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Biomedical subjects

A Schmidt

Publications and source records attributed to A Schmidt.

At least 199 records · Page 11Linked to original sources
PubMed1999

Sublocalization of putative tumor suppressor gene loci on chromosome arm 14q in neuroblastoma.

RFLP and microsatellite analysis with 23 polymorphic markers spanning the entire long arm of chromosome 14 in 108 neuroblastomas showed allelic loss in 19 out of 107 (18%) informative tumors, placing 14q among the most frequently affected chromosomal regions in neuroblastoma. One minimal deletion region could be sublocalized in 17 of 19 cases between markers D14S1 and D14S16, and a second one between markers D14S17 and D14S23 in band 14q32. Furthermore, breakpoints in bands 14q23 and 14q12 were detected. These results suggest the presence of at least two putative tumor suppressor gene loci on chromosome 14. Survival analyses revealed no prognostic impact of allelic loss of 14q in neuroblastoma. Genes Chromosomes Cancer 26:40-46, 1999.

Chromosome Mapping↗
PubMed1999

[Performance of different CT angiography imaging modalities in detecting renal artery stenoses].

PURPOSE: Purpose of the study was to compare the sensitivity and specificity of various display modalities in the detection of renal artery stenosis. In particular, the difference between hard-copy reading and interactive analysis at the workstation was assessed. MATERIALS AND METHODS: Selected patients (n = 31) with expected suboptimal conditions for CT angiography due to long-standing hypertension and compromised renal function were included. Six radiologists evaluated independently a total of 77 renal arteries with 49 renal artery stenoses proven by angiography. Image analysis included: mode A: interactive display, analysis of multiplanar reformats and axial sections at the workstation, mode B: visualization of MIPs in the coronal and axial planes as hard copies only, mode C: visualization of MIPs and axial sections as hard copies only. RESULTS: The following sensitivities and specificities were found for internal readers and (readers from outside institutions): mode A: 94.8%, 87.9%, mode B: 97.7% (95.1%), 80.3% (75.4%); mode C: 97.0% (95.3%), 78.8% (76.6%). The differences were statistically not significant (p > 0.05). CONCLUSIONS: Standardized hard copies of MIPs plus axial CT provide sufficient accuracy to detect renal artery stenosis compared to interactive imaging even in this highly selected group of patients.

Adult↗
PubMed1998

The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease.

The Saccharomyces cerevisiae targets of rapamycin, TOR1 and TOR2, signal activation of cell growth in response to nutrient availability. Loss of TOR or rapamycin treatment causes yeast cells to arrest growth in early G1 and to express several other physiological properties of starved (G0) cells. As part of this starvation response, high affinity amino acid permeases such as the tryptophan permease TAT2 are targeted to the vacuole and degraded. Here we show that the TOR signalling pathway phosphorylates the Ser/Thr kinase NPR1 and thereby inhibits the starvation-induced turnover of TAT2. Overexpression of NPR1 inhibits growth and induces the degradation of TAT2, whereas loss of NPR1 confers resistance to rapamycin and to FK506, an inhibitor of amino acid import. NPR1 is controlled by TOR and the type 2A phosphatase-associated protein TAP42. First, overexpression of NPR1 is toxic only when TOR function is reduced. Secondly, NPR1 is rapidly dephosphorylated in the absence of TOR. Thirdly, NPR1 dephosphorylation does not occur in a rapamycin-resistant tap42 mutant. Thus, the TOR nutrient signalling pathway also controls growth by inhibiting a stationary phase (G0) programme. The control of NPR1 by TOR is analogous to the control of p70 s6 kinase and 4E-BP1 by mTOR in mammalian cells.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Tumors of the retinal pigment epithelium metastasize to inguinal lymph nodes and spleen in tyrosinase-related protein 1/SV40 T antigen transgenic mice.

