Mapping thyrotropin β subunit gene in man and mouse

Thyrotropin (TSH) is composed of two subunits: and . Previously, we have mapped the TSH gene to human chromosome 6 and mouse chromosome 4. In this study we have located the human TSH gene on chromosome 1 and the mouse TSH gene to chromosome 3. These data suggest that the TSH gene lies in a conserved linkage group with the genes for amylase 1 and 2, nerve growth factor, and the protooncogene Nras.     

Further observations on the morphogenesis of human rotavirus in MA 104 cells

Summary Human rotavirus KUN strain was cultivated in a fetal rhesus monkey kidney cell line, MA 104 cells. Four types of virus particles in cells infected with KUN strain were clearly identified: nucleoid cores, single-shelled particles, double-shelled particles, and membrane band, enveloped particles. Enveloped particles were found only in the thin sections of infected cells. When first visible, the virus precursors appeared at the ribosome free membrane of rough endoplasmic reticulum (RER), increasing in size while simultaneously being coated with nucleocapsid, inner shell. Single-shelled particles were also synthesized within bundles of filaments of viroplasm in the cytoplasma. During subsequent virus maturation two types of budding processes were observed. Double-shelled particles arising at the RER membrane entered the cisternae of the RER through an exocytosis-like process. In contrast, the enveloped particles developed in the cisternae by being completely enclosed with RER membrane, and later during cytolysis released the single-shelled particles. These enveloped virus particles appeared to be the result of inefficient virus maturation at the last stage of outer capsid formation.With 11 Figures     

The role of interferon β in human cytomegalovirus-mediated inhibition of HLA DR induction on endothelial cells

Summary Human cytomegalovirus (HCMV), a member of the virus familyHerpesviridae that is associated with extensive worldwide morbidity and mortality in immunocompromised hosts, inhibits interferon- (IFN)-mediated induction of human leukocyte antigen (HLA) class II antigens on endothelial cells. In this study, the ability of HCMV-infected endothelial cells to synthesize interferon- (IFN), and the role of IFN in HCMV-mediated inhibition of HLA class II induction, was investigated. As detemined by an encephalomyocarditis virus protection assay, HCMV-infected endothelial cell culture supernatants contained 240 IU/ml of IFN type I activity, of which 99.9% was IFN, as compared to the absence of IFN in mock-infected culture supernatants. UV-irradiated supernatants from HCMV-infected cultures inhibited induction of HLA class II in noninfected cultures by 24%. This inhibition could be abolished with 500 NU/ml of anti-IFN antibody. Addition of anti-IFN antibody directly to HCMV-infected cultures mitigated but did not abolish HLA class II antigen inhibition. Dual immunohistochemistry for HCMV and HLA DR demonstrated that infected cells, in contrast to noninfected cells, were rarely induced to express HLA class II even in the presence of anti-IFN antibody. These findings suggest that HCMV inhibits induction of HLA class II antigens by IFN dependent and independent mechanisms.     

Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K⁺ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K⁺ channel blocker barium (10 nmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mol/l), by cobalt (100 mol/l), zinc (100 mol/l), gadolinium (100 mol/l), verapamil (10 mol/l) and diltiazem (10 mol/l), but are abolished in the presence of 100 mol/l lanthanum, 1 mmol/l cadmium, 10 mol/l nifedipine, 25 mol/l SK & F 96365 and 200 mol/l TMB-8. Stimulation of calcium entry by 10 mol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.     

