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Biomedical subjects

H H Schmidt

Publications and source records attributed to H H Schmidt.

At least 19 recordsLinked to original sources
PubMed2004

RT-PCR and FISH analysis of acute myeloid leukemia with t(8;16)(p11;p13) and chimeric MOZ and CBP transcripts: breakpoint cluster region and clinical implications.

The translocation t(8;16)(p11;p13) is associated with acute myeloid leukemia displaying monocytic differentiation (AML FAB M4/5) and fuses the MOZ (also named MYST3) gene (8p11) with the CBP (also named CREBBP) gene (16p13). Detection of the chimeric RNA fusions has proven difficult; only three studies have described successful amplification of the chimeric MOZ-CBP and CBP-MOZ fusions by reverse transcriptase-polymerase chain reaction (RT-PCR). We analyzed four cases of AML M4/5 with t(8;16)(p11;p13) by RT-PCR and fluorescence in situ hybridization (FISH) and characterized the reciprocal RNA fusions from three cases. We cloned both genomic translocation breakpoints from one case by long-range PCR and successfully applied RT-PCR to monitor minimal residual disease (MRD) between clinical complete remission and relapse. In three cases, the genomic breakpoints occurred in MOZ intron 16 and CBP intron 2. In one case, no fusion transcript was detected. The available data suggest clustering of t(8;16)(p11;p13) breakpoints in these introns leading to reciprocal in-frame MOZ exon 16/CBP exon 3 and in-frame CBP exon 2/MOZ exon 17 chimeric transcripts in the majority of cases. The described RT-PCR strategy may be valuable both for the routine detection of the t(8;16)(p11;p13) as well as for monitoring of MRD in this prognostically unfavorable patient group.

Acetyltransferases↗
PubMed2004

Intrapleural instillation of rituximab for the treatment of malignant pleural effusions in NHL.

A patient with CD20(+) leukaemic lymphoplasmacytic Non-Hodgkin's lymphoma (NHL) presented with bilateral malignant pleural effusions. Systemic chemotherapy, repeated percutaneous drainage and bilateral continous chest tube drainage were unable to control the effusions. Rituximab was instilled in a dose-escalating manner via the chest tubes into both pleural spaces, within two weeks the effusions resolved, and the patient has stayed free of symptoms for eight months ongoing. Rituximab may be a promising novel treatment option for malignant effusions in CD20(+) NHL.

Antibodies, Monoclonal↗
PubMed2003

Titer determination of Ad5 in blood: a cautionary note.

Recombinant adenoviruses are presently the most efficient in vivo gene transfer system available. Targeting single organs or large tumors by adenoviral vectors requires an intravascular route of application. During the first pass of viral particles through the vascular bed of the target tissue, virus uptake is not quantitative and indefinite amounts of particles leak into circulation. To determine the amount of leaking particles and to calculate organ-specific uptake (in-/outflow ratio), it is necessary to titrate virus particles directly in blood. In preclinical and clinical trials titration is currently mostly done with blood plasma instead of full blood. However, this technique provides valid results only as long as there is no affinity between adenovirus particles and erythrocytes. In this study we demonstrate that Ad5 particles, as mostly employed for gene therapy, have a strong affinity to human erythrocytes. At 60 min after coincubation of human erythrocytes and Ad5 particles, more than 98% of the particles are attached to the surface of erythrocytes. Therefore, ignoring the amount of red cell bound particles by performing titration in plasma leads to severe miscalculation of organ-specific transfer rates or virus circulation half-life. The biological impact of an increased affinity between virus particles and erythrocytes will be discussed.

Adenoviridae↗
PubMed2001

Substance P and nitric oxide signaling in cerebral cortex: anatomical evidence for reciprocal signaling between two classes of interneurons.