The pigment epithelium of the retina (RPE) is derived from the optic cup and is essential for function and development of the eye. We produced a transgenic mouse line that expresses simian virus (SV40) transforming sequences under control of the 1.4 kb tyrosinase-related protein 1 (TRP-1) promoter, targeting expression of T antigen (Tag) to the RPE. In transgenic embryos, RPE cells proliferated in the anterior part of the eye and near the optic nerve. This resulted in formation of tumors, which were pigmented and of epithelial origin. In 3 months-old mice, pigmented cells were detected in spleen and inguinal lymph nodes. In spleen, tyrosinase, TRP-1 and SV40 Tag were expressed and tyrosinase was enzymatically active. Pigmented regions were positive for an epithelial marker, cytokeratin. Cell lines were established from tumor and metastases and kept in culture for more than 2 months. These were pigmented, and maintained expression of tyrosinase, TRP-1, cytokeratin and SV40 Tag. This demonstrates that RPE tumor cells metastasize to lymph node and spleen. In conclusion, the metastasis from TRP-1/Tag RPE tumors towards spleen and lymph nodes serves as potential tool to investigate biology and metastasis of tumors derived from the pigment epithelium.

Animals↗
PubMed1998

On the analysis of iodine-129 and iodine-127 in environmental materials by accelerator mass spectrometry and ion chromatography.

Based on a review of literature about the abundances of 129I (T1/2 = 15.7 Ma) in the environment we show that there is a severe lack of knowledge, in particular about natural, pre-nuclear levels. Among the two analytical techniques which are sensitive enough to investigate 129I in environmental materials, namely radiochemical neutron activation analysis (RNAA) and accelerator mass spectrometry (AMS), only AMS is capable of covering the natural, pre-nuclear levels. Since such AMS measurements require chemical separation of iodine from the matrix, a wide variety of separation schemes are necessary for environmental analyses. We report here on such schemes for the analysis of soils, plants and soft tissue. They are applied exemplarily to analyses of soils from the vicinity of Chernobyl. For chemical separations prior to analysis, contamination control and blank analyses are essential. Here, we discuss quality control procedures in detail, both for RNAA and AMS. In the case of AMS we use ion-chromatography (IC) for the determination of stable iodine. The IC analysis is included in the separation schemes for environmental materials. First AMS-analyses of terrestrial biospheric materials demonstrate that natural environmental levels of 129I are lower than previously deduced from investigations using RNAA, but higher than expected from model calculations. AMS is capable of providing the missing knowledge about the radioecology of 129I.

Air Pollutants, Radioactive↗
PubMed1998

Influence of the angiotensin II antagonist valsartan on left ventricular hypertrophy in patients with essential hypertension.

BACKGROUND: Left ventricular hypertrophy (LVH) represents an independent risk factor in patients with essential hypertension. Because reversal of LVH may be associated with an improvement of prognosis, the influence of new antihypertensive compounds, such as angiotensin II AT1 receptor antagonists, on LVH should be determined. METHODS AND RESULTS: In a randomized, double-blind trial, 69 predominantly previously untreated hypertensive patients with echocardiographically proven LVH, ie, left ventricular mass index (LVMI) >134 g/m2 in men and >110 g/m2 in women and/or end-diastolic septal thickness >12 mm, received either the angiotensin II antagonist valsartan or atenolol for 8 months. Echocardiographic data of 58 patients were available. After 8 months of valsartan treatment (n=29), LVMI decreased from 127+/-23 to 106+/-25 g/m2 (ratio [R]=0.83; 95% CI, 0.79 to 0.87; P<0.0001 versus baseline). Under atenolol (n=29), LVMI decreased to a smaller extent, from 127+/-25 to 117+/-27 g/m2 (R=0.92; 95% CI, 0.86 to 0.98; P=0.0082 versus baseline). The mean reduction of LVMI came to 21 g/m2 under valsartan and only to 10 g/m2 under atenolol (R=0.91; 90% CI, 0.85 to 0.97 versus atenolol). Baseline mean blood pressure values were determined to be 163+/-12/101+/-6 mm Hg before treatment with valsartan and 160+/-14/103+/-6 mm Hg before atenolol treatment. After 8 months of treatment, mean blood pressure decreased to 146+/-13/90+/-7 mm Hg with valsartan and to 147+/-18/90+/-7 mm Hg with atenolol. Nine patients in the valsartan group and 8 patients in the atenolol group required additional medication with hydrochlorothiazide. CONCLUSIONS: Antihypertensive treatment with the angiotensin II antagonist valsartan for 8 months produced a significant regression of LVH in predominantly previously untreated patients with essential hypertension. The drug may be safely administered in this subset of hypertensive patients; however, the long-term benefit in terms of risk reduction has still to be evaluated in further trials.

