Allosteric activation of the protein kinase PDK1 with low molecular weight compounds.

Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation.

Prostacyclin in the cardiovascular system: new aspects and open questions.

Several indications exist that prostacyclin (PGI(2)) release in the cardiovascular system might be affected by cyclooxygenase (COX)-2-specific inhibitors. This could reflect an inhibition of PGI(2) synthesis in the endothelium although in these cells mainly COX-1 is expressed. Inflammation and stress induce COX-2 in smooth muscle cells which could have happened in patients with cardiac diseases.

ERj1p has a basic role in protein biogenesis at the endoplasmic reticulum.

ERj1p is a membrane protein of the endoplasmic reticulum (ER) that can recruit the ER lumenal chaperone BiP to translating ribosomes. ERj1p can also modulate protein synthesis at initiation and is predicted to be a membrane-tethered transcription factor. Here we attribute the various functions of ERj1p to distinct regions within its cytosolic domain.

BAG-2 acts as an inhibitor of the chaperone-associated ubiquitin ligase CHIP.

Cellular protein quality control involves a close interplay between molecular chaperones and the ubiquitin/proteasome system. We recently identified a degradation pathway, on which the chaperone Hsc70 delivers chaperone clients, such as misfolded forms of the cystic fibrosis transmembrane conductance regulator (CFTR), to the proteasome. The cochaperone CHIP is of central importance on this pathway, because it acts as a chaperone-associated ubiquitin ligase.

Specific detection and semi-quantitative analysis of TRPC4 protein expression by antibodies.

In mouse tissues two variants of the transient receptor potential (canonical) (TRPC) 4 protein are expressed: the "full-length" TRPC4 protein and a slightly smaller variant, called TRPC4Delta(761-864), which lacks 84 amino acid residues. Although the presence of mRNA encoding the TRPC4 protein in mammalian cells and the detection of the heterologously expressed TRPC4 protein by Western blot analysis have been reported, the unequivocal detection of endogenous TRPC4 proteins has proven difficult. In the present study we compared polyclonal antibodies for the detection of TRPC4 proteins in mouse tissues and monitored their specificity and reliability by analysing corresponding tissues from TRPC4-deficient mice.

p53 autoantibodies from patients with head and neck cancer recognise common epitopes on the polypeptide chain of p53.

In the present study, we analysed p53 autoantibodies from patients with head and neck cancer by ELISA, by Western blot using C- and N-terminal fragments of p53 and with peptide libraries of p53. We found that 8.2% of the patients with head and neck cancer developed antibodies against p53.

Characterization of pancreatic ERj3p, a homolog of yeast DnaJ-like protein Scj1p.

We have previously identified in the human EST sequence data base four overlapping clones that could be aligned with both a predicted protein sequence, deduced from the C. elegans genomic sequence, and partial amino acid sequences, obtained for a protein from canine pancreatic microsomes. We suggested that these proteins are homologs of yeast microsomal and DnaJ-like protein Scj1p and termed them ERj3p.

Differential expression of electrogenic NBC1 (SLC4A4) variants in rat kidney and pancreas.

The purpose of this study was to determine expression and localization of NH(2)-terminal variants of the electrogenic Na(+)-HCO(3)(-) co-transporter NBC1 (SLC4A4) in the rat kidney and pancreas. We generated two anti-peptide antibodies: alpha333 against the "mste" start (kidney; kNBC1) and alpha332 against the "mede" start (pancreas; pNBC1). Transcripts for both NBC1 variants were detected in kidney and pancreas by RT-PCR, though kNBC1 was more prominent in the kidney and pNBC1 was more prominent in the pancreas.

Epitope analysis of the MAb 1AD9 antibody detection site in human protein kinase CK2alpha-subunit.

1AD9 is a murine monoclonal antibody (MAb) that was produced against the recombinant human alpha-subunit of protein kinase CK2, a pleiotropic and constitutively active serine/threonine protein kinase. We have further characterized this antibody, which is suitable for Western blot, immunoprecipitation and enzyme-linked immunosorbant assay tests. Using an overlapping peptide library, we have identified the epitope targeted by MAb1AD9 characterized by the following sequence: (319)MEHPYF(324).

The TRPV6 gene, cDNA and protein.

The mouse TRPV6 gene is localized on chromosome 6 and extends over 15.66kb. The encoded protein comprises 727 amino acid residues with a calculated relative molecular mass of 83,210Da.

A short-chain peptide toxin isolated from Centruroides sculpturatus scorpion venom inhibits ether-à-go-go-related gene K(+) channels.

From the venom of the American scorpion Centruroides sculpturatus Ewing we have isolated a minute peptide fraction (named CsEKerg1) which reversibly inhibits the current through ERG (ether-à-go-go-related gene) K(+) channels. Isolation was done by CM-cellulose column chromatography and reversed phase high-performance liquid chromatography. To test for an effect on ERG channels we used NG108-15 neuroblastomaxglioma hybrid cells voltage-clamped in the whole-cell mode.

A novel type of co-chaperone mediates transmembrane recruitment of DnaK-like chaperones to ribosomes.

Recently, the homolog of yeast protein Sec63p was identified in dog pancreas microsomes. This pancreatic DnaJ-like protein was shown to be an abundant protein, interacting with both the Sec61p complex and lumenal DnaK-like proteins, such as BiP. The pancreatic endoplasmic reticulum contains a second DnaJ-like membrane protein, which had been termed Mtj1p in mouse.