Parvalbumin-containing fast-spiking interneurons in the cerebral cortex exhibit widespread electrical coupling, as do somatostatin-containing low-threshold spiking interneurons. Besides the classical neurotransmitter gamma-aminobutyric acid, these cortical interneurons may also release various neuropeptides including substance P (SP), as well as the freely diffusible messenger nitric oxide (NO). To investigate whether these two networks of interneurons might interact via these nonclassical messengers, we performed immunocytochemistry for SP and NO signaling pathways in rat somatic sensory cortex. SP was found in a subset of parvalbumin-positive cells concentrated in layers IV and V, whereas its receptor, NK1, was found in a subset of somatostatin-containing neurons (and also, at much lower levels, in a disjoint subset of parvalbumin-containing neurons). Only 4% of SP-containing axon terminals were apposed to NK1-positive dendrites, suggesting that in the cerebral cortex, SP may act predominantly as a paracrine neuromediator. Nitric oxide synthase-I (NOS-I), the synthetic enzyme for NO, was found almost exclusively in NK1-positive neurons; 95% of intensely somatostatin/NK1-positive neurons were also positive for NOS-I, and 94% of NOS-positive neurons were also positive for NK1. Immunoreactivity for soluble guanylyl cyclase (the NO receptor) was at high levels in the apical dendrites of layer V pyramidal neurons and in parvalbumin/SP-positive neurons. These data point to a novel reciprocal chemical interaction between two inhibitory networks in the rat neocortex.

Animals↗
PubMed2001

Upregulation of the vascular NAD(P)H-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo.

In different cardiovascular disease states, oxidative stress decreases the bioavailability of endothelial NO, resulting in endothelial dysfunction. An important molecular source of reactive oxygen species is the enzyme family of NAD(P)H oxidases (Nox). Here we provide evidence that the vascular Nox isoforms Nox1 and Nox4 appear to be involved in vascular oxidative stress in response to risk factors like angiotensin II (Ang II) in vitro as well as in vivo. Nox mRNA and protein levels were quantified by real-time RT-PCR and Western blotting, respectively. Nox1 and Nox4 were expressed in the vascular smooth muscle cell (VSMC) line A7r5 and aortas and kidneys of rats. Upon exposure of A7r5 cells to Ang II (1 microM, 4 h), Nox1 and Nox4 mRNA levels were increased 6-fold and 4-fold, respectively. Neither the vasoconstrictor endothelin 1 (up to 500 nM, 1-24 h) nor lipopolysaccharide (up to 100 ng/ml, 1-24 h) had any effect on Nox1 and Nox4 expression in these cells. Consistent with these observations made in vitro, aortas and kidneys of transgenic hypertensive rats overexpressing the Ren2 gene [TGR(mRen2)27] had significantly higher amounts of Nox1 and Nox4 mRNA and of Nox4 protein compared to tissues from normotensive wild-type animals. In conclusion, Nox4 and Nox1 are upregulated by the renin-angiotensin system. Increased superoxide production by upregulated vascular Nox isoforms may diminish the effectiveness of NO and thus contribute to the development of vascular diseases. Nox1 and Nox4 could be targeted therapeutically to reduce vascular reactive oxygen species production and thereby increase the bioavailability of NO.

Angiotensin II↗
PubMed2001

Structural basis for pterin antagonism in nitric-oxide synthase. Development of novel 4-oxo-pteridine antagonists of (6R)-5,6,7,8-tetrahydrobiopterin.

Pathological nitric oxide (NO) generation in sepsis, inflammation, and stroke may be therapeutically controlled by inhibiting NO synthases (NOS). Here we targeted the (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)Bip)-binding site of NOS, which, upon cofactor binding, maximally increases enzyme activity and NO production from substrate l-arginine. The first generation of H(4)Bip-based NOS inhibitors employed a 4-amino pharmacophore of H(4)Bip analogous to antifolates such as methotrexate. We developed a novel series of 4-oxo-pteridine derivatives that were screened for inhibition against neuronal NOS (NOS-I) and a structure-activity relationship was determined. To understand the structural basis for pterin antagonism, selected derivatives were docked into the NOS pterin binding cavity. Using a reduced 4-oxo-pteridine scaffold, derivatives with certain modifications such as electron-rich aromatic phenyl or benzoyl groups at the 5- and 6-positions, were discovered to markedly inhibit NOS-I, possibly due to hydrophobic and electrostatic interactions with Phe(462) and Ser(104), respectively, within the pterin binding pocket. One of the most effective 4-oxo compounds and, for comparisons an active 4-amino derivative, were then co-crystallized with the endothelial NOS (NOS-III) oxygenase domain and this structure solved to confirm the hypothetical binding modes. Collectively, these findings suggest (i) that, unlike the antifolate principle, the 4-amino substituent is not essential for developing pterin-based NOS inhibitors and (ii), provide a steric and electrostatic basis for their rational design.

