Search PubMed⌕ Search

Biomedical subjects

K Weber

Publications and source records attributed to K Weber.

At least 253 records · Page 14Linked to original sources
PubMed1993

Abnormal RNA processing associated with a novel tRNA mutation in mitochondrial DNA. A potential disease mechanism.

A patient with a mitochondrial myopathy and biochemically proven profound complex I deficiency has a new mutation in mtDNA. This A-to-G transition at position 3302, involving the aminoacyl stem of tRNA(Leu(UUR)), is associated with abnormal mitochondrial RNA processing. Northern analysis demonstrates marked accumulation of a polycistronic RNA precursor containing sequence for 16 S rRNA, tRNA(Leu(UUR)), and ND1. Comparison of skeletal muscle and skin fibroblasts suggests that the processing error may be quantitatively less severe in this tissue, and biochemical analysis shows that fibroblasts do not express a biochemical defect despite containing the mutation. Important qualitative differences in the processing of this RNA precursor were found when comparing muscle and skin fibroblasts. In muscle, processing appears to occur first at the 5'-end of the tRNA, generating 16 S rRNA plus a tRNA + ND1 intermediate. In fibroblasts, processing occurs at the 3'-end of the tRNA, generating a 16 S rRNA + tRNA intermediate. We suggest that the mutation at position 3302 induces abnormal mitochondrial RNA processing that is linked to the biochemical defect (profound loss of complex I activity), either by qualitative or quantitative abnormalities in the ND1 message. The restriction to skeletal muscle of both the processing error and the biochemical defect suggests that the observed tissue differences in RNA processing play a protective role in skin fibroblasts.

Adult↗
PubMed1993

Clathrin assembly protein AP180: primary structure, domain organization and identification of a clathrin binding site.

Binding of AP180 to clathrin triskelia induces their assembly into 60-70 nm coats. The largest rat brain cDNA clone isolated predicts a molecular weight of 91,430 for AP180. Two cDNA clones have an additional small 57 bp insert. The deduced molecular weight agrees with gel filtration results provided the more chaotropic denaturant 6 M guanidinium thiocyanate is substituted for the weaker guanidinium chloride. The sequence and the proteolytic cleavage pattern suggest a three domain structure. The N-terminal 300 residues (pI 8.7) harbour a clathrin binding site. An acidic middle domain (pI 3.6, 450 residues), interrupted by an uncharged alanine rich segment of 59 residues, appears to be responsible for the anomalous physical properties of AP180. The C-terminal domain (166 residues) has a pI of 10.4. AP180 mRNA is restricted to neuronal sources. AP180 shows no significant homology to known clathrin binding proteins, but is nearly identical to a mouse phosphoprotein (F1-20). This protein, localized to synaptic termini, has so far been of unknown function.

Adaptor Proteins, Vesicular Transport↗
PubMed1993

Recombinant immunoblot in the serodiagnosis of Lyme borreliosis. Comparison with indirect immunofluorescence and enzyme-linked immunosorbent assay.

A recombinant immunoblot was developed for detection of IgM and IgG antibodies in patients with Lyme borreliosis. The recombinant antigens were the chromosomal-encoded Borrelia burgdorferi proteins p100, the flagellin and an internal flagellin fragment thereof as well as the plasmid-encoded outer surface proteins A (OspA) and C (OspC). A panel of 144 sera from patients with Lyme borreliosis (erythema migrans, n = 31; neuroborreliosis state II, n = 60; Lyme arthritis, n = 24 and acrodermatitis chronica atrophicans, n = 19) have been investigated and the results have been compared to the immunofluorescence absorption test (IFA-ABS) and to two different enzyme-linked immunosorbent assays [the flagellin ELISA and a newly developed ELISA (OGP-ELISA)]. The two ELISAs were comparable in sensitivity, whereas the IFA-ABS was less sensitive for IgM antibody but equally sensitive for IgG antibody detection. Immunoblot analysis revealed that IgG antibodies are mainly reactive with p100 and the internal flagellin fragment (sensitivity 51% and 32%, respectively) and rarely with OspC (14%). All patients with late Lyme borreliosis had IgG antibodies against the p100. IgM antibodies were predominantly directed against OspC (43%) and in a lower extent against the internal flagellin fragment and p100 (15% and 13%, respectively). The complete flagellin was not useful due to a high number of unspecific reactions with control sera and the OspA was only exceptionally reactive in Lyme borreliosis patients. The sensitivity of IgM antibody detection could be increased in cases with early Lyme borreliosis from 46% to 65% when the OspC blot was performed in addition to the flagellin ELISA, or from 56% to 65% when performed in addition to the OGP-ELISA. The recombinant blot is, therefore, a valuable diagnostic test to increase sensitivity of early antibody detection and is regarded as a valuable confirmatory test also in late disease.

