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K Weber

Publications and source records attributed to K Weber.

At least 289 records · Page 16Linked to original sources
PubMed1991

Polyisoprenylation of the CAAX motif--an in vitro protein synthesis study.

A number of proteins, including most nuclear lamins, certain fungal mating pheromones, G-protein gamma-subunits and ras proteins, contain a C-terminal cysteine-aliphatic-aliphatic-undefined amino acid (CAAX) motif which is thought to be a roughly defined consensus sequence capable of directing a series of posttranslational events, beginning with the addition of a polyisoprene moiety to the cysteine. So far such a motif has been found in every protein known to have this type of modification. We have utilized the rabbit reticulocyte lysate translation system, which is capable of carrying out the polyisoprene modification in vitro, to investigate features of the C-terminal motif which affect its suitability as a substrate. We demonstrate that a cysteine is only isoprenylated when situated at position -4 from the C-terminus. We further show that the presence of a glycine at position -3 or a terminal aromatic residue, features typical of some G-protein alpha subunits, cause a reduction and abolition respectively of isoprenylation.

Amino Acid Sequence↗
PubMed1991

Characterization of a Ca(2+)-binding site in human annexin II by site-directed mutagenesis.

Annexin II, a major cytoplasmic substrate of the src tyrosine kinase, is a member of the annexin family of Ca2+/phospholipid-binding proteins. It is composed of a short N-terminal tail (30 residues) followed by four so-called annexin repeats (each 70-80 residues in length) which share sequence homologies and are thought to form (a) new type(s) of Ca(2+)-binding site(s). We have produced wild-type and site specifically mutated annexin II molecules to compare their structure and biochemistry. The recombinant wild-type annexin II displays biochemical and spectroscopical properties resembling those of the authentic protein purified from mammalian cells. In particular, it shows the Ca(2+)-induced blue shift in fluorescence emission which is typical for this annexin. Replacement of the single tryptophan in annexin II (Trp-212) by a phenylalanine abolishes the fluorescence signal and allows the unambiguous assignment of the Ca(2+)-sensitive spectroscopic properties to Trp-212. This residue is located in the third annexin repeat in a highly conserved stretch of 17 amino acids which are also found in the other repeats and known as the endonexin fold. To study the precise architecture of the Ca2+ site which must reside in close proximity to Trp-212, we changed several residues of the endonexin fold in repeat 3 by site-directed mutagenesis. An analysis of these mutants by fluorescence spectroscopy and Ca(2+)-dependent phospholipid binding reveals that Gly-206 and Thr-207 seem indispensible for a correct folding of this Ca(2+)-binding site.

Amino Acid Sequence↗
PubMed1991

Characterization of a discontinuous epitope on annexin II by site-directed mutagenesis.

Recombinant annexin II mutants were generated to identify amino acids involved in the formation of the discontinuous epitope of the monoclonal antibody H28. Analysis of the various mutant proteins by immunoblotting and enzyme-linked immunosorbent assay revealed that residues Lys27, Arg62, Glu65, and Arg67 are indispensable for H28 reactivity. Residues in equivalent positions are also in close proximity in the recently determined X-ray structure of annexin V, a different member of the same family of Ca2+/lipid-binding proteins. Thus annexins II and V show a similar three-dimensional folding in this region of the molecule. Consequently, the Ca2+ binding sites and the residues phosphorylated by pp60src (Tyr23) and protein kinase C (Ser25) most likely reside on opposite sides of the annexin II molecule.

Amino Acid Sequence↗
PubMed1991

Primary structure of human, chicken, and Xenopus laevis p11, a cellular ligand of the Src-kinase substrate, annexin II.

The p11 protein is a member of the S-100 family of Ca(2+)-binding proteins and serves within the cell as a ligand of the tyrosine kinase substrate, annexin II. To obtain more structural information on this molecule, we have isolated and characterized p11 cDNA clones from several different species. A comparison of the deduced amino acid (aa) sequences reveals that mammalian and avian p11 are highly similar (at least 90% identical at the aa level), whereas p11 from Xenopus laevis shows a considerable degree of sequence variation (the aa sequence identity drops to approx. 60% when compared to mammalian or chicken p11). Interestingly, the C-terminal 18 aa, which are unique to p11 within the S-100 family, show a relatively high conservation among species. This high evolutionary conservation is in line with a structurally and/or functionally important role of this C terminus, e.g., in annexin II binding.

Amino Acid Sequence↗
PubMed1991

Age, cardiovascular risk factors and coronary heart disease as determinants of platelet function in men. A multivariate approach.