Animals↗
PubMed2001

Regional and age-dependent expression of the nitric oxide receptor, soluble guanylyl cyclase, in the human brain.

Nitric oxide (NO), synthesized by neuronal NO synthase (NOS-I), plays essential physiological roles in the brain. The major molecular target for NO is soluble guanylyl cyclase (sGC), a heterodimeric hemoprotein composed of a larger alpha and a smaller beta subunit. Both subunits of sGC are needed to generate the second messenger cyclic GMP (cGMP). Here we show using subunit-specific antibodies and Western blot analysis that sGCalpha1 and sGCbeta1 protein subunits are present in all examined human brain regions. The relative distribution of the two subunits was similar and also correlated well with the known distribution of NOS-I. The highest expression levels of sGC were found in cortex, basal ganglia and the limbic system. These regions display the most prominent biochemical and histological changes during ageing. In cortex, a negative correlation between the amounts of sGC and age was found, while sex and post-mortem delay time did not affect sGC levels significantly. Our data suggest that sGCalpha1 and sGCbeta1 subunits are widely distributed in human brain, consistent with a major role in NO signaling. Moreover, the NO/cGMP pathway appears to be affected by ageing in the human brain.

Aged↗
PubMed2001

Pterin interactions with distinct reductase activities of NO synthase.

Besides oxidizing L-arginine, neuronal NO synthase (NOS) NADPH-dependently reduces various electron acceptors, including cytochrome c and tetrazolium salts. The latter NADPH diaphorase reaction is used as a NOS-specific histochemical stain. Both reductase activities have been utilized to analyse electron transfer mechanisms within NOS. Basal L-arginine turnover by homodimeric NOS is enhanced by exogenous tetrahydrobiopterin, and the intra-subunit electron flow may include intermediate trihydrobiopterin. In the present work we have investigated the possible role of the tetrahydrobiopterin binding site of NOS in its reductase activities by examining the effects of anti-pterin type (PHS) NOS inhibitors. Although the type I anti-pterin, PHS-32, which does not affect basal dimeric NOS activity, also had no effect on either reductase activity, the type II anti-pterin, PHS-72, which inhibits basal NOS activity, inhibited both reductase activities and the NADPH diaphorase histochemical stain. Pterin-free NOS monomers catalysed both cytochrome c and tetrazolium salt reduction. Our data suggest that both NOS reductase activities are independent of tetrahydrobiopterin. However, occupation of an exosite near the pterin site in NOS by type II anti-pterins may interfere with the electron flow within the active centre, suggesting that steric perturbation of the pterin binding pocket or reductase interaction contribute to the mechanism of inhibition by this class of NOS inhibitors.

Biopterins↗
PubMed2001

Nitroxyl oxidizes NADPH in a superoxide dismutase inhibitable manner.

Nitric oxide synthases (NOS) convert L-arginine and N(omega)-hydroxy-L-arginine to nitric oxide (*NO) and/or nitroxyl (NO(-)) in a NADPH-dependent fashion. Subsequently, *NO/superoxide (O(2-)-derived peroxynitrite (ONOO(-)) consumes one additional mol NADPH. The related stoichiometry of NO(-) and NADPH is unclear. We here describe that NO(-) also oxidizes NADPH in a concentration-dependent manner. In the presence of superoxide dismutase (SOD), which also converts NO(-) to *NO, nitrite accumulation was almost doubled and no oxidation of NADPH was observed. Nitrate yield from NO(-) was low, arguing against intermediate ONOO(-) formation. Thus, biologically formed NO(-) may function as an effective pro-oxidant unless scavenged by SOD and affect the apparent NADPH stoichiometry of the NOS reaction.

Free Radicals↗
PubMed2001

Immunohistochemical localization of nitric oxide synthase and soluble guanylyl cyclase in the ventral cochlear nucleus of the rat.

