from an IP3-sensitive intracellular pool by Thapsigargin (2 microM) prevented Carb-evoked Ca2+ release (100 and 60%, respectively). Therefore, glomerular cells possess functional cholinergic receptors which stimulate contraction via IP3-mediated intracellular Ca2+ release.


Role of prostacyclin in the renal response to acetylcholine. Renal arterial infusion of acetylcholine (ACh; 40 micrograms/min) in control dogs produced a sustained rise in urinary sodium excretion (UNa V) and in renal plasma flow (RPF). When prostaglandin (PG) synthesis was inhibited with indomethacin (5 mg/kg), ACh produced only a transient rise in UNa V and RPF followed by a progressive decline in UNa V and RPF. Renal arterial infusion of PGI2 (0.2 microgram/min) restored the response to ACh to normal in indomethacin-treated dogs. The RPF was 84 +/- 9 ml/min during control and increased to 132 +/- 19 ml/min with the infusion of PGI2 (p < 0.01). RPF increased further to 188 +/- 18 ml/min at 20-min infusion of ACh and was maintained at 189 +/- 14 ml/min at 100-min infusion of ACh (p < 0.01). Our data suggest that the initial rise in RPF by ACh is independent of the PG system, whereas maintenance of the rise in RPF by ACh requires an intact synthesis of PGs, presumably PGI2 synthesized in the vascular tissues.