Biomedical subjects

W Bloch

Publications and source records attributed to W Bloch.

At least 73 records · Page 4Linked to original sources

PubMed1999

LMR spectroscopy: a new sensitive method for on-line recording of nitric oxide in breath.

Laser magnetic resonance spectroscopy (LMRS) is a sensitive and isotope-selective technique for determining low concentrations of gaseous free radicals with high time resolution. We used this technique to analyze the nitric oxide (NO) concentration profile while simultaneously measuring the flow and expired volume during several single breathing cycles. Eight healthy, nonallergic volunteers were investigated. An initial NO peak was found in all breathing cycles before the NO concentration dropped to a relatively stable plateau in the late phase of expiration. The nasal NO peak was significantly higher than the oral NO peak. The nasal NO plateau was always higher than the oral NO plateau. The height of the initial nasal and oral NO peak rose with increasing duration of breath hold, whereas the late expiratory NO plateau changed only little for either the nasal or the oral breathing cycles. Our findings demonstrate, in line with other reports using other techniques, that the nose is the primary source for NO within the airways.

Adult↗

PubMed1999

Establishment of ionic channels and signalling cascades in the embryonic stem cell-derived primitive endoderm and cardiovascular system.

The first organ system to be established in early embryogenesis is the cardiovascular system which develops upon interaction with hypoblastic cells of the primitive endoderm. Here we focus on recent work on embryoid bodies derived from pluripotent embryonic stem (ES) cells. Ca(2+) oscillations and Ca(2+) signalling pathways during the differentiation of primitive endodermal cell layers are reported. Furthermore, the development-dependent expression of ion channels and the buildup of signalling cascades involved in the modulation of voltage-dependent L-type Ca(2+) channels during early cardiomyogenesis and the formation of functional vascular structures in the process of vasculogenesis and angiogenesis are reviewed. We also report on the use of green fluorescent protein reporter gene expression under the control of cardiac-specific promoters, e.g. the human cardiac alpha-actin promoter, which enables the identification and in vivo characterization of cardiomyocytes at very early stages of cardiomyogenesis.

Animals↗

PubMed1999

Effectiveness of oral L-arginine in first-line treatment of erectile dysfunction in a controlled crossover study.

BACKGROUND AND AIMS: Relaxation of cavernous smooth muscle is a parasympathetic and non-adrenergic, non-cholinergic mediated process which requires nitric oxide (NO). NO is synthesized from L-arginine by NO synthase (NOS). Some studies report good clinical results under oral L-arginine medication in the treatment of erectile dysfunction. We examined the effectiveness and safety of L-arginine in the treatment of mixed-type impotence. METHODS: 32 patients (mean age 51.6 years) with mixed-type impotence diagnosed according to the results of sexual history and urological examination were enrolled in a randomized, placebo-controlled, crossover comparison of an oral placebo with 3 x 500 mg L-arginine/day. A validated questionnaire (KEED) was used to define the grade of impotence with a score. The treatment consisted of two 17-day courses (50 tablets). After a 7-day washout period the patients who initially received the placebo for 17 days were switched to L-arginine and vice versa. We assessed the efficacy with the validated questionnaire at the end of each drug period. RESULTS: 30 patients (94%) completed the whole treatment schedule. Five (17%) patients reported a significant improvement in erectile function at the end of the L-arginine phase and 6 (20%) patients after the placebo period. 17 (56%) patients showed little improvement with L-arginine and 13 (43%) with placebo. In 8 patients (27%) of the verum group there was either no change in the ED score or even a slight worsening. No statistical difference in the impotence scores were found. No drug-related adverse effects occurred with L-arginine treatment. CONCLUSION: Oral L-arginine 3 x 500 mg/day is not better than placebo as a first-line treatment for mixed-type impotence.

Administration, Oral↗

PubMed1998

Functional characteristics of ES cell-derived cardiac precursor cells identified by tissue-specific expression of the green fluorescent protein.

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions. In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac alpha-actin promoter. First, fluorescent areas in ES cell-derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1-2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs. Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1998

Cardiac specific expression of the green fluorescent protein during early murine embryonic development.