Older age, the cardiovascular risk factors and arteriosclerosis have been reported to be associated with stimulated platelet function. To evaluate the relative importance of these factors in determining platelet function, a cross-sectional multivariate study in 191 men, 113 healthy subjects and 78 patients with angiographically documented coronary heart disease, was performed. In healthy subjects, stepwise multiple linear regression identified age to be a major determinant of platelet aggregability. After induction with both ADP and collagen the platelet aggregatory response markedly increased with age. In the patients, platelet function was not age dependent. In multivariate analysis of variance, neither smoking status nor hypercholesterolemia (greater than or equal to 240 mg/dl) were determinants of platelet function in either group. An increase in systolic blood pressure was associated with slightly more inhibited ADP induced aggregation in both healthy subjects and patients with coronary heart disease. In patients compared to healthy subjects, aggregation after induction with ADP and collagen was markedly enhanced and the in vitro formation of thromboxane after collagen stimulation increased. Thus, by multivariate analysis, age and the presence or absence of coronary heart disease were found to be major determinants of platelet function. In contrast, the cardiovascular risk factors smoking, hypercholesterolemia and hypertension were associated with only minor or no alterations of platelet function.

Adult↗
PubMed1991

Reduction of nocturnal diuresis and natriuresis during treatment of obstructive sleep apnea (OSA) with nasal continuous positive air pressure (nCPAP) correlates to cGMP excretion.

In ten patients with severe obstructive sleep apnea (OSA) profound changes in renal function could be demonstrated at night during nCPAP therapy. Natriuresis and diuresis decreased by about 50% while creatinine excretion rate and urinary osmolality did not change. We found parallel changes in the excretion of ANP's second messenger cyclic guanosine monophosphate (cGMP) in a dose-response-related manner to natriuresis respectively diuresis. These data are in agreement with recently demonstrated decrease of nocturnal plasma levels of atrial natriuretic peptide (ANP) during nCPAP therapy in apneic patients. This may be an indicator for an increased cardiac volume load during obstructive apnea. The decrease of diuresis, natriuresis and cGMP excretion demonstrate the beneficial effects of nCPAP treatment on the cardiovascular system. Therefore measurements of cGMP excretion may be a useful parameter to assess the cardiovascular function of apneic patients before and during treatment.

Adult↗
PubMed1991

Characterization of the cell-cycle-regulated protein calcyclin from Ehrlich ascites tumor cells. Identification of two binding proteins obtained by Ca2(+)-dependent affinity chromatography.

The nearly complete amino acid sequence obtained for murine calcyclin from Ehrlich ascites tumor cells reveals a very strong similarity with the rat and human sequences previously deduced from corresponding cDNA clones. While mouse and rat calcyclins are identical, the human protein shows at three positions a conservative amino acid replacement. Using a mouse calcyclin affinity matrix, two proteins with molecular masses of about 36 kDa have been purified from Ehrlich ascites tumor cells. The interaction between these two proteins and the immobilized calcyclin is strictly Ca2(+)-dependent. Immunological criteria and partial sequence data identify the two calcyclin-binding proteins as the phospholipid-binding protein annexin II (p36) and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. These observations suggest that calcyclin may exert its physiological function by a Ca2(+)-dependent interaction with cellular targets, e.g. annexin II or glyceraldehyde-3-phosphate dehydrogenase.

Amino Acid Sequence↗
PubMed1991

A 210 kDa nuclear matrix protein is a functional part of the mitotic spindle; a microinjection study using SPN monoclonal antibodies.

Six monoclonal antibodies identify a 210 kDa polypeptide which shows a cell cycle specific redistribution from the nucleus to the mitotic spindle. In interphase cells this polypeptide was localized in the nucleus and behaved during differential cell extraction as a component of the nuclear matrix. It accumulated in the centrosome region at prophase, in the pole regions of the mitotic spindle at metaphase and in crescents at the poles in anaphase, and reassociated with the nuclei as they reformed in telophase. Due to its staining pattern we call the protein the Spindle Pole-Nucleus (SPN) antigen. The localization of SPN antigen during mitosis was dependent on the integrity of the spindle since treatment of cells with nocodazole resulted in the dispersal of SPN antigen into many small foci which acted as microtubule organizing centres when the drug was removed. The SPN antigen was present in nuclei and mitotic spindles of all human and mammalian cell lines and tissues so far tested. When microinjected into the cytoplasm or nuclei of HeLa cells, one antibody caused a block in mitosis. Total cell number remained constant or decreased slightly after 24 h. At this time, about half the cells were arrested in a prometaphase-like state and revealed aberrant spindles. Many other cells were multinucleate. These results show that the SPN antigen is a protein associated with mitotic spindle microtubules which has to function correctly for the cell to complete mitosis.

Antibodies, Monoclonal↗
PubMed1991

Versatile controlling system for cryopreparation techniques in electron microscopy.