Angiotensin II↗
PubMed1998

The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin cytoskeleton.

In Saccharomyces cerevisiae, the phosphatidylinositol kinase homologue Tor2 controls the cell-cycle-dependent organisation of the actin cytoskeleton by activating the small GTPase Rho1 via the exchange factor Rom2 [1,2]. Four Rho1 effectors are known, protein kinase C 1 (Pkc1), the formin-family protein Bni1, the glucan synthase Fks and the signalling protein Skn7 [2,3]. Rho1 has been suggested to signal to the actin cytoskeleton via Bni1 and Pkc1; rho1 mutants have never been shown to have defects in actin organisation, however [2,4]. We have further investigated the role of Rho1 in controlling actin organisation and have analysed which of the Rho1 effectors mediates Tor2 signalling to the actin cytoskeleton. We show that some, but not all, rho1 temperature-sensitive (rho1ts) mutants arrest growth with a disorganised actin cytoskeleton. Both the growth defect and the actin organisation defect of the rho1-2ts mutant were suppressed by upregulation of Pkc1 but not by upregulation of Bni1, Fks or Skn7. Overexpression of Pkc1, but not overexpression of Bni1, Fks or Skn7, also rescued a tor2ts mutant, and deletion of BNI1 or SKN7 did not prevent the suppression of the tor2ts mutation by overexpressed Rom2. Furthermore, overexpression of the Pkc1-controlled mitogen-activated protein (MAP) kinase Mpk1 suppressed the actin defect of tor2ts and rho1-2ts mutants. Thus, Tor2 signals to the actin cytoskeleton via Rho1, Pkc1 and the cell integrity MAP kinase cascade.

Actins↗
PubMed1998

Monitoring of scFv selected by phage display using detection of scFv-pIII fusion proteins in a microtiter scale assay.

We describe here a method for the efficient and rapid analysis of antigen binding characteristics of recombinant antibodies (ab) selected by phage display. This novel approach combines the bacterial production of soluble single chain ab (scFv)-pIII fusion proteins on a microtiter scale with the detection of these fusion proteins via a pIII-specific ab. It facilitates the parallel analysis of large numbers of clones and is more efficient than current analysis protocols. Applying this technique, we analysed phage display selection of tetanus toxoid (TTX) specific scFv with respect to: (i) the productive expression of fusion proteins; (ii) the enrichment of specific scFv in subsequent rounds of phage display selection on a polyclonal level; (iii) the antigen specificity of individual scFv clones; (iv) the antigen binding affinity of a selected scFv. A TTX-specific scFv (clone 4.3) was further examined in a mono- and bivalent form by surface plasmon resonance analysis. ScFv 4.3 possesses a subnanomolar affinity and a low off rate constant.

Animals↗
PubMed1998

Receptors and effects of the inhibitory neuropeptide somatostatin in microglial cells.