We demonstrate the establishment of transgenic mice, where the expression of the green fluorescent protein (GFP) is under control of the human cardiac alpha-actin promoter. These mice display cardiac specific GFP expression already during early embryonic development. Prominent GFP fluorescence was observed at the earliest stage of the murine heart anlage (E8). Cardiomyocytes of different developmental stages proved GFP positive, but the intensity varied between cells. We further show that contractions of single GFP positive cardiomyocytes can be monitored within the intact embryo. At later stages of embryonic development, the skeletal musculature was also GFP positive, in line with the known expression pattern of cardiac alpha-actin. The tissue specific labeling of organs is a powerful new tool for embryological as well as functional investigations in vivo.

Actins↗

PubMed1998

Nitric oxide synthase in the vestibulocochlear system of mice.

The exact distribution of nitric oxide-synthases (NOS) and the NO-target enzyme soluble guanylyl cyclase (sGC) in the cochlea and vestibular organ is an issue of current discussion. The existence of NOS-isoforms in the cochlea of the guinea pig has been described recently, while information about the vestibular system are still rare and non-satisfying. In order to gain more information, immunostaining was performed, using specific antibodies to NOS I-III and to sGC, on paraffin sections of complete temporal bones from mice. NOS III could be detected in cochlea and vestibular ganglion cells, in nerve fibres, in outer hair cells of the cochlear and in the sensory epithelium of the maculae. Also, the spiral ligament and the limbus epithelium was positive to NOS III. NOS I was found in the sensory epithelium of the maculae and cristae ampullares, outer and inner hair cells of the cochlea, in nerve fibres and in ganglion cells. In contrast to that NOS II could not be detected at all. Furthermore, a strong NOS I immunoreaction was displayed on the endosteum of the bone, while the periosteum was lacking of NOS. NOS detection was accompanied by immunoreactivity to sGC. The findings imply that NOS I and III-generated NO is involved in neurotransmission and other regulative processes in the vestibulocochlear system.

Animals↗

PubMed1998

Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects.

The pleiotrophic but overlapping functions of the cytokine family that includes interleukin (IL)-6, IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin 1 are mediated by the cytokine receptor subunit gp130 as the common signal transducer. Although mice lacking individual members of this family display only mild phenotypes, animals lacking gp130 are not viable. To assess the collective role of this cytokine family, we inducibly inactivated gp130 via Cre-loxP-mediated recombination in vivo. Such conditional mutant mice exhibited neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects, demonstrating the widespread importance of gp130-dependent cytokines.

Animals↗

PubMed1998

Localisation of the nitric oxide (NO)/cGMP-pathway in the vestibular system of guinea pigs.

The exact distribution of nitric oxide-synthases (NOS) in the vestibular system has not been described satisfying yet. Immunostaining, using specific antibodies to the three known NOS-isoforms, to cyclic guanosine monophosphate (cGMP) and soluble guanylyl-cyclase (sGC), the second messenger system of nitric oxide (NO), was performed on paraffin sections of temporal bone from guinea pigs. eNOS could be detected in vestibular ganglion cells and in nerve fibres, including the calyces, surrounding the type 1 hair cells (HC). bNOS was found in the sensory epithelium, ganglion cells and in bone, while iNOS could not be found. NOS-detection was accompanied by reactivity to sGC and to cGMP. This finding implies that b- and eNOS-generated NO is involved in regulative processes in neurotransmission and regulation of blood flow.

Animals↗

PubMed1998

Beta1 integrin deficiency impairs migration and differentiation of mouse embryonic stem cell derived neurons.

Cell-matrix interaction plays an important role during neuronal development, which is demonstrated by comparing wild type (D3)- and beta1 integrin-deficient (G201) embryonic stem cell derived neurons. In D3 preparations complex networks of functionally coupled neurons with bi- and multipolar morphologies develop. In contrast, neuronal differentiation is retarded in G201 derived neurons, recognised by limited migration and restricted morphological differentiation. Furthermore, beta1 integrin deficiency causes a delay in expression of major neurotransmitters like GABA and glutamate as well as of synaptophysin. These findings indicate a prominent role of beta1 integrin for both morphological and chemical differentiation.