MGEA6 is tumor-specific overexpressed and frequently recognized by patient-serum antibodies.

Tumorigenesis of meningioma has been associated with chromosome 22, most notably the NF2 gene, but additional genes have also been implicated in meningioma development. Previously, we have cloned the cDNAs for the meningioma expressed antigen 6 (MGEA6) and its splice variant MGEA11. Here, we show that antibodies against recombinantly expressed MGEA6/11 are found in 41.

Expression, cellular distribution and protein binding of the glioma amplified sequence (GAS41), a highly conserved putative transcription factor.

The glioma amplified sequence 41 (GAS41) was previously isolated by microdissection mediated cDNA capture from the glioblastoma multiforme cell line TX3868 and shown to be frequently amplified in human gliomas. We determined the complete cDNA sequence of the GAS41 gene, demonstrated that the GAS41 protein is evolutionarily conserved, specifically at the N-terminus, and identified the yeast transcription factor tf2f domain within the GAS41 sequence. A human multiple-tissue Northern blot revealed ubiquitous expression of GAS41 with the highest expression in human brain.

PHF3-specific antibody responses in over 60% of patients with glioblastoma multiforme.

Glioblastoma multiforme (GBM), a malignant astrocytic tumour, represents the most frequent tumour of the human brain. Nevertheless, its molecular pathology is not well understood. We utilized the immune system, which contributes to cancer protection, to help identify new GBM-related genes.

The di-aromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein PEX14.

PEX5 functions as a mobile import receptor for peroxisomal matrix proteins with a peroxisomal targeting signal 1 (PTS1). A critical step within the PTS1-import pathway is the interaction between PEX5 and the peroxisome membrane-associated protein PEX14. Based on two-hybrid analyses in mammalian cells and complementary in vitro binding assays, we demonstrate that the evolutionarily conserved pentapeptide repeat motifs, WX(E/D/Q/A/S)(E/D/Q)(F/Y), in PEX5 bind to PEX14 with high affinity.

Homologs of the yeast Sec complex subunits Sec62p and Sec63p are abundant proteins in dog pancreas microsomes.

Cotranslational protein transport into dog pancreas microsomes involves the Sec61p complex plus a luminal heat shock protein 70. Posttranslational protein transport into the yeast endoplasmic reticulum (ER) involves the so-called Sec complex in the membrane, comprising a similar Sec61p subcomplex, the putative signal peptide receptor subcomplex, and the heat shock protein 40-type subunit, Sec63p, plus a luminal heat shock protein 70. Recently, human homologs of yeast proteins Sec62p and Sec63p were discovered.

Immunodetection of alpha1E voltage-gated Ca(2+) channel in chromogranin-positive muscle cells of rat heart, and in distal tubules of human kidney.

The calcium channel alpha1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of alpha1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies.

TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells.

Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca(2+) entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive.

A Scj1p homolog and folding catalysts present in dog pancreas microsomes.

Dog pancreas microsomes represent the key components of the established model system for the analysis of protein transport into the mammalian endoplasmic reticulum. More recently, these microsomes were also employed in cell-free systems which address questions related to protein folding and protein degradation in the mammalian endoplasmic reticulum. In order to get at a complete picture of these undoubtedly related processes in the in vitro system we need to know all the proteins we are dealing with, and their respective stoichiometries.

Identification of a CK2 phosphorylation site in mdm2.

Mdm2 is a cellular oncoprotein the most obvious function of which is the down-regulation of the growth suppressor protein p53. It represents a highly phosphorylated protein but only little is yet known about the sites phosphorylated in vivo, the kinases that are responsible for the phosphorylation or the functional relevance of the phosphorylation status. Recently, we have shown that mdm2 is a good substrate for protein kinase CK2 at least in vitro.

A comparative study of rat and human Tmp21 (p23) reveals the pseudogene-like features of human Tmp21-II.

Tmp21 (p23) is involved in biosynthetic transport from the endoplasmic reticulum to the Golgi complex. We have recently characterized two cDNA-variants of human Tmp21, the Tmp21-isoforms-I and -II. Because of the lack of cDNA sequence data and protein expression data, it was not clear if Tmp21-II encodes a functional Tmp21-protein.

Protein kinase CK2 interacts with a multi-protein binding domain of p53.

p53 is one of the most powerful negative regulators of growth. To manage this in an efficient way it has to interact with a set of different cellular proteins. Most contacts with the cellular environment occur in the N- or the C-terminal domain of the protein.

Intracellular localization and in vivo trafficking of p24A and p23.

Recently, p24A and p23 (also termed Tmp21), two members of the p24 protein family, have been proposed to function as integral receptors for the COPI-vesicle coat. This study describes the intracellular localization and trafficking of p24A in comparison to p23. For immunolocalization of p24A and p23, strong reduction and denaturation conditions were necessary to allow antibody interaction.

Production and characterization of a rabbit monoclonal antibody against human CDC25C phosphatase.

We produced a rabbit monoclonal antibody (MAb) against human CDC25C phosphatase. The antibody reacts with a minimal epitope between amino acids 291-295 in the highly conserved C-terminal region of CDC25C. The antibody recognizes denatured CDC25C of recombinant and mammalian origin in Western blot analysis.