Antibodies, Bacterial↗
PubMed1993

Transduction of Ca2+ signals upon fertilization of eggs; identification of an S-100 protein as a major Ca(2+)-binding protein.

A transient increase in the level of free cytosolic Ca2+ is observed upon fertilization of the eggs of many species and is thought to represent a key event in the initiation of development. To identify components in the egg which could be involved in mediating such Ca2+ signals we searched for Ca(2+)-binding proteins in eggs of the fresh-water fish Misgurnus fossilis (loach). We show that loach eggs contain two major Ca(2+)-binding proteins which can be purified through their Ca(2+)-dependent interaction with a hydrophobic matrix. Protein sequencing revealed that the larger 18 kDa protein is calmodulin, while the smaller polypeptide of 10 kDa is a member of the S-100 protein family. This is the first report of the presence of an S-100 protein in vertebrate eggs and shows that this protein is found in two fold higher concentration than calmodulin. Since the 10 kDa protein shares 68% sequence identity with S-100 alpha from bovine brain, it can be considered as the loach homologue of mammalian S-100 alpha. During early embryonic development, de novo protein synthesis of calmodulin is observed at the earliest stages analyzed (mid-blastula), while de novo protein synthesis of the S-100 alpha homologue begins with the mid-gastrula stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Characterization of the tubulin-tyrosine ligase.

The sequence of tubulin-tyrosine ligase (TTL), the enzyme catalyzing the ATP-dependent posttranslational addition of a tyrosine to the carboxyterminal end of detyrosinated alpha-tubulin, has been determined. TTL from bovine and porcine brain was purified by immunoaffinity chromatography and extensively characterized by protein sequencing. Oligonucleotides derived from the protein sequence were synthesized and partial cDNA sequences were obtained using reversed transcribed brain mRNA in polymerase chain reactions. Polymerase chain reaction fragments were used to isolate a full-length cDNA clone from a randomly primed lambda gt10 cDNA library obtained from embryonic porcine brain mRNA. Porcine TTL is encoded by 1,137 nucleotides corresponding to 379 amino acid residues. It has a molecular weight of 43,425 and a calculated isoelectric point of 6.51. Northern blot analysis revealed a surprisingly long mRNA (approximately 6 kb in embryonic porcine brain). The protein sequence of TTL shares no extended homology with the sequences in the data banks. TTL contains a potential serine phosphorylation site for cAMP-dependent protein kinase (RKAS at positions 73 to 76). Residues 244 to 258 lie at the surface of the molecule. A rabbit antibody raised against a synthetic peptide corresponding to this sequence binds to native TTL. The same sequence contains the cleavage site for endoproteinase Glu-C (residue 248) previously shown to convert TTL into a nicked derivative in which the two fragments still form a tight complex but don't display enzymatic activity.

Amino Acid Sequence↗
PubMed1993

Phosphorylation on protein kinase C sites inhibits nuclear import of lamin B2.

The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.

Amino Acid Sequence↗
PubMed1993

SF-assemblin, the structural protein of the 2-nm filaments from striated microtubule associated fibers of algal flagellar roots, forms a segmented coiled coil.

The microtubule associated system I fibers of the basal apparatus of the flagellate green alga Spermatozopsis similis are noncontractile and display a 28-nm periodicity. Paracrystals with similar periodicities are formed in vitro by SF-assemblin, which is the major protein component of system I fibers. We have determined the amino acid sequence of SF-assemblin and show that it contains two structural domains. The NH2-terminal 31 residues form a nonhelical domain rich in proline. The rod domain of 253 residues is alpha-helical and seems to form a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. The distinct cluster of acidic residues at the COOH-terminal end of the motifs (periodicity about 4 nm) may be related to tubulin binding of SF-assemblin and/or its self assembly. A similar structure has been predicted from cDNA cloning of beta-giardin, a protein of the complex microtubular apparatus of the sucking disc in the protozoan flagellate Giardia lamblia. Although the rod domains of SF-assemblin and beta-giardin share only 20% sequence identity, they have exactly the same length and display 42% sequence similarity. These results predict that system I fibers and related microtubule associated structures arise from molecules able to form a special segmented coiled coil which can pack into 2-nm filaments. Such molecules seem subject to a strong evolutionary drift in sequence but not in sequence principles and length. This conservation of molecular architecture may have important implications for microtubule binding.

Amino Acid Sequence↗
PubMed1993

A functional homologue of the RNA1 gene product in Schizosaccharomyces pombe: purification, biochemical characterization, and identification of a leucine-rich repeat motif.