In situ expression of β1, β3 and β4 integrin subunits in non-neoplastic endothelium and vascular tumours

Endothelial cells play an important role in adhesive interactions between circulating cells and extracellular matrix proteins. In vitro studies have shown that many of these processes are mediated by a superfamily of heterodimeric transmembrane glycoproteins called integrins. The distribution patterns of 1, 3 and 4 integrin subunits in endothelial cells (EC) in situ were examined immunohistochemically on serial forzen sections of a wide range of non-neoplastic tissues and of vascular tumours, both benign and malignant. Expression of the 1 subunit was a constitutive feature of EC. Among the 1-associated subunits, 5 and 6 were broadly distributed in EC, irrespective of vessel size and microenvironment. The 3 subunit displayed intermediate levels of expression with a slight preference for small vessel EC. Presence of 1 was confined to EC of capillaries and venules/small veins. Expression of 2 in EC was inconsistent. With rare exceptions, the 4 chain was absent in EC. The 3 and v subunits were expressed in most EC, though not always concomitantly. In contrast to the 1 chain, however, these integrin subunits were absent in EC of glomerular capillaries and were expressed variably in sinusoidal EC. The 4 chain was evenly present in the great majority of EC, except for those of large vessels. In vascular tumours, the patterns of 1 and 1 to 6 subunit expression generally corresponded to those found in their non-neoplastic counterparts. Expression of 3, v and 4 chains, however, decreased in neoplasia, especially in angiosarcomas. These data show that EC dispose of broad and at the same time differential repertoires of integrin subunits that presumably reflect vessel-type associated functional differences among these cells. In vascular tumours, the orthologous distribution patterns of 1 and 1 to 6 chains are conserved in most instances while the amounts of 3, v and 4 subunits expressed in EC tend to decrease in the course of malignant transformation.Dedicated to Prof. Dr. med. Dres. h.c. Wilhelm Doerr on the occasion of his 80th birthday     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Evidence for a direct relationship between mitochondrial genome organization and regeneration ability in hexaploid wheat somatic tissue cultures

Summary Embryogenic and non-embryogenic long-term callus cultures of hexaploid wheat exhibit differences in the organization of their mitochondrial genome. Embryogenic and non-embryogenic fractions of callus cultures initiated from immature embryos of the wheat cultivar Chinese Spring have been isolated and subsequently subcultured. DNA-DNA hybridization experiments using labelled cloned wheat mitochondrial DNA fragments have shown that the mitochondrial DNA organization of embryogenic subcultures derived from embryogenic parts of Chinese Spring calli is closely related to that of the initial Chinese Spring calli, while non-embryogenic subcultures derived from non-embryogenic fragments of Chinese Spring calli exhibit a mitochondrial DNA organization similar to that found in non-embryogenic calli derived from cultivar Aquila. In addition, somatic tissue cultures initiated from three other non-embryogenic wheat cultivars (Talent, Thésée and Capitole) display mitochondrial DNA arrangements similar to those found in cultivar Aquila. These results strongly suggest that, in wheat callus cultures, a particular mitochondrial genome organization is correlated with the ability of cultured cells to regenerate whole plants.Abbreviations mtDNA mitochondrial DNA - ctDNA chloroplast DNA - rRNA ribosomal RNA - kb kilobase pair - cv cultivar - 2,4-D 2,4-dichlorophenoxyacetic acid     

Molecular organisation of an A mating type factor of the basidiomycete fungus Coprinus cinereus

Summary The A3 and A3 genes, which together constitute the A42 mating type factor of Coprinus cinereus, were isolated from a cosmid genomic library by walking 50 kb, a map distance of 0.5 units, from the closely linked metabolic gene pab-1. Cosmid clones having A gene function were identified by transformation into compatible A6 (22) and A5 (11) host cells where either 3 or 3 was expected to elicit the A factor — regulated development of unfused clamp cells. DNAs were digested with various enzymes before transformation in order to identify the smallest fragments containing an active 3 or 3 gene. Two non-overlapping fragments were identified as containing the 3 and 3 genes respectively. Southern hybridisation analyses showed that these two cloned genes had no detectable sequence homology, and that there was little or no hybridisation to the and gene alleles that constitute the A5 and A6 factors. 3 and 3 were shown to be less than 2.0 kb apart and embedded in a DNA sequence extending over 9.0 kb which was unique to our A42 strain and may contain a third A factor gene.     

