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Biomedical subjects

C Klein

Publications and source records attributed to C Klein.

At least 91 records · Page 5Linked to original sources
PubMed2001

The importance of gene dosage studies: mutational analysis of the parkin gene in early-onset parkinsonism.

Early-onset parkinsonism (EOP) may be associated with different mutations in the parkin gene, including exon deletions and duplications. To test for gene dosage alterations, we developed a new method of quantitative duplex PCR using the fluorescence resonance energy transfer technique on the LightCycler (Roche Diagnostics). In 21 patients with EOP, three mutations (a single base pair substitution in exon 3 and small deletions in exon 9) were detected by conventional mutational screening (single-strand conformation polymorphism and sequence analysis), while alterations of gene dosage were found in seven patients. We identified heterozygous and compound heterozygous deletions of exons 2, 3, 5 and 7. The latter was also found in the homozygous state. In addition, two heterozygous duplications of exon 4 were observed. Remarkably, two patients carried more than two parkin mutations. This is the first study systematically screening all 12 exons of parkin by real-time, kinetic quantification and clearly shows that mutational analysis of the parkin gene should include gene dosage studies. Furthermore, our method of quantitative PCR is easily applicable to any other gene to be screened for deletions or duplications of whole exons.

Age of Onset↗
PubMed2001

High thermostability and lack of cooperative DNA binding distinguish the p63 core domain from the homologous tumor suppressor p53.

The p53 protein is the major tumor suppressor in mammals. The discovery of the p53 homologs p63 and p73 defined a family of p53 members with distinct roles in tumor suppression, differentiation, and development. Here, we describe the biochemical characterization of the core DNA-binding domain of a human isoform of p63, p63-delta, and particularly novel features in comparison with p53. In contrast to p53, the free p63 core domain did not show specific binding to p53 DNA consensus sites. However, glutathione S-transferase-fused and thus dimerized p63 and p53 core domains had similar affinity and specificity for the p53 consensus sites p21, gadd45, cyclin G, and bax. Furthermore, the fold of p63 core was remarkably stable compared with p53 as judged by differential scanning calorimetry (T(m) = 61 degrees C versus 44 degrees C for p53) and equilibrium unfolding ([urea](50%) = 5.2 m versus 3.1 m for p53). A homology model of p63 core highlights differences at a segment near the H1 helix hypothetically involved in the formation of the dimerization interface in p53, which might reduce cooperativity of p63 core DNA binding compared with p53. The model also shows differences in the electrostatic and hydrophobic potentials of the domains relevant to folding stability.

Amino Acid Sequence↗
PubMed2001

Tumor necrosis factor-alpha-induced adipose-related protein (TIARP), a cell-surface protein that is highly induced by tumor necrosis factor-alpha and adipose conversion.

Tumor necrosis factor-alpha (TNF alpha) is involved in the physiological and biological abnormalities found in two opposite metabolic situations: cachexia and obesity. In an attempt to identify novel genes and proteins that could mediate the effects of TNFalpha on adipocyte metabolism and development, we have used a differential display technique comparing 3T3-L1 cells exposed or not to the cytokine. We have isolated a novel adipose cDNA encoding a TNF alpha-inducible 470-amino acid protein termed TIARP, with six putative transmembrane regions flanked by a large amino-terminal and a short carboxyl-terminal domain, a structure reminiscent of channel and transporter proteins. Commitment into the differentiation process is required for cytokine responsiveness. The differentiation process per se is accompanied by a sharp emergence of TIARP mRNA transcripts, in parallel with the expression of the protein at the plasma membrane. Transcripts are present at high levels in white and brown adipose tissues, and are also detectable in liver, kidney, heart, and skeletal muscle. Whereas the biological function of TIARP is presently unknown, its pattern of expression during adipose conversion and in response to TNF alpha exposure as a transmembrane protein mainly located at the cell surface suggest that TIARP might participate in adipocyte development and metabolism and mediate some TNF alpha effects on the fat cell as a channel or a transporter.

3T3 Cells↗
PubMed2001

[The genetics of Parkinson syndrome].

A genetic contribution to the etiology of Parkinson's disease was first suspected by Charcot and later confirmed by case control, family, and twin studies, as well as by the description of large parkinsonian families with Mendelian inheritance of the disease. Recent progress in the field of molecular neurogenetics has led to the identification of several Parkinson disease genes and gene loci. Mutations in the alpha-Synuclein gene (PARK1) and in the gene for the ubiquitin C-terminal hydrolase I (PARK5), along with two gene loci harboring currently unknown genes (PARK3 and PARK4), have been linked to very rare autosomal dominantly inherited parkinsonian syndromes. Mutations in the parkins gene (PARK2), causing autosomal recessive early-onset parkinsonism, are much more common and therefore of clinical relevance. A second gene locus for an autosomal dominantly inherited Parkinsonian syndrome was recently localized on chromosome 1 (PARK6). All three parkinson genes identified thus far imply the involvement of the ubiquitin pathway of protein degradation in the pathogenesis of Parkinson's disease.

