VEGF-PLCgamma1 pathway controls cardiac contractility in the embryonic heart

The strength of the heart beat can accommodate in seconds to changes in blood pressure or flow. The mechanism for such homeostatic adaptation is unknown. We sought the cause of poor contractility in the heart of the embryonic zebrafish with the mutation dead beat. We find through cloning that this is due to a mutation in the phospholipase C gamma1 (plcgamma1) gene. In mutant embryos, contractile function can be restored by PLCgamma1 expression directed selectively to cardiac myocytes. In other situations, PLCgamma1 is known to transduce the signal from vascular endothelial growth factor (VEGF), and we show here that abrogation of VEGF also interferes with cardiac contractility. Somewhat unexpectedly, FLT-1 is the responsible VEGF receptor. We show that the same system functions in the rat. Blockage of VEGF-PLCgamma1 signaling decreases calcium transients in rat ventricular cardiomyocytes, whereas VEGF imposes a positive inotropic effect on cardiomyocytes by increasing calcium transients. Thus, the muscle of the heart uses the VEGF-PLCgamma1 cascade to control the strength of the heart beat. We speculate that this paracrine system may contribute to normal and pathological regulation of cardiac contractility.     

Proteomic and postproteomic characterization of keratan sulfate-glycanated isoforms of thyroglobulin and transferrin uniquely elaborated by papillary thyroid carcinomas

Previous studies have suggested that surface components of papillary thyroid carcinoma (PTC) cells may be aberrantly glycanated, but the precise nature of these molecules has not been unveiled nor documented to be of clinical relevance. A monoclonal antibody was raised against a unique keratan sulfate (KS) determinant and used to differentially screen benign and malignant thyroid tissue for the expression of components carrying these moieties. In a total of 349 cases of benign and malignant thyroid lesions, 100% of the 115 PTC cases examined (including various histological subtypes) were found to contain KS-bearing molecules, whereas these were virtually absent from benign tissues and other thyroid tumors, with the exception of 21% of the follicular carcinoma cases analyzed. A composite immunoaffinity chromatography, immunochemistry, and mass spectrometric approach revealed that the PTC-specific KS-bearing macromolecules were unique glycoforms of thyroglobulin and transferrin. Combined, reciprocal immunoprecipitation and Western blotting further indicated that the former glycoform predominated and that most of the transferrin produced by PTC was glycanated with KS moieties. Fluorescent keratanase II-based fingerprinting of the KS moieties bound to these isoforms further demonstrated several PTC-specific peculiarities: 1) that a considerable portion of the moieties was covalently attached via a novel core protein linkage structure; 2) they had an unusual extended average length; 3) an unusual relative ratio of highly sulfated disaccharides terminating with alpha (2-3)-linked N-acetylneuraminic acid capping residues; and 4) a novel unidentified oligosaccharide moiety at the nonreducing terminus. Comparative analysis of the relative distribution of transferrin in benign versus PTC tissues highlighted a marked malignancy-associated abundance of the molecule, with a >75% frequency in expression in PTC. These findings demonstrate that PTC cells synthesize unique post-translationally modified thyroglobulin and transferrin variants in situ that may be directly exploitable for diagnosis, through histological and noninvasive cytological procedures; for devising novel strategies for antibody-guided imaging of this tumor in vivo; and for postsurgery follow-up of PTC patients.     

Generation, annotation, evolutionary analysis, and database integration of 20,000 unique sea urchin EST clusters

Together with the hemichordates, sea urchins represent basal groups of nonchordate invertebrate deuterostomes that occupy a key position in bilaterian evolution. Because sea urchin embryos are also amenable to functional studies, the sea urchin system has emerged as one of the leading models for the analysis of the function of genomic regulatory networks that control development. We have analyzed a total of 107,283 cDNA clones of libraries that span the development of the sea urchin Strongylocentrotus purpuratus. Normalization by oligonucleotide fingerprinting, EST sequencing and sequence clustering resulted in an EST catalog comprised of 20,000 unique genes or gene fragments. Around 7000 of the unique EST consensus sequences were associated with molecular and developmental functions. Phylogenetic comparison of the identified genes to the genome of the urochordate Ciona intestinalis indicate that at least one quarter of the genes thought to be chordate specific were already present at the base of deuterostome evolution. Comparison of the number of gene copies in sea urchins to those in chordates and vertebrates indicates that the sea urchin genome has not undergone extensive gene or complete genome duplications. The established unique gene set represents an essential tool for the annotation and assembly of the forthcoming sea urchin genome sequence. All cDNA clones and filters of all analyzed libraries are available from the resource center of the German genome project at http://www.rzpd.de.     

Separate and variably shaped chromosome arm domains are disclosed by chromosome arm painting in human cell nuclei

Fluorescence in situ hybridization (FISH) with microdissection probes from human chromosomes 3 and 6 was applied to visualize arm and subregional band domains in human amniotic fluid cell nuclei. Confocal laser scanning microscopy and quantitative three-dimensional image analysis showed a pronounced variability of p- and q-arm domain arrangements and shapes. Apparent intermingling of neighbouring arm domains was limited to the domain surface. Three-dimensional distance measurements with pter and qter probes supported a high variability of chromosome territory folding.     