A new setup for freeze-substitution and a versatile controlling system has been developed. Our goal was to build a simple system allowing precise control of the physical parameters of freeze-substitution experiments to learn more about their influences on the cellular ultrastructure and immunoreactivity of macromolecules. An improved apparatus for freeze-substitution, based on liquid nitrogen cooling, and a universal software for controlling the complex preparation protocols from cryofixation to final polymerization are described. This controlling system has the following advantages: it allows precise control and registration of temperature profiles, reconstruction of each individual step of previous experiments, and optimization of working conditions. The setup of the freeze-substitution apparatus is designed to run many different substitution media in parallel; freeze-substitution (cryostat), embedding (working platform), and polymerization are carried out at separate places; therefore, more experiments can be done simultaneously. The ergonomic working platform allows exchange of media at controlled temperature and easy handling; survey of the temperature in individual tubes is possible, and the system is protected from water condensation and uncontrolled warming by the deep freezer.

Computers↗
PubMed1991

Influence of antihypertensive drug therapy on sleep pattern and sleep apnea activity.

Sleep apnea, which affects 10% of men in the mean age group, is a common illness, and arterial hypertension one of its early symptoms. For the large group of, mainly young, patients with mild to moderate sleep apnea and arterial hypertension it is important to have a drug treatment available which will effectively control blood pressure without exacerbating symptoms of sleep apnea. We studied the effects of antihypertensive agents on blood pressure, sleep and sleep apnea in a randomized double-blind study of 24 patients with a sleep apnea activity of more than 10 apnea phases per hour of sleep and arterial hypertension with diastolic blood pressure values in the sitting position greater than or equal to 95 mm Hg. Mean age was 51 (range: 33-69) years, mean body mass index 31.4 (24.9-40.6) kg/m2. The study protocol envisaged two baseline measurements in the sleep laboratory, after which the medication was administered for 8 days. On the last 2 days of the treatment, polysomnographic leads were once again recorded in the sleep laboratory. The patients received either the beta-blocker metoprolol (1 x 100 mg/day) or the angiotensin-converting enzyme inhibitor cilazapril (1 x 2.5 mg/day). Systolic and diastolic blood pressure were decreased by both substances as expected. Total sleep time was 358 (233-425) min vs. 332 (255-383) min in the metoprolol group and 368 (295-424) min vs. 341 (265-434) min in the cilazapril group which is statistically not different between the two groups nor between the proportions of non-REM and REM sleep.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Purification and physical properties of nematode mini-titins and their relation to twitchin.

We have isolated mini-titin from the nematodes Ascaris lumbricoides and Caenorhabditis elegans under native conditions using a modification in the procedure to prepare this protein from insect muscle. The proteins have an apparent molecular weight of 600,000 and appear in oriented specimens as flexible thin rods with a length around 240-250 nm. The circular dichroism spectrum of the Ascaris protein is dominated by beta-structure. The proteins react with antibodies to insect mini-titin and also with antibodies raised against peptides contained in the sequence predicted for twitchin, the product of the Caenorhabditis elegans unc-22 gene. Antibodies to insect mini-titin decorate the body musculature as well as the pharynx of wild-type C. elegans in immunofluorescence microscopy. In the twitchin mutant E66 only the pharynx is decorated. We conclude that the mini-titins of invertebrate muscles defined earlier by ultrastructural criteria are very likely to be twitchins, i.e. molecules necessary for normal muscle contraction. We discuss the molecular properties of the proteins in the light of the sequence established for twitchin.

Amino Acid Sequence↗
PubMed1991

Modulation of keratin intermediate filament assembly by single amino acid exchanges in the consensus sequence at the C-terminal end of the rod domain.

All known intermediate filament (IF) proteins display -8 -4 -1 a consensus sequence TYRKLLEGE at the carboxyl end of the rod domain. To analyse the contribution of this sequence to the formation of IF we have changed two of the invariant positions of this motif by site-directed mutagenesis. We produced three mutant keratins, each containing a single point mutation. Tyrosine at position -8 was changed to alanine in keratin K8 (K8Y----A-8) and keratin K18 (K18Y----A-8) and leucine at position -4 was changed to glycine in keratin K18 (K18L----G-4). Mutant keratins were expressed in Escherichia coli, purified and analysed for their filament-forming capacity in vitro using either the complementary wild-type keratin or the corresponding mixture of mutant keratins. In standard filament buffer (50 mM Tris-HCl, pH7.5), assembly involving any of the mutants leads to large electron-dense aggregates instead of normal IF. In order to explain this effect, we studied the process of filament formation in more detail. Whereas the formation of tetramers in buffers containing 4M urea is unaffected, the elongation process seems slowed down. In buffer of lower ionic strength (10 mM Tris-HCl, pH7.5) mutant keratins K8Y----A-8 plus K18Y----A-8 become able to form long filaments, although short filaments and protofilamentous material are still detected. The filaments formed differ from normal keratin IF by their remarkable tendency to aggregate into thick cables. Assemblies involving K18L----G-4 can only form short IF lengths. The dense aggregates formed in standard filament buffer are able to dissociate into IF and their fragments upon dialysis into 10 mM Tris-HCl, pH7.5. The results show that the consensus sequence is needed for IF formation under normal conditions and that already one mutation per heterodimer affects the assembly.