Glucocorticoids modulate TGF-beta production by human fetal lung fibroblasts

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-1, -2, or -3. Post-culture media were collected for ELISA assays of TGF-1, -2, and -3 . TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and TGF-2 production and reduced expression of the up-regulated TGF-1 and TGF-2 mRNA induced by exogenous TGF-1, -2, or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Die Lymphocytentransformierbarkeit von Gesunden mit einer „Hodgkin-HL-A-Konstellation“

Zusammenfassung Gefundene Korrelationen zwischen HL-A Muster und Morbus Hodgkin lassen vermuten, daß genetische Faktoren, nämlich HL-A Lokus assoziierte Immunresponsegene, in der Pathogenese eine Rolle spielen. In der vorliegenden Arbeit wurde davon ausgegangen, daß gesunde Probanden mit einer HL-A-Hodgkin-Konstellation möglicherweise ein gestörtes Immunverhalten zeigen, welches sich durch Lymphocytenstimulierbarkeit mit Mitogenen und allogenen Zellen aufdecken läßt. 12 Personen aus dieser Gruppe zeigten jedoch im Vergleich mit einer Kontrollgruppe kein abweichendes Verhalten in den in-vitro-Testen.Assistent des Psychologischen Instituts Heidelberg, für die statistischen Analysen verantwortlich     

Evidence for respiratory interneurones in the C3-C5 cervical spinal cord in the decorticate rabbit

Summary In mammals, it has long been considered that the bulbo-spinal inspiratory drive provided a direct (monosynaptic) excitation of phrenic motoneurones (Phr Mns). Although such connections have been demonstrated, recent indirect data strongly suggested that the main inspiratory drive is polysynaptic. We tried to directly demonstrate relay respiratory interneurones at the C3–C6 spinal cord level where the Phr Mn pool is located. The experiments were performed on decorticate, unanaesthetized, bilaterally vagotomized and curarized rabbits and the firing pattern of spinal interneurones was compared to the phrenic bursting. Dorsally and dorso-medially to the Phr Mn pool, different classes of inspiratory (54%) and expiratory (46%) interneurones could be identified in the ventral horn. Three classes of inspiratory interneurones were characterized and classified as I all (26%), I late (43%) and I tonic (29%) according to the terminology used by other authors for the bulbospinal inspiratory neurones which drive the spinal respiratory motoneurones. The expiratory interneurones could also be divided into 3 classes: E all (48%), E late (10%) and E tonic (41%). This first direct evidence of inspiratory interneurones at the C3–C6 spinal cord levels can account for the major polysynaptic excitation of the Phr Mns while the presence of numerous expiratory interneurones at this level suggests a polysynaptic bulbo-spinal inhibitory action onto the Phr Mns. These classes of inspiratory and expiratory interneurones did not always coincide with the bulbo-spinal classes of neurones described elsewhere. Unless these discrepancies are due to the different experimental conditions, they may indicate that some of these interneurones are not just relay target cells and they suggest that they might behave as integrative operators between the medullary drive and the Phr Mns.     

Two specific feedback pathways to the central afferent terminals of phasic and tonic mechanoreceptors

Summary In five types of mechanoreceptor afferents of the cat's hind foot, the primary afferent depolarization (PAD) induced by mechanical skin stimulation was measured by testing the excitability of their terminations in the dorsal horn. Two types of skin stimuli were used to set up activity in distinct populations of rapidly and slowly adapting mechanoreceptors respectively.The experiments revealed that two systems exist to generate PAD in cutaneous afferents, both being of negative feedback character. One system is activated by impulses from rapidly adapting low threshold receptors and preferentially depolarizes the terminals of such afferents, and correspondingly, the other system is activated by and operates on the slowly adapting units.In both PAD systems the size of the depolarization is graded depending on the stimulus strength. Further, the tonic system displays a surround pattern of organization similar to that of the phasic system which has already been described (Schmidt et al. 1967b).In the discussion the operational relationships of both systems and their functional implications are outlined.This work was supported by the Deutsche Forschungsgemeinschaft.     