Differences in cytokine secretion by intestinal mononuclear cells, peripheral blood monocytes and alveolar macrophages from HIV-infected patients.

Mononuclear cells of the lamina propria (LpMNC), isolated from endoscopically taken biopsies of the large bowel from AIDS patients, were analysed for their ability to secrete tumour necrosis factor-alpha (TNF-alpha), IL-1 beta and IL-6. Stimulation of LpMNC from normal controls with pokeweed mitogen (PWM) led to a time- and dose-dependent enhancement of TNF-alpha, IL-1 beta and IL-6 secretion. In contrast, PWM stimulation of LpMNC from AIDS patients resulted in only a small increase in TNF-alpha release. Constitutive secretion of IL-1 beta and IL-6 in these patients was already increased to the concentration range of stimulated cells from normal controls and could not be further increased, probably due to maximal in vivo stimulation. Secretion of TNF-alpha, IL-1 beta and IL-6 by peripheral blood monocytes (PBM) and alveolar macrophages from AIDS patients was elevated with or without stimulation compared with normal controls. Obviously, the regulation of TNF-alpha secretion is dependent on the microenvironment. Since it is known that interferon-gamma (IFN-gamma) may induce the production of TNF-alpha, the secretion of this cytokine was examined. Release of IFN-gamma was constitutively and under stimulation lowered in LpMNC from AIDS patients compared with normal controls. Addition of IFN-gamma to LpMNC did not result in enhanced TNF-alpha secretion. Our data indicate a defective function of intestinal mononuclear cells in AIDS patients as shown by the diminished TNF-alpha secretion.     

Serodiagnosis of Helicobacter hepaticus infection in mice by an enzyme-linked immunosorbent assay.

Helicobacter hepaticus is a newly recognized bacterium associated with chronic active hepatitis, hepatic carcinoma, and inflammatory bowel disease in mice. Currently, fecal or tissue PCR, fecal culture, or histologic examination of silver-stained liver sections is used to diagnose H. hepaticus infection. In this report, we describe an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of H. hepaticus infection in mice with a membrane digest preparation of H. hepaticus as the antigen. Sera from mice positive for H. hepaticus by PCR or histologic examination (n = 88), positive for Helicobacter bilis by PCR (n = 13), positive for other helicobacters (not identifiable to species level) by PCR (n = 25), or negative for all Helicobacter species by PCR (n = 162) were used to evaluate the ELISA. Results indicated that ELISA provided 93.2% sensitivity, 94% specificity, 87.2% positive predictive value, and 96.9% negative predictive value. Cross-reactive antibodies were detected in some mice infected with helicobacters not identifiable to species level. To further define ELISA sensitivity and specificity, groups of 10 C57BL/6 mice were inoculated per os with H. hepaticus, Helicobacter muridarum, or H. bilis. Sera were collected and examined by the ELISA. H. hepaticus-infected mice seroconverted by 2 weeks and maintained ELISA reactivity throughout the 18-week study, while mice infected with H. muridarum and H. bilis were negative by ELISA. These results indicate that this reported ELISA is highly sensitive and specific for the serodiagnosis of H. hepaticus infection in mice.     

ICAM-1, VCAM-1, and MAdCAM-1 are expressed on choroid plexus epithelium but not endothelium and mediate binding of lymphocytes in vitro.

The expression of cell adhesion molecules (CAMs) in the choroid plexus was studied in normal brain and during experimental autoimmune encephalomyelitis (EAE) in the SJL/J mouse during inflammation induced by intracerebral injection of killed Corynebacterium parvum in the C3H/He mouse. Both ICAM-1 and VCAM-1, but not MAdCAM-1, were constitutively expressed on choroid plexus epithelium but not on the fenestrated capillary endothelial cells within the choroid plexus. During EAE, we observed an up-regulation of ICAM-1 and VCAM-1 and de novo expression of MAdCAM-1 on choroid plexus epithelial cells. In contrast, endothelial cells in the choroid plexus were not induced to express any of the investigated CAMs. In in situ hybridization analysis we demonstrated that ICAM-1, VCAM-1, and MAdCAM-1 were locally synthesized and that the amount of their mRNAs increased in the inflamed choroid plexus. In vitro, primary choroid plexus epithelial cells could be induced to express ICAM-1, VCAM-1, and MAdCAM-1 on their surface after treatment with proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1, interferon-gamma, and lipopolysaccharide. To investigate the functional status of the expressed CAMs we performed Stamper-Woodruff binding assays on frozen sections of inflamed and naive brains. ICAM-1, VCAM-1, and MAdCAM-1 expressed in choroid plexus epithelial cells mediated binding of lymphocytes via their known ligands LFA-1 and alpha4-integrin, respectively. The expression of ICAM-1, VCAM-1, and MAdCAM-1 on choroid plexus epithelial cells together with the lack of their expression on the fenestrated choroid plexus endothelium raises the possibility that the epithelial blood-cerebrospinal-fluid barrier plays an important role in the immunosurveillance of the central nervous system.     