The RNA1 gene from Saccharomyces cerevisiae is defined by the temperature-sensitive rna1-1 mutation that interferes with the maturation and/or nucleocytoplasmic transport of RNA. We describe the purification of a 44-kDa protein from the evolutionary distant fission yeast Schizosaccharomyces pombe and the cloning and sequence analysis of the corresponding gene. Although this protein shares only 42% sequence identity with the RNA1 gene product, it represents a functional homologue because the expression of the S. pombe gene in S. cerevisiae complements the rna1-1 defect. Disruption in S. pombe of the gene encoding the 44-kDa protein, for which we propose the name S. pombe rna1p, reveals that it is essential for growth. Our analysis of purified S. pombe rna1p represents the first biochemical characterization of an RNA1 gene product and reveals that it is a monomeric protein of globular shape. Cell fractionation and immunofluorescence microscopy indicate that rna1p is a cytoplasmic protein possibly enriched in the nuclear periphery. We identify a sequence motif of 29 residues, which is rich in leucine and repeated eight times both in S. pombe and in S. cerevisiae rna1p. Similar leucine-rich repeats present in a series of other proteins, e.g., the mammalian ribonuclease/angiogenin inhibitor, adenylyl cyclase from S. cerevisiae, the toll protein from Drosophila melanogaster, and the sds22 protein phosphatase regulatory subunit from S. pombe, are thought to be involved in protein-protein interactions. Thus rna1p may act as a scaffold protein possibly interacting in the nuclear periphery with a protein ligand that could be associated with exported RNA.

Amino Acid Sequence↗
PubMed1993

US-guided hydrostatic reduction of intussusception in children.

Over a period of 3 3/4 years, hydrostatic reduction of ileocolic intussusception was performed under sonographic guidance in 47 cases (a total of 41 consecutive patients). The enema consisted of a mixture of isotonic electrolyte solution and water-soluble contrast medium (9:1 ratio) and was given at a hydrostatic pressure of 80-100 cm water. Reduction was successful in 83% of cases (n = 39). Even initially remaining ileo-ileal intussusception could finally be reduced in two cases. No complications were observed. Since, in comparison with other conservative methods of reduction, the success rate was not substantially worse (versus that of pneumatic reduction) or was even higher (versus that of barium enema reduction), the method described can be recommended for wider use because of the substantial reduction in radiation exposure.

Child↗
PubMed1993

Microinjection of a monoclonal antibody against SPN antigen, now identified by peptide sequences as the NuMA protein, induces micronuclei in PtK2 cells.

Several high molecular mass proteins which relocate from the interphase nucleus to the spindle poles during mitosis have been defined by antibodies. Microinjection experiments have shown that at least the antigen defined by SPN antibody plays a functional role during mitosis. Recently the cDNA sequence for human NuMA antigen was established and epitopes for antibodies to centrophilin, and to 1F1 and 1H1 antigens were found to be included in the NuMA protein. Here we show that immunoprecipitated SPN antigen reacts with an autoimmune human NuMA serum. In addition three peptides derived from immunoprecipitated human SPN by cyanogen bromide cleavage and covering more than fifty amino acids show a perfect fit with the sequence predicted for NuMA protein. Thus SPN antigen and NuMA are the same protein. Injection of SPN-3 antibody into interphase or mitotic PtK2 cells results in cells with micronuclei. For cells injected in prophase, prometaphase or metaphase 90%, 78% and 77% display defective cytokinesis or yield daughter cells with micronuclei. In contrast only 16% of cells injected in anaphase are abnormal. Thus SPN/NuMA antigen may be required during early, but not during later, stages of mitosis. Surprising parallels are seen between the effects of microinjecting SPN-3 antibody and treatment with colcemid and taxol of PtK2 and HeLa cells. Our results identify an important role during mitosis for the SPN/NuMA antigen.

Amino Acid Sequence↗
PubMed1993

The globular head domain of titin extends into the center of the sarcomeric M band. cDNA cloning, epitope mapping and immunoelectron microscopy of two titin-associated proteins.