Chromosome Aberrations↗
PubMed2001

Disorders of peroxisome biogenesis due to mutations in PEX1: phenotypes and PEX1 protein levels.

Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD) are clinically overlapping syndromes, collectively called "peroxisome biogenesis disorders" (PBDs), with clinical features being most severe in ZS and least pronounced in IRD. Inheritance of these disorders is autosomal recessive. The peroxisome biogenesis disorders are genetically heterogeneous, having at least 12 different complementation groups (CGs). The gene affected in CG1 is PEX1. Approximately 65% of the patients with PBD harbor mutations in PEX1. In the present study, we used SSCP analysis to evaluate a series of patients belonging to CG1 for mutations in PEX1 and studied phenotype-genotype correlations. A complete lack of PEX1 protein was found to be associated with severe ZS; however, residual amounts of PEX1 protein were found in patients with the milder phenotypes, NALD and IRD. The majority of these latter patients carried at least one copy of the common G843D allele. When patient fibroblasts harboring this allele were grown at 30 degrees C, a two- to threefold increase in PEX1 protein levels was observed, associated with a recovery of peroxisomal function. This suggests that the G843D missense mutation results in a misfolded protein, which is more stable at lower temperatures. We conclude that the search for the factors and/or mechanisms that determine the stability of mutant PEX1 protein by high-throughput procedures will be a first step in the development of therapeutic strategies for patients with mild PBDs.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2001

H reflex behavior in Parkinson's disease patients and patients with extrapyramidal and pyramidal signs combined.

The H reflex was elicited in 21 normal subjects, 48 patients with Parkinson's disease (PD) and 22 patients with pyramidal and extrapyramidal signs combined (PESC). In most normal subjects (90.5%), in 29.2% of PD patients and 54.5% of patient with PESC the threshold for sensory fibers was lower than for motor fibers, and the H reflex was obtained before the M response for all duration stimuli in both legs. In 9.5% of normal subjects, 39.6% of PD patients with mild and moderate rigidity (according to the motor part of UPDRS) and 31.8% of patients with PESC, the threshold for the H reflex and M response was the same or the M response threshold was lower in at least one of the legs for short stimulus duration (0.1-0.2 ms). In 31.2% of PD patients (most of them with severe rigidity) and 13.7% of patients with PESC, the threshold for M response was lower for all stimulus duration in at least one of the legs, and it was obtained before H reflex. These very significant differences in behavior of the H reflex in PD patients (Fisher exact test, p<0.0001) that almost disappear in patients with PESC, could be possibly explained by changes in agonist-antagonist inhibition.

Adult↗
PubMed2001

Up-regulation of transporters of the MRP family by drugs and toxins.

Expression of a variety of ABC efflux pumps including certain conjugate transporters of the multidrug resistance protein (MRP) subfamily is inducible in primate and rodent tissues, and in a variety of cell lines and primary cells in culture. In human cell lines (HepG2, MCF-7), we studied the inducibility of MRPs 1-5. Similar to the rat mrp2 gene, human mrp2 is inducible by the chemical carcinogen 2-AAF, the chemotherapeutic drug cisplatin and the barbiturate phenobarbital, as demonstrated in Northern and Western Blots. Furthermore, the antibiotic rifampicin was identified as MRP2 inducer in HepG2 cells. MRP1 and 4 mRNAs being expressed in human liver at a very low level could not be detected in HepG2 cells after treatment with various agents. However, MRP3 and 5 mRNAs were detected in addition to MRP2 and their expression was found to be increased by 2-AAF, cisplatin and rifampicin. MRP1 expression was studied in MCF-7 cells where the chemotherapeutic drug vinblastine and tert-butyl hydroquinone but not the MRP2 inducing agents described above acted as inducers.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

Distribution in ocular structures and optic pathways of immunocompetent and glial cells in an experimental allergic encephalomyelitis (EAE) relapsing model.