The expression of receptors for the neuropeptide somatostatin was investigated in cultured immunocytochemically pure rat microglial cells. By the reverse transcriptase-polymerase chain reaction, the mRNAs for the receptor subtypes sst2, sst3 and sst4, but not sst1 and sst5 could be detected. To show that these receptors were functionally active, the effects of somatostatin and the metabolically stable, receptor subtype (2, 3 and 5) selective derivative octreotide (SMS 201-995, Sandostatin) on protein phosphorylation and proliferation were evaluated. Somatostatin induced the tyrosine phosphorylation of a 95 kDa protein in microglia. Furthermore, somatostatin or octreotide inhibited the basal as well as the GM-CSF-(granulocyte macrophage colony-stimulating factor) or the IL-3-(interleukin-3)-stimulated proliferation of microglial cells. This effect was dose-dependent, with a half maximum activity of about 0.2-0.3 nM. Somatostatin was relatively stable in the cultures due to protease inhibitors in the serum. The results indicate that microglial cells are targets for the widespread neuropeptide somatostatin and that its receptors can transduce complex signals to microglia.

Animals↗
PubMed1998

Nuclear Magnetic Resonance Detection of Two Distinctly Different Chains in the Orthorhombic Crystalline Phase of Polyethylenes.

Several commercial and noncommercial, high- and low-density and ultraoriented polyethylene samples, as well as polyethylene samples with inorganic fillers, have been investigated by inversion-recovery cross-polarization magic angle spinning carbon-13 nuclear magnetic resonance (NMR). In all these samples two types of all-trans chains in orthorhombic crystalline domains are detected, which give two overlapping carbon-13 lines with different line widths and different relaxation times. From the NMR relaxation parameters we conclude that one type of the crystalline chains, which composes 60-90% of the crystalline fraction in all samples, can execute at room temperature 180 degrees flips with a frequency in the kilohertz domain. The other crystalline chains are more rigid and probably are found in more perfect structures in which such chain flips do not occur or occur on a much slower time scale. Adding kaoline filler particles to polyethylene enhances the contribution of the more mobile crystalline chains. The presence of the two distinctly different types of crystalline environments is found in all polyethylene samples investigated so far (more than 25 samples).

Journal Article↗
PubMed1998

Plant polyketide synthases: a chalcone synthase-type enzyme which performs a condensation reaction with methylmalonyl-CoA in the biosynthesis of C-methylated chalcones.

Heterologous screening of a cDNA library from Pinusstrobus seedlings identified clones for two chalcone synthase (CHS) related proteins (PStrCHS1 and PStrCHS2, 87.6% identity). Heterologous expression in Escherichia coli showed that PStrCHS1 performed the typical CHS reaction, that it used starter CoA-esters from the phenylpropanoid pathway, and that it performed three condensation reactions with malonyl-CoA, followed by the ring closure to the chalcone. PstrCHS2 was completely inactive with these starters and also with linear CoA-esters. Activity was detected only with a diketide derivative (N-acetylcysteamine thioester of 3-oxo-5-phenylpent-4-enoic acid) that corresponded to the CHS reaction intermediate postulated after the first condensation reaction. PstrCHS2 performed only one condensation, with 6-styryl-4-hydroxy-2-pyrone derivatives as release products. The enzyme preferred methylmalonyl-CoA against malonyl-CoA, if only methylmalonyl-CoA was available. These properties and a comparison with the CHS from Pinus sylvestris suggested for PstrCHS2 a special function in the biosynthesis of secondary products. In contrast to P. sylvestris, P. strobus contains C-methylated chalcone derivatives, and the methyl group is at the position predicted from a chain extension with methylmalonyl-CoA in the second condensation of the biosynthetic reaction sequence. We propose that PstrCHS2 specifically contributes the condensing reaction with methylmalonyl-CoA to yield a methylated triketide intermediate. We discuss a model that the biosynthesis of C-methylated chalcones represents the simplest example of a modular polyketide synthase.

Acyl Coenzyme A↗
PubMed1998

Cell wall integrity modulates RHO1 activity via the exchange factor ROM2.