The diffusible messenger nitric oxide (NO) is implicated in auditory processing. It acts in the brain largely through activation of soluble guanylyl cyclase (sGC), a heterodimer comprised of alpha and beta subunits. The authors used immunohistochemistry to study the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway in the cochlear nucleus of Sprague-Dawley rats. Central fibers of the cochlear nerve were stained for neuronal nitric oxide synthase (NOS-I) but not for sGCbeta. Within the ventral cochlear nucleus, a large fraction of principal cells were immunopositive for both NOS-I and sGCbeta; these cells could be seen at times receiving contacts from NOS-I-positive fibers. sGC staining of somatic cytoplasm extended into the distal dendritic tree. At variance with this pattern, NOS-I was concentrated mainly in somata. Double-labeling experiments showed that most of the principal neurons expressed both antigens. By contrast, in the granule cell domain, small cells that were immunopositive for NOS-I rarely corresponded to those that were immunopositive for sGC. To assess whether NOS-I and sGC immunoreactivities colocalize with their respective catalytic activities, the authors performed multiple labeling with L-citrulline (a by-product of the formation of NO from L-arginine) and cGMP, respectively. L-citrulline was restricted to NOS-I-positive elements, and the large majority of NOS-expressing neurons were positive for citrulline. Multiple labeling revealed that almost all sGC-positive neurons also accumulated cGMP both in the ventral cochlear nucleus and in the granule cell domain. These data suggest that NO is a signaling molecule in the cochlear nucleus, perhaps functioning in both a paracrine manner and an autocrine manner.

Animals↗
PubMed2001

Replacement of calcineurin inhibitors with mycophenolate mofetil in liver-transplant patients with renal dysfunction: a randomised controlled study.

BACKGROUND: Renal dysfunction is a major complication of long-term immunosuppressive therapy with calcineurin inhibitors (CNI) in liver-transplant recipients. We undertook a randomised study to assess the safety and efficacy of CNI withdrawal and replacement by mycophenolate mofetil. METHODS: 28 people who had had renal dysfunction attributable to suspected CNI toxicity after liver transplantation participated in the study. We replaced CNI with mycophenolate mofetil in a stepwise pattern in half the group (study patients); the other half (controls) stayed on CNI immunosuppression. Renal function, blood pressure, uric acid, and blood lipids were measured before and 6 months after study entry. Side-effects of medication and graft function were recorded throughout the study. FINDINGS: At the end of the study, mean (SD) serum creatinine had fallen by 44.4 (48.7) micromol/L in study patients compared with 3.1 (14.3) micromol/L in controls; a mean difference of 41.3 micromol/L (95% CI 12.4-70.2). Moreover, systolic and diastolic blood pressure, and serum uric acid decreased significantly in the study group but not in the control group (mean [95% CI] between group differences 10.8 mm Hg [3.0-18.6], 5.0 mm Hg [0.9-9.2], and 83.1 micromol/L [12.7-153.6], respectively). There were no changes in cholesterol or triglyceride concentrations in either group. Side-effects were reported by eight of the study patients. Three reversible episodes of acute graft rejection occurred in study patients during mycophenolate mofetil monotherapy, whereas none occurred in the control group. INTERPRETATION: Substitution of CNI by mycophenolate mofetil can improve renal function, blood pressure, and uric acid concentration of liver-transplant patients, but there is an increased rejection risk with mycophenolate mofetil monotherapy.

Blood Pressure↗
PubMed2001

Interphase fluorescence in situ hybridization assay for the detection of 3q21 rearrangements in myeloid malignancies.

In myeloid malignancies, chromosome rearrangements involving band 3q21 are associated with a particularly poor prognosis of the disease. Their sensitive and unequivocal detection is therefore of great clinical importance. In this report, we describe the establishment of an interphase fluorescence in situ hybridization (FISH) assay that complements classical cytogenetic analysis in the diagnosis of such aberrations. PACs that map centromeric and telomeric of known 3q21 breakpoints were labeled with different fluorescent dyes, and the separation of the normally colocalizing signals was used as an indicator of the presence of a 3q21 rearrangement. Two cell lines and 10 primary samples from myeloid leukemia and myelodysplastic syndrome (MDS) patients with 3q21 rearrangements were investigated using the newly established method. The rate of false positivity was determined in 27 control samples from patients with various types of myeloid malignancies. In addition to providing a sensitive and rapid test for the detection of 3q21 aberrations, the interphase FISH assay yields preliminary information about the localization of individual breakpoints. Six of the 10 breakpoints in the patient samples map to an only recently described breakpoint cluster region (BCR) 60 kb centromeric of the originally reported 3q21 BCR. These findings may contribute to the understanding of the molecular basis of the clinical features associated with 3q21 rearrangements.

Adult↗