Relapsing experimental allergic encephalomyelitis (EAE) was induced in DA rats and the ocular pathologic events were examined at the various phases of the illness. About 80% of EAE rats presented anterior uveitis (AU), even after complete EAE recovery. We studied the phenotype and localization of immunocompetent cells, the major histocompatibility complex (MHC) class I and II antigen expression, as well as the chemokine monocyte chemoattractant protein-1 (MCP-1) appearance. In control animals, there were many glial fibrillary acidic protein (GFAP)(+) cells and OX42(+) cells in the ciliary body, retina, optic nerve and chiasma. Except in retina, we observed constitutive MHC class I and II expression. During the EAE acute phase, there was up-regulation of MHC class II and GFAP antigens in iris, ciliary body, limbus, and optic pathways. MHC class I and ED2 antigens were expressed in meninges and in the prechiasmatic cisterna, by cells which could have a role in immune surveillance. MCP-1 mRNA was highly expressed in optic pathways during the acute phase and the protein was expressed by astrocytes, macrophages, and lymphocytes. During the relapsing phase, MCP-1 was weakly expressed to disappear almost completely during the final recovery phase. The expression of MHC class II on astrocytes was increased during the relapsing and final recovery phase in which the inflammatory lesions persisted. These findings suggest that ocular areas and optic pathways, mainly optic chiasma, are important targets in the relapsing EAE.

Animals↗
PubMed2001

Mineralocorticoid receptor-mediated signaling regulates the ion gated sodium channel in vascular endothelial cells and requires an intact cytoskeleton.

The PCR analysis followed by sequence alignment showed that both the mineralocorticoid receptor (MCR) and the epithelial sodium channel (ENaC) genes were expressed in the human vascular endothelial cell line (ECV). The growth and multiplication of the ECV in culture were influenced by both aldosterone and the MCR-specific antagonist ZK 91587. Following double labelled immunofluorescence recorded by confocal microscopy, both the MCR and the ENaC were found to colocalize with the tubulin filaments in ECV cells in situ; no association was observed with cellular actin. ZK 91587 not only eliminated the basal expression, but it also impaired the transactivation of the ENaC gene by aldosterone. The disruption of actin and tubulin by cytochalasin D and colchicine, respectively, resulted in the total elimination of ENaC induction by aldosterone. These studies suggest that (i) the transcriptional regulation of the ENaC gene by the MCR-mediated signalling is not restricted to epithelial cells and requires cytoskeleton integrity in ECV cells in situ, (ii) tubulin may form a new and novel mediator in cell regulation, and (iii) the vascular tone may actually be regulated via transactivation of the ion gated sodium channel in the endothelial cell of the blood vessels under direct, receptor-mediated action of aldosterone.

Actins↗
PubMed2001

Chalcone derivatives antagonize interactions between the human oncoprotein MDM2 and p53.

The oncoprotein MDM2 inhibits the tumor suppressor protein p53 by binding to the p53 transactivation domain. The p53 gene is inactivated in many human tumors either by mutations or by binding to oncogenic proteins. In some tumors, such as soft tissue sarcomas, overexpression of MDM2 inactivates an otherwise intact p53, disabling the genome integrity checkpoint and allowing cell cycle progression of defective cells. Disruption of the MDM2/p53 interaction leads to increased p53 levels and restored p53 transcriptional activity, indicating restoration of the genome integrity check and therapeutic potential for MDM2/p53 binding antagonists. Here, we show by multidimensional NMR spectroscopy that chalcones (1,3-diphenyl-2-propen-1-ones) are MDM2 inhibitors that bind to a subsite of the p53 binding cleft of human MDM2. Biochemical experiments showed that these compounds can disrupt the MDM2/p53 protein complex, releasing p53 from both the p53/MDM2 and DNA-bound p53/MDM2 complexes. These results thus offer a starting basis for structure-based drug design of cancer therapeutics.

Antineoplastic Agents↗
PubMed2001

Comparison of magnetic resonance real-time imaging of left ventricular function with conventional magnetic resonance imaging and echocardiography.

This study analyzes the accuracy of a new real-time magnetic resonance imaging (MRI) technique (acquisition duration, 62 ms/image) and echocardiography for the determination of left ventricular (LV) end-diastolic volume, end-systolic volume, ejection fraction, and muscle mass when compared with turbo gradient echo imaging as the reference standard. Thirty-four patients were examined with digital echocardiography, standard, and real-time MRI. A close correlation was found between the results of real-time imaging and the reference standard for end-diastolic volume, end-systolic volume, and ejection fraction (r >0.95), with a lower correlation for LV muscle mass (r = 0.81). Correlations between echocardiography and the reference standard were lower for all parameters. Real-time MRI enables the acquisition of high-quality cine loops of the entire heart in minimal time without electrocardiographic triggering or breath holding. Thus, patient setup and scan time can be reduced considerably. Results are similar to the reference standard and superior to echocardiography for determining LV volumes and ejection fraction. This technique is a valid alternative to current approaches and can form the basis of every cardiac MRI examination.