Three-dimensional reconstruction of transmitter-identified central neurons by “en bloc” immunofluorescence histochemistry and confocal scanning microscopy

Summary A new method for three-dimensional reconstruction of transmitter-identified neurons is presented which involves en bloc immunofluorescence histochemistry and confocal scanning microscopy. The technique was applied to different types of neurons in the rat brain and lamprey spinal cord. Thick sections or tissue blocs (50–200 m thick) were incubated with antisera against neuropeptides or monoaminergic markers, followed by fluorescent secondary antibodies. Three-dimensional reconstructions were obtained by scanning the preparations in sequential focal planes with a thin laser beam, while sampling the emitted light in each focal plane. The method is convenient and can be applied to a wide variety of neuron types. The reconstructions obtained are accurate since the optical serial sections of the specimen are perfectly aligned, and optic disturbances such as halo phenomena do not occur.     

Analysis of the effect of barium ions on isolated medullated nerve fibers

Zusammenfassung An der isolierten markhaltigen Nervenfaser wird nach Ba⁺⁺-Zusatz eine Verschiebung der Elektrotonuskurve nach rechts und unten sowie eine Erhöhung des durch einen Einwärtsstrom bestimmter Größe bewirkten Elektrotonuspotentiales gemessen.Diese Ergebnisse werden im Zusammenhang mit früheren Messungen so gedeutet, daß Barium eine sofortige und anhaltende, der Calciumwirkung vergleichbare Erhöhung der Schwelle bewirkt und daß sich dieser Wirkung eine langsam zunehmende Schwellenerniedrigung überlagert, die durch die Abnahme der Kalium permeabilität verursacht wird.Mit 4 Textabbildungen.     

The use of local anaesthetic microinjections to identify central pathways: a quantitative evaluation of the time course and extent of the neuronal block

Summary The time course and extent of local anaesthetic blocks within the spinal cord of cats were evaluated. A monopolar stimulation electrode with the tip lowered into the dorsal columns (DC) 1000 m below cord surface was used to activate antidromically DC fibers at the T13 level and evoke cord dorsum potentials at the level of the lumbar spinal cord. The amplitude of the negative deflection, the N-wave, was determined for various stimulation intensities (stimulation-response-function, SRF). Lidocaine (1%) was microinjected in volumes of 0.5 or 1.0 l into the DC from a glass micropipette 1 mm caudal to the stimulation site. Conduction block was characterized by a reversible shift of the SRFs to higher stimulation intensities. The diameter of the blocked area in the transverse plane was evaluated from threshold intensities and was found to be 0.9±0.1 mm 4 to 30 min after the injection of 0.5 l lidocaine and 1.6±0.36 mm 10 to 45 min after the injection of 1.0 l lidocaine. In the sagittal plane, the diameter of the blocked area following 1.0 l lidocaine was found to be up to 2.8 mm. The DC-block was reversible within 92 min following injection of 1.0 l and 69 min after the injection of 0.5 l lidocaine. The application of the present findings for blocks in other CNS structures is discussed.     

Cortico-cortical connections of two electrophysiologically identified arm representations in the mesial agranular frontal cortex

Summary Neuronal tracers (diamidino yellow or wheat germ agglutinin conjugated with horseradish peroxidase) were injected in the arm representations of area 6a (mesial surface, area F3), in the arm representation of area 6a (mesial surface) as well as in the eye field of area 6a (dorso-medial surface). The results showed that the arm representation of area F3 receives topographically organized afferents from motor and premotor areas (areas F1, F2, F4 and F5). A further connection was found with that part of cingulate cortex that sends projections to the spinal cord. In contrast, the arm representation of area 6a receives afferents chiefly from area F5, the prefrontal cortex and that part of cingulate sulcus which has few, if any, connections with the spinal cord. No connections were found with the precentral motor cortex (area F1). The area 6a eye field receives afferents mostly from the frontal eye field. Further connections are with the prefrontal cortex and cingulate gyrus. It is suggested that the so called low level motor functions of supplementary motor area are due to the activity of area F3, whereas the so called high level motor functions depend upon an independent area located in area 6a.     