Amino Acid Sequence↗
PubMed1991

Immunocytochemistry in fine needle aspirates of small cell-, round-, blue-cell malignant tumors of childhood (neuroblastoma, nephroblastoma, lymphoma, Ewing's sarcoma, rhabdomyosarcoma).

Immunocytochemistry with the use of monoclonal antibodies to vimentin, keratin, desmin, neurofilaments and leukocyte common antigen (LCA) was applied to 53 fine needle aspirates of small cell-, round-, blue-cell malignant tumors of childhood (neuroblastomas, nephroblastomas, non-Hodgkin's lymphomas, Ewing's sarcomas and rhabdomyosarcomas). The results revealed the following immunophenotypes: neuroblastomas were positive for neurofilaments. Nephroblastomas contained vimentin (in blastema cells), keratin (in epithelial and blastema cells) Non-Hodgkin's lymphomas were vimentin and LCA positive, keratin negative. Ewing's sarcomas displayed multidirectional differentiation. Rhabdomyosarcomas were desmin and vimentin positive. It is concluded that immunocytochemistry in fine needle aspirates should be used as an objective evidence to support morphologic differential diagnosis of small cell-, round-, blue cell malignant tumors of childhood.

Biopsy, Needle↗
PubMed1991

High molecular weight components are main constituents of Mallory bodies isolated with a fluorescence activated cell sorter.

Mallory bodies (MBs) are cytoplasmic filamentous aggregates containing cytokeratin (CK) material. They occur in hepatocytes of patients with alcoholic liver disease (i.e., alcoholic hepatitis) and can also be induced experimentally in mice by chronic griseofulvin intoxication. To further investigate components and mechanisms involved in MB formation, a new method for MB purification was established. MBs present in a liver homogenate of griseofulvin-fed mice were labeled with a murine monoclonal antibody specific for MBs and a second fluorescein isothiocyanate-conjugated (anti-mouse IgG and IgM) antibody and subsequently isolated by two sequential sorting procedures using a fluorescence activated cell sorter (FACS). Purity of MB isolates was over 90% as revealed by computer analysis of sorting signals and fluorescence and electron microscopy. Electrophoretic separation on sodium dodecyl sulfate-polyacrylamide gels revealed three MB-related polypeptides with apparent molecular masses of 48, 55, and 65 kilodaltons but most of the highly purified MB material did not enter the gel or remained at the interphase between stacking and resolving gels. Western blotting with CK-specific antibodies showed the presence of CK epitopes in the high molecular weight MB material, which has a similar amino acid composition as normal liver CKs. These results establish that very high molecular weight material is the main constituent of MBs and suggest that a post-translational modification of CKs by covalent crosslinks is a principal mechanism of MB pathogenesis.

Amino Acids↗
PubMed1991

Cloning of the non-neuronal intermediate filament protein of the gastropod Aplysia californica; identification of an amino acid residue essential for the IFA epitope.

We describe the isolation and characterization of a full-length cDNA corresponding to the larger non-neuronal (nn) intermediate filament (IF) protein of the gastropod Aplysia californica. Comparison of the sequences of the nn-IF proteins from Aplysia californica and Helix aspersa shows a strong evolutionary drift. At a 72% sequence identity level, the IF proteins of Opisthobranchia and Pulmonata show a larger distance than vimentins from Xenopus and mammals. The sequence comparison of the two snail proteins provides an important step in understanding the epitope of the monoclonal antibody IFA mapped by previous studies to the consensus sequence at the carboxy-terminal end of the rod domain of IF proteins. We identify for the first time in a naturally occurring IF protein a single amino acid exchange which leads to the loss of the epitope. The consensus sequence YRKLLEGEE present in IFA-positive proteins such as the Helix IF protein is changed in the IFA-negative Aplysia protein only by the conservative substitution of the arginine (R) by a lysine (K). Thus, the IFA epitope is not a necessity of IF structure, and its presence or absence on different IF proteins reflects only small changes in an otherwise conserved consensus sequence. Consequently, lack of IFA reactivity does not exclude the presence of IF. This result predicts that IF are much more universally expressed in lower eukaryotes than currently expected from immunological results with the monoclonal antibody IFA.

Amino Acid Sequence↗