Adult↗
PubMed2001

Epithelial sodium channel and the mineralocorticoid receptor in cultured rat Müller glial cells.

Müller glial cells are the major non-neuronal cells of the retina. They are involved in retinal function and exert a profound influence on the function of retinal neurons. We present an in vitro study of the localization of the mineralocorticoid receptor (MCR) and of the epithelial sodium channel (ENaC) in rat Müller glial cells isolated from rat retina, using respectively, a polyclonal antiserum raised against the rat purified MCR, and a rabbit polyclonal antibody against the 14-amino acid (aa) peptide QGLGKGDKREEQGL, which corresponds to the N-terminal region (44-58aa) of the alpha-subunit of the ENaC. In an immunocytochemical study using anti-MCR and anti-ENaC antibodies, the MCR was detected as a protein present in the cytoplasm and in the nucleus, whereas ENaC was detected as a membrane-bound protein. Reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers, 5'-CTGCCTTTATGGATGATGGT-3' (sense), 5'-GTTCAGCTCGAAGAAGA-3' (antisense) for ENaC and 5'-AGGCTACCACAGTCTCCCTG-3' (sense) and 5'-GCAGTGTAAAATCTCCAGTC-3' (antisense) for MCR, showed expression of the ENaC and MCR genes in Müller cells. The presence of ENaC and MCR was detected as the predicted bands of 520 bp and 843 bp, respectively. In both cases, 100% identity was observed between the sequences of rat Müller cell (RMC) PCR products and rat kidney. Interestingly, the basal levels of ENaC were increased in vitro by the MCR-specific hormone, aldosterone. Thus, our results strongly suggest that the Müller glial cells may play a role in the regulation of extracellular Na+ concentration, which could be regulated by steroid-mediated sodium uptake.

Aldosterone↗
PubMed2001

Multi-slice dynamic imaging: complete functional cardiac MR examination within 15 seconds.

A new magnetic resonance (MR) sequence was developed to acquire real-time images in a multi-slice dynamic imaging mode to cover the complete heart in 15 seconds without the need for electrocardiogram (ECG) triggering and multiple breath holds. In 34 patients, left ventricular function was assessed with the new technique and a standard technique. The new technique proved to be feasible and accurate for functional cardiac examinations.

Adult↗
PubMed2001

Magnetic resonance flow measurements in real time: comparison with a standard gradient-echo technique.

Ultrafast gradient systems and hybrid imaging sequences offer the opportunity to acquire phase contrast flow data in real time. In a 1.5-Tesla magnetic resonance (MR)-tomograph, peak velocity and volume flow were assessed in 36 large vessels (aorta) and 33 medium-sized vessels (carotid and iliac artery) using a real-time (segmented k-space turbo gradient-echo planar imaging sequence) in comparison with a gradient-echo technique. With the real-time technique, the matrix was reduced from 116 to 64, and temporal resolution changed from 30 msec to 124 msec. Measurements of peak velocity correlated in large (r = 0.88) and medium-sized vessels (r = 0.81). Volume flow measurements correlated in large vessels (r = 0.87), however, a poor correlation (r = 0.64) was found in medium-sized vessels. Thus, scan time can be significantly reduced and images acquired without electrocardiogram (ECG)-triggering. Flow volume can only be determined in large vessels with sufficient accuracy, mainly due to reduced spatial resolution in smaller vessels.

Blood Flow Velocity↗
PubMed2001

NMR monitoring of accumulation and folding of 15N-labeled protein overexpressed in Pichia pastoris.

Postgenomic studies have led to an increasing demand for isotope-labeled proteins. We present a method for producing large quantities of truly native (15)N-labeled protein. Based on the secretion capabilities of the yeast Pichia pastoris, the recombinant protein is easily purified in a single step as it is secreted. Control of all nitrogen sources permits very high labeling yields. As a result, accumulation and folding of the recombinant protein can be monitored by heteronuclear NMR without purification. Comparison of sample spectra with the spectrum of the purified recombinant protein allows detection of the secreted protein in the culture and monitoring of its folding, from the start of the induction phase. The detection limit for a (15)N-labeled protein is estimated as 20 microM and corresponds, for a 10-kDa protein, to a load of 40 mg/liter in the fermentor. This concentration is reached by most reported preparations in P. pastoris. Further concentration by ultrafiltration would compensate for lower production. This procedure may be useful in many structural genomics and combinatorial chemistry screening projects where most protein productions meet the requirements for this method.

Antigens, Plant↗