Expression of estrogen receptors-α and -β in primary breast neoplasms and tumors exposed to neoadjuvant hormonal therapy

We studied the content and expression of mRNA for estrogen receptors receptors- and - in breast tumors before and after 3-month neoadjuvant hormone therapy with antiestrogen tamoxifen and/or aromatase inhibitors. Expression of estrogen receptors- and - was most often detected in ER⁺PR⁺ tumors and most significantly decreased in these neoplasms after exemestane therapy. Immunocytochemical and radioligand assays showed that tamoxifen and anastrozole have little effect on the number of estrogen receptors- The number of progesterone receptors in tumors decreased by the end of anastrozole therapy. Estrogen receptors- were immunocytochemically revealed in 50% primary breast tumors. Anastrozole slightly decreased, while tamoxifen increased the incidence of these receptors. Interruption of signaling through estrogen receptors and suppression of estrogen biosynthesis had different effects on the receptor status of neoplasms and distribution of estrogen receptors- and -.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 11, pp. 559–562, November, 2004     

Phosphorylation-regulated low-conductance Cl− channels in a human pancreatic duct cell line

A low-conductance Cl^– channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mol/l), dibutyryl-adenosine 3,5-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3,5-cyclic monophosphate (8-BrcAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mol/l) and forskolin (10 mol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23°C and 12 pS at 37°C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl^–>I^–>HCO₃ ^–>gluconate. In insideout excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg²⁺). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl^– channel was 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS, 100 mol/l) and 4-acetamido-4-isothiocyanatostilbene-2, 2-disulphonic acid (SITS, 100 mol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mol/l). These results demonstrate that the apical low-conductance Cl^– channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases. They provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel.     

Glucocorticoids and TGF-β1 Synergize in Augmenting Fibroblast Mediated Contraction of Collagen Gels

TGF- plays a central role in the initiation and progression of pulmonary fibrosis. Glucocorticoids are frequently used to treat fibrotic diseases, but beneficial effects are often modest. Both TGF- and glucocorticoids have been reported to increase fibroblast contraction of native collagen gels, a model of fibrotic tissue remodeling. Therefore, we sought to determine how glucocorticoids interact with TGF- in this system. In this study, human fetal lung fibroblasts (HFL-1) were pretreated with or without TGF- for 72 h before they were cast into type I collagen gels. Various concentrations of glucocorticoids (budesonide or hydrocortisone) were added at the time of casting. Gel size was then monitored at different times after gel release. The surrounding media were collected for the assay of prostaglandin E₂ (PGE₂) and the cell lysates were analyzed for cyclooxygenase (COX) expression by immunoblot. Glucocorticoids alone significantly enhanced fibroblast-mediated contraction of collagen gels (P < 0.01) and dose-dependently inhibited PGE₂ release by HFL-1 fibroblasts. TGF- significantly augmented gel contraction but also induced a 30% increase in PGE₂ release and increased the expression of COX-1. Glucocorticoids inhibited TGF-1 induced-PGE₂ release, and enhanced TGF- augmented gel contraction without significantly affecting TGF- augmented COX-1 expression. Indomethacin, a COX inhibitor, increased TGF- augmented gel contraction but had no further effect when added together with glucocorticoids. Thus, glucocorticoids can synergize with TGF- in augmenting fibroblast mediated collagen gel contraction through the inhibition of PGE₂ production. Such interactions between glucocorticoids and TGF- may account, in part, for the lack of response of fibrotic diseases to glucocorticoids.     

Zur flammenphotometrischen Calciumbestimmung im Urin

Zusammenfassung Für die Calciumbestimmung im Urin wird eine einfache und rasche flammenphotometrische Methode ohne vorherige Fällung des Calciums beschrieben. Der Kationenfehler kann dabei durch das Korrekturverfahren weitgehend ausgeschaltet werden, der Phosphat- und Sulfatfehler durch das Parallelverfahren, während der Bicarbonatfehler infolge Ansäuerung entfällt.Der mittlere Gesamtfehler der Methode beträgt etwa ± 7% (± 2).