The essential phosphatidylinositol kinase homologue TOR2 of Saccharomyces cerevisiae controls the actin cytoskeleton by activating a GTPase switch consisting of RHO1 (GTPase), ROM2 (GEF) and SAC7 (GAP). We have identified two mutations, rot1-1 and rot2-1, that suppress the loss of TOR2 and are synthetic-lethal. The wild-type ROT1 and ROT2 genes and a multicopy suppressor, BIG1, were isolated by their ability to rescue the rot1-1 rot2-1 double mutant. ROT2 encodes glucosidase II, and ROT1 and BIG1 encode novel proteins. We present evidence that cell wall defects activate RHO1. First, rot1, rot2, big1, cwh41, gas1 and fks1 mutations all confer cell wall defects and suppress tor2(ts). Second, destabilizing the cell wall by supplementing the growth medium with 0.005% SDS also suppresses a tor2(ts) mutation. Third, disturbing the cell wall with SDS or a rot1, rot2, big1, cwh41, gas1 or fks1 mutation increases GDP/GTP exchange activity toward RHO1. These results suggest that cell wall defects suppress a tor2 mutation by activating RHO1 independently of TOR2, thereby inducing TOR2-independent polarization of the actin cytoskeleton and cell wall synthesis. Activation of RHO1, a subunit of the cell wall synthesis enzyme glucan synthase, by a cell wall alteration would ensure that cell wall synthesis occurs only when and where needed. The mechanism of RHO1 activation by a cell wall alteration is via the exchange factor ROM2 and could be analogous to signalling by integrin receptors in mammalian cells.

Cell Cycle Proteins↗
PubMed1998

A minimally replicative HIV-2 live-virus vaccine protects M. nemestrina from disease after HIV-2(287) challenge.

M. nemestrina immunized with an apathogenic HIV-2 molecular clone (HIV-2KR) were protected from CD4 decline and disease upon challenge with HIV-2(287), after any immunizing virus could be detected. Higher but not lower inocula of HIV-2KR were protective against intravenous inoculation of either 10(5) or 10(1) TCID50 of HIV-2(287). Protected animals displayed substantial reductions in PBMC proviral burden (1-3 logs), viral titers (1-2 logs), and plasma viral RNA (2-4 logs) compared to unprotected or naive animals as early as 1 week postinfection. Plasma viral RNA became undetectable after 24 weeks in protected animals, but remained high in unprotected animals. No viral RNA was present in the spleen of the protected animal necropsied more than a year after challenge (though viral DNA was still present). No neutralizing responses could be demonstrated, but CTL activity was detected sooner and at higher levels after challenge in protected than in unprotected macaques. In this novel HIV-2 vaccine model, protection was clearly dose-dependent, and clearance of challenge virus RNA from the plasma did not require detectable ongoing replication of the immunizing virus at the time of challenge.

AIDS Vaccines↗
PubMed1998

Characterization of a molecular clone of HIV type 2 infectious for Macaca nemestrina.

A lambda phage clone containing a full-length HIV-2 provirus, designated HIV-2KR, was obtained from the genomic DNA of Molt4 clone 8 (Molt4/8) lymphoblastic cells infected with the HIV-2PEI2 strain. HIV-2KR is genetically distinct from known HIV-2 isolates, possessing both a unique deletion in the LTR promoter region, and a long rev reading frame. It is replication competent in vitro after transfection into Molt4/8 cells, replicates in a variety of established human T lymphoblastic (Molt-3, Molt4/8, SupT1, H9, C8166) and myelomonocytic (U937) cell lines, and displays prominent cytopathic effects on infection of Molt4/8 cells, reflecting usage of both CCR5 and CXCR4 coreceptors. In addition, HIV-2KR was found to be infectious for human and Macaca nemestrina peripheral blood lymphocytes, and primary human monocyte-macrophage cultures. Intravenous inoculation of cell-free virus into M. nemestrina resulted in infection characterized by transient, low-level viremia and modest temporary decline in CD4 lymphocyte numbers, making HIV-2KR the first HIV-2 molecular clone reported to be infectious for this primate species.

Amino Acid Sequence↗
PubMed1998

Structure of the xylanase from Penicillium simplicissimum.