Animals↗

PubMed1998

Selective expression of inducible nitric oxide synthase in human prostate carcinoma.

BACKGROUND: Nitric oxide (NO) is synthesized by inducible nitric oxide synthase (iNOS) and plays an important role in tumor growth and angiogenesis. NO generation by iNOS also influences the cytotoxicity of macrophages and tumor-induced immunosuppression. Before now, the expression of iNOS in prostate carcinoma tissue had not been determined. METHODS: In this study, tissue sections from 16 patients with prostate carcinoma were studied immunohistochemically and compared with tissue specimens from 10 patients with benign hyperplasia. RESULTS: Positive iNOS immunostaining was detected in all sections from patients with prostate carcinoma. The malignant epithelial cells were highly positive. The antibody against iNOS also marked round cells, which had the same cell shape as that observed for macrophages. These cells were located in stroma and epithelium adjacent to tumor islets. However, round cells in benign tissue stained negative for iNOS. None of the benign hyperplasia specimens stained positive for iNOS immunohistochemically. CONCLUSIONS: Prostate carcinoma tissue had a high iNOS content, whereas benign tissue did not. The authors suggest that epithelial iNOS expression can be used as a specific immunohistochemical marker for prostate carcinoma. NO generation by iNOS may play multiple roles in the development of this disease.

Adenocarcinoma↗

PubMed1998

Deficiency of beta 1 integrins in teratoma interferes with basement membrane assembly and laminin-1 expression.

Subcutaneous injection of beta 1 integrin-deficient embryonic stem cells in mice causes the formation of teratomas although they occur with a lower frequency and are smaller than wild-type cells. Immunofluorescence analysis of these deficient tumors indicates a disorganized deposition of several basement membrane proteins. This was confirmed by electron microscopy which demonstrated frequent gaps in cell-associated basement membranes or loss of close contacts to the cells. Further aberrant features were multilaminar structures and amorphous deposits, indicating a strong impairment of correct basement membrane assembly. Quantitative radioimmunoassays were used to determine the levels of specific proteins in successive tissue extracts with neutral buffer in the absence and presence of EDTA and with 6 M guanidine. This demonstrated a more than 90% decrease in the content of laminin-1 (alpha 1 beta 1 gamma 1) and a 70% decrease in nidogen in the beta 1 integrin-deficient teratomas. No significant changes were detected for other matrix proteins (perlecan, fibronectin, fibulins). This selective change impaired the formation of laminin-nidogen complex and enhanced nidogen degradation. Northern blots also demonstrated a distinctly reduced expression of laminin alpha 1, beta 1, and gamma 1 chains. Similar reductions were also observed in cultured embryonic stem cells prior to any differentiation. No or only smaller changes were observed for laminin alpha 2 and beta 2 chain, nidogen, and perlecan mRNA. These data emphasize a distinct role of beta 1 integrins in the correct assembly of basement membranes which may occur through direct ligand binding and/or regulatory events at the transcriptional level.

Animals↗

PubMed1998

Unchanged protein expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin in terminally failing human myocardium.