Gestörte alpha2-Adrenozeptorfunktion bei Hämodialysepatienten mit renaler Anämie — eine mögliche Ursache der Blutdrucksteigerung unter rekombinantem humanem Erythropoietin?

Summary Nine patients on maintenance hemodialysis and transfusion-demanding renal anemia (group A) were treated with rHuEPO 120 IU/kg i.v. three times per week. Hemoglobin-content was raised from 7.2±0.9 to 10.4±0.8 g/dl. In all patients blood pressure rose, three patients developed arterial hypertension. Mean diastoloic blood pressure was 66±12 and 78±16 mmHg (p<0.001) before and after rHuEPO. Rise in blood pressure was accompanied by a significant fall in plasma-noradrenaline-levels (from 498±100 to 383±75 pg/ml;p<0.05) and alpha₂-adrenoceptor-density (from 574±76 to 384±49;p<0.05). Compared to nine patients on maintenance hemodialysis and hematocrit over 30% (group B), patients with severe renal anemia (group A before treatment) had higher densities of alpha₂-adrenoceptors (574±76 vs. 218±32;p<0.001) despite higher plasma-noradrenaline-levels (498±100 vs. 399±63; n.s.). We suppose a anemia-related disturbance of alpha₂-receptor-function with the result of abolished receptor down-regulation and impaired vascular reagibility to vasoconstricting stimuli. With the correction of anemia receptor-function improves, receptor down-regulation as well as vascular reagibility is re-established resulting in augmented vascular resistance and higher blood pressure.

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Abkürzungen rHuEPO rekombinantes humanes Erythropoietin - teMAP mittlerer arterieller Blutdruck - RR Blutdrucknach RIVA-ROCCI - RBF regionaler Blutfluß - RPR regionaler peripherer Widerstand     

Effect of the serotonin agonist buspirone on behaviour and hypothalamic-pituitary-adrenal axis in confident and fearful mink

Behavioural and hypothalamic-pituitary-adrenal (HPA) axis responses were investigated in farm mink (Mustela vison) selected for either confident or fearful behaviour for nine generations.Two groups of 2-year-old confident (n=12) and fearful (n=12) female mink were given the serotonin (5-HT) 1A receptor agonist buspirone (1.25 mg/kg/day), whereas two other groups of 2-year-old confident (n=12) and fearful (n=12) female mink were given saline, continuously for 5 weeks via osmotic minipumps. Behavioural reactions towards a novel object and towards humans were tested after 19-25 days, and HPA axis reactivity [adrenocorticotropic hormone (ACTH), cortisol] was measured after 28-31 days of treatment. Confident mink were more exploratory than fearful mink towards humans and a novel object. Confident mink spent more time in contact with the object than did fearful mink during saline-but not during buspirone-treatment. buspirone increased approach-withdrawal conflict behaviour towards a object in fearful mink only. The chronic dose of buspirone did not reduce fear towards humans and did not affect latencies to reaction, number of contacts, number and duration of manipulations, and stereotypic behaviour in a Novel Object test. Different HPA axis responses have emerged between confident and fearful mink, together with a different degree of fear-related behaviour. Fearful mink have a higher cortisol combined with a lower ACTH secretion than confident mink in response to capture and blood sampling. The central serotonergic system may be involved, and even though the precise underlying mechanisms are presently unknown, treatment with a 5-HT(1A) receptor agonist reduces the difference between confident and fearful mink in HPA axis reactivity.     

Multilocus haplotype analyses reveal association between 5 novel IL-15 polymorphisms and asthma

BACKGROUND: IL-15 is a T(H)1-related cytokine that is involved in the inflammatory response in various infectious and autoimmune diseases. IL-15 has recently been shown to be upregulated in T-cell-mediated inflammatory disorders. The observations suggest a potential role for this cytokine in a variety of pathologic conditions, including T(H)1-mediated and T(H)2-mediated inflammatory diseases. OBJECTIVE: In this study, we searched for single nucleotide polymorphisms in the whole IL-15 gene and investigated their association with inflammatory and/or atopic phenotypes. METHODS: The screening for single nucleotide polymorphisms was performed by single-strand conformation polymorphism analysis. Genotyping of the identified polymorphisms was performed by restriction fragment length polymorphism. Genotypic association analysis used the Armitage trend test. Haplotype frequency estimation and subsequent testing for differences between cases and controls were performed by using the programs FASTEHPLUS and FAMHAP. RESULTS: We identified 5 novel noncoding nucleotide sequence variants, all of which were typed in our asthmatic, our atopic, and our control population. According to the Armitage trend test, none of the 5 polymorphisms is associated with the phenotype bronchial asthma or atopy. However, multilocus haplotype analysis based on simulations to find out whether the haplotype frequencies differed between cases and controls by using the program FAMHAP yielded a P value of 6.1 x 10(-5) in the asthmatic versus the control population, which is highly significant. Furthermore, we obtained a nominally significant result of P=.0232 for the atopic versus the control population by using FAMHAP. CONCLUSION: These results strongly underscore previous findings that suggest a potential role of this cytokine in allergic diseases.