Despite its relatively low pH and temperature optimum, the xylanase from Penicillium simplicissimum performs exceedingly well under conditions of paper bleaching. We have purified and characterized this enzyme, which belongs to family 10 of glycosyl hydrolases. Its gene was cloned, and the sequence of the protein was deduced from the nucleotide sequence. The xylanase was crystallized from ammonium sulfate at pH 8.4, and X-ray data were collected at cryo-temperature to a crystallographic resolution of 1.75 A. The crystal structure was solved by molecular replacement using the catalytic domain of the Clostridium thermocellum xylanase as a search model, and refined to a residual of R = 20% (R(free) = 23%) for data between 10 and 1.75 A. The xylanase folds in an (alpha/beta)8 barrel (TIM-barrel), with additional helices and loops arranged at the "top" forming the active site cleft. In its overall shape, the P. simplicissimum xylanase structure is similar to other family 10 xylanases, but its active site cleft is much shallower and wider. This probably accounts for the differences in catalysis and in the mode of action of this enzyme. Three glycerol molecules were observed to bind within the active site groove, one of which interacts directly with the catalytic glutamate residues. It appears that they occupy putative xylose binding subsites.

Amino Acid Sequence↗
PubMed1998

Plasmodium falciparum: asexual erythrocytic stages synthesize two structurally distinct free and protein-bound glycosylphosphatidylinositols in a maturation-dependent manner.

Glycosylphosphatidylinositols represent the predominant class of glycolipids synthesized by the asexual, intraerythrocytic stages of Plasmodium falciparum. These glycolipids have been implicated as malarial toxins involved in parasite-induced release of cytokines, such as tumor necrosis factor-alpha and interleukin-1. Two potential glycosylphosphatidylinositol membrane-anchor precursors with the structures ethanolamine phosphate (mannose-alpha 1,2)mannose-alpha 1,2-mannose-alpha 1,6-mannose-alpha 1,4-glucosamine-inositol(acyl)phosphate diacylglycerol (P.f.alpha) and ethanolamine-phosphate-mannose-alpha 1,2-mannose-alpha 1,6-mannose-alpha 1,4-glucosamine-inositol(acyl)phosphate diacylglycerol (P.f.beta) have been described in P. falciparum. Only one (P.f.alpha) has been demonstrated to serve as an anchor for merozoite surface protein-1 and merozoite surface protein-2. In this report we present data showing that asexual, intraerythrocytic stages of P. falciparum use both glycosylphosphatidylinositols to anchor proteins. The synthesis of the two glycosylphosphatidylinositol membrane anchor precursors and the protein-bound glycosylphosphatidylinositol anchors is tightly regulated and varies throughout the intraerythrocytic development of the asexual stages of P. falciparum. The glycosylphosphatidylinositol membrane-anchor precursor P.f.beta is synthesized and transferred to protein predominantly in trophozoite stages (about 30 h).

Animals↗
PubMed1998

Biogenesis of synaptic-like microvesicles in perforated PC12 cells.

We have established a system that reconstitutes the biogenesis of synaptic-like microvesicles (SLMVs) in perforated cells of the rat neuroendocrine cell line PC12. The system is based on the biotinylation of synaptophysin, a marker of synaptic vesicles and SLMVs. Biotinylation is performed at 18 degrees C, a temperature at which formation of SLMVs is blocked and biotinylated synaptophysin accumulates in the SLMV donor compartment. The biotinylated PC12 cells are then perforated by scraping and incubated at 37 degrees C in the presence of ATP and cytosolic proteins, conditions required for SLMV biogenesis. After the perforated-cell reaction, the newly formed SLMVs are isolated by differential centrifugation followed by either glycerol gradient centrifugation or a simple single-glycerol-step centrifugation. The latter allows the analysis of many perforated-cell reactions in parallel and, hence, the dissection of the molecular machinery mediating SLMV biogenesis. Using this system, we have found that clathrin, dynamin, phosphatidylinositol transfer protein, and SH3P4 are involved in SLMV biogenesis.

Adenosine Triphosphate↗