The enhanced diastolic Ca2+ levels observed in cardiac myocytes from patients with idiopathic dilated cardiomyopathy (DCM) may be either a consequence of functional impairment of sarcoplasmic reticulum calcium-ATPase (SERCA 2) and its regulator protein phospholamban or due to a reduction in the number of SERCA 2 proteins. As different myocardial membrane preparations may lead to different accumulation of proteins, the present study evaluated two different membrane preparations, in human failing and nonfailing myocardium for comparison of SERCA 2 activity and the protein expression of SERCA 2 and phospholamban. Crude membranes and tissue homo-genates without any centrifugation steps were prepared from human nonfailing hearts (donor hearts, NF, n=18) and terminally failing hearts (heart transplant, DCM, n=18). Calsequestrin protein expression was used as an internal control for overall protein expression. In both crude membranes and homogenates maximal SERCA 2 activity (Vmax) was significantly reduced in failing heart preparations (NF crude membranes, 130+/-8; DCM crude membranes, 102+/-5 nmol ATP/mg protein per minute). In contrast, the protein expression of SERCA 2 (NF crude membranes, 488+/-35; DCM crude membranes, 494+/-42; P=0.92), phospholamban (NF crude membranes, 497+/-51; DCM crude membranes, 496+/-45; P=0.98) and calsequestrin (NF crude membranes, 109+/-06; DCM crude membranes, 107+/-08; P=0.84) was unchanged in NF and DCM hearts in both preparation methods. This was also the case when the protein expression was normalized to calsequestrin protein levels. Preparation of sarcoplasmic reticulum in crude membranes led to enhanced purification and consequently higher SERCA 2, phospholamban, and calsequestrin protein levels in crude membranes than in the homogenates, which was paralleled by an increase in SERCA 2 enzyme activity. In conclusion, the altered Ca2+ handling in DCM may be a consequence of reduced SERCA 2 enzyme activity and not the result of differences in protein expression of the Ca2+ regulating proteins SERCA 2, phospholamban, and calsequestrin in human myocardium. The present study emphasizes the importance of different myocardial membrane preparations with respect to quantitative investigations of protein expression and function.

Adult↗

PubMed1998

Evidence for the involvement of endothelial nitric oxide synthase from smooth muscle cells in the erectile function of the human corpus cavernosum.

Nitric oxide (NO) is an important mediator in the relaxation of cavernosal smooth muscle. The present study examines the existence and location of the constitutive isoform eNOS (endothelial NO synthase) accompanying the already substantiated neurogenic NOS (nNOS) in the human corpus cavernosum of men with and without erectile dysfunction. Activities of NOS enzymes were examined in specimens of 11 potent and nine long-term impotent patients by means of light and electron microscopy using NADPH-diaphorase staining and immunohistochemical eNOS-specific, smooth muscle actin-specific and nNOS-specific markers. Cavernosal smooth muscle shows a distinct expression of eNOS. In contrast to the weaker expression of eNOS and nitrinergic innervation found in larger veins, the small intracavernosal helicine arteries express large quantities of eNOS and possess a dense nitrinergic innervation. Long-term impotent patients display a broad heterogeneity in eNOS expression and nitrinergic innervation while no overall correlation between NOS expression and erectile function was observed. The expression of eNOS indicates eNOS as a main source of NO alongside nNOS. The differentiated localization of eNOS supports at least a role of this isoform in vascular regulation.

Erectile Dysfunction↗

PubMed1998

In vitro expression of inducible nitric oxide synthase in the nasal mucosa of guinea pigs after incubation with lipopolysaccharides or cytokines.

In order to demonstrate the involvement of nitric oxide synthases (NOS)--in particular the inducible isoform (iNOS)--in inflammatory processes within the nasal airways, we used organ-bath incubation to study isolated inferior turbinates and mucosa of the maxillary sinus of guinea pigs. The pattern of the expression in various substructures of the nasal mucosa was of special interest. Mucosa was incubated for 6 h with lipopolysaccharides (LPS) produced by E. coli, interleukin II (IL-2) or tumor necrosis factor-alpha (TNF-alpha). Saline was used as the control solution. Following incubation the specimens were fixed in buffered 4% formaldehyde solution over a period of 4 h. Tissues were next exposed to nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase-reaction and immunostained with specific antibodies to iNOS. Results then showed a clearly increased or initiated expression of iNOS in epithelium, glands, leucocytes and blood vessels of treated tissues in comparison to the control specimens. The inflammatory mediator LPS and the cytokines Il-2 or TNF-alpha alone were found to be capable of increasing the expression of iNOS, although the effects of LPS clearly exceeded those of the cytokines. This finding implicates iNOS-generated nitric oxide as a key factor for causing nasal swelling, secretion and obstruction during nasal infections and allergic episodes.

Animals↗

PubMed1997

Beta 1 integrin is essential for teratoma growth and angiogenesis.

Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of beta 1 integrin during teratoma formation, we compared teratomas induced by normal and beta1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, beta 1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of beta 1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in beta1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface. Normal teratomas had large blood vessels with a smooth inner surface, containing both host- and ES cell-derived endothelial cells. In contrast, beta 1-null teratomas had small vessels that were loosely embedded into the connective tissue. Furthermore, endothelial cells were always of host-derived origin and formed blood vessels with an irregular inner surface. Although beta 1- deficient endothelial cells were absent in teratomas, beta 1-null ES cells could differentiate in vitro into endothelial cells. The formation of a complex vasculature, however, was significantly delayed and of poor quality in beta1-null embryoid bodies. Moreover, while vascular endothelial growth factor induced proliferation of endothelial cells as well as an extensive branching of blood vessels in normal embryoid bodies, it had no effect in beta 1-null embryoid bodies.

Animals↗

PubMed1997

Distribution of nitric oxide synthase implies a regulation of circulation, smooth muscle tone, and secretory function in the human prostate by nitric oxide.

BACKGROUND: Nitric oxide (NO) is suggested as a mediator involved in the regulation of smooth muscle tone, blood flow, and secretory function of the genitourinary tract and originates from different NO synthase (NOS) isoforms located in endothelial, neuronal, and epithelial structures. The aim of the present study was to determine the location of endothelial and neuronal NOS in the human prostate. METHODS: Histochemical NADPH-diaphorase (NADPH-d) staining, ultrastructural NADPH examination, and NOS immunohistochemistry were performed on histologically verified nonmalignant prostate tissue from normal nonobstructive and hyperplastic obstructive human prostates. RESULTS: In the prostatic tissue, NADPH-d staining and immunohistochemistry with bNOS antibody revealed the existence of a dense nitrinergic innervation of glandular epithelium, fibromuscular stroma, and blood vessels. NADPH-d reaction in glandular epithelium was not confirmed by ecNOS or bNOS immunohistochemistry. In benign prostatic hyperplasia (BPH), the nitrinergic innervation is reduced. The vascular distribution of ecNOS provides evidence for a segmental differentiation of the NO-mediated vascular regulation. CONCLUSIONS: NO plays an important role in the autonomic innervation of all compartments of prostatic tissue. In obstructive BPH, the nitrinergic innervation is reduced compared to that in normal prostate tissue. Further studies are necessary to elucidate the complex role of NO in the prostate.

Aged↗

PubMed1997

NO synthase-II is transiently expressed in embryonic mouse olfactory receptor neurons.

Among three NO synthase (NOS) isoforms only the inducible NOS-II was localized in developing olfactory receptor neurons of the mouse. First NOS-II immunoreactive receptor cells including their processes were detected by embryonic day 11 when the olfactory pit starts to invaginate. Cellular staining lasted until embryonic day 16, and was reduced during the next few days. At embryonic day 20 no reactivity was found in the olfactory epithelium, whereas centripetal nerve fibers remained positive. This transient expression of NOS-II implies a role for the differentiation of early olfactory receptor neurons and synaptic plasticity.

Animals↗

PubMed1997

[Pattern of distribution of constitutive isoforms of NO synthase in the normal prostate and obstructive prostatic hyperplasia].

Nitric oxide (NO) is suggested as an important mediator for the regulation of biological processes. In the present study we tried to determine histochemically and immunohistochemically the localization and distribution of the constitutive NO-synthase Isoforms (bNOS and eNOS) of 14 normal non-obstructive and 12 hyperplastic obstructive human prostates. Differentiated nitrinergic innervation was shown for the prostate glands, fibromuscular stroma and blood vessels by NADPH-diaphorase staining and immunohistochemically with specific NOS antibodies. In the specimens with benign prostatic hyperplasia nitrinergic innervation seems to be distinctly reduced. The vascular distribution of NOS provides evidence for segmental differentiation of NO-mediated vascular regulation. The NADPH-diaphorase reaction was not confirmed immunohistochemically by the specific NOS antibody in the glandular epithelium. The distribution of NO synthase shows the importance of nitric oxide in the regulation of smooth muscle tone, blood flow and secretory function in the normal and hyperplastic human prostate.

Adult↗