Immunoelectron microscopical results have shown that the Z and M bands of the sarcomere are interconnected by the long titin molecules. Here we have characterized by monoclonal antibodies, cDNA cloning and immunoelectron microscopy the two titin-associated proteins (190 and 165 kDa proteins), which seem responsible for the formation of a head structure on one end of the 0.9 micron long titin string. The human 165 kDa (1465 residues) and 190 kDa (1451 residues) proteins have unique N-terminal domains some 110 residues in length. Both proteins show 12 repeat domains with strong homology to either fibronectin type III (motif I) or immunoglobulin C2 (motif II) domains, which are arranged in the order II-II-I-I-I-I-I-II-II-II-II-II. Over these repeat domains the two proteins share 50% sequence identity (70% similarity). Epitopes situated in the C-terminal 138 or in the preceding 206 residues of the 165 kDa protein locate in immunoelectron microscopy to stripes situated 18 or 15 nm from the center of the M band. An epitope situated 277 to 129 residues prior to the C-terminus of the 190 kDa protein (i.e. repeats 10 and 11) locates to the center of the M band. Thus the head structure of the titin molecule extends into the center of the M band. Microsequence data on peptides from the titin-associated bovine 165 kDa protein and from conventionally purified bovine M-protein argue together with the reactivity of the antibodies that 165 kDa protein and M-protein are identical. The integrating structure of the sarcomere, which is based on titin and its side-on (C-protein and 86 kDa protein) or end-on (190 kDa protein and 165 kDa protein) associated proteins arises from muscle-specific members of the superfamily of immunoglobulin-like proteins.

Amino Acid Sequence↗
PubMed1993

In vitro assembly properties of vimentin mutagenized at the beta-site tail motif.

The intermediate filament (IF) proteins vimentin, desmin and peripherin share a 9-residue sequence motif (beta-site) located near the end of their COOH-terminal tail domain. Peptide inhibition experiments have previously suggested that the beta-site is involved in interactions that limit the lateral growth of IFs and prevent inappropriate filament-filament associations. To investigate this question further, we have constructed and expressed, in Escherichia coli, hamster vimentin bearing different mutations in the beta-site. We show here that a single exchange of glycine 450 with a valine residue, or an internal deletion of amino acids 444-452, strongly interferes with the normal assembly of IFs under in vitro conditions. These mutants polymerize into irregular fibrils that have a strong tendency to anastomose and laterally aggregate under isotonic conditions. In contrast, a non-conservative substitution of arginine 448 for glutamic acid does not significantly interfere with filament structure and yields subunits that polymerize into long, smooth filaments that show a slight aberration in thickness. All mutant proteins are soluble in low salt and form oligomers similar to the ones formed by wild-type vimentin. On the basis of these findings and on related observations, we propose that the tail domain of type III IF proteins contains important structural elements involved in lateral protofilament-protofilament interactions.

Amino Acid Sequence↗
PubMed1993

A nuclear lamin of the nematode Caenorhabditis elegans with unusual structural features; cDNA cloning and gene organization.

This report describes the characterization of the nuclear lamin CeLam-1 of the nematode Caenorhabditis elegans by molecular analysis of the corresponding complete cDNA and gene sequences. The primary structure of CeLam-1, representing only the third non-vertebrate lamin sequence currently known, follows essentially the features displayed by the B-type lamins of vertebrates and Drosophila. The nematode lamin shows, however, some exceptional properties. First, it lacks the SPTR sequence in front of the coil 1a domain which constitutes the major mitotic cdc2 kinase phosphorylation site. Second, two prominent deletions occur in the CeLam-1 sequence. One eliminates 14 amino acid residues from the coil 2 domain. A larger deletion of approximately 25 residues results in the shortest lamin tail domain documented so far. The latter corresponds to a region which varies considerably in sequence from highly acidic in vertebrate B-type lamins to rather basic in Drosophila lamin Dmo. CeLam-1 is encoded by a single 2.3 kb mRNA which is abundantly expressed in mixed-stage worm populations. The 5'-end of the mRNA is generated by trans-splicing to the SL1 leader sequence. The CeLam-1 gene extending over 2.7 kb is located on chromosome I. The gene is composed of 6 exons and 5 short introns, which all interrupt the coding sequence. Surprisingly, none of the intron positions has a counterpart in either the Drosophila lamin Dmo or the vertebrate lamin genes.

Amino Acid Sequence↗
PubMed1993

[Long-term theophylline therapy in sleep apnea--follow-up over a period of 12 months].

Among all the sleep-related respiratory disorders, sleep apnoea is clinically the most important one because of its high incidence and potential cardiovascular sequelae. Treatment depends upon the risk profile of the individual patient; in severe cases or in risk patients nasal continuous superpressure ventilation is employed, whereas in milder cases theophylline has been successfully used alongside general measures. 103 patients were followed up during treatment on an outpatient basis for 2 to maximum 12 months. Initially a significant reduction of the apnoea index by a 50% average was achieved. This therapeutical effect remained largely constant with a slight average deterioration at the end of the observation period, whereas the subjective feeling tone also improved in most patients. The treatment course was prematurely discontinued in 34 patients. Good to very good success was achieved in a total of 43 patients, whereas 26 patients were non-responders either initially or as the treatment proceeded. Even though theophylline treatment is now well-tried in mild forms of sleep apnoea, predictors for its use in certain groups of patients must be determined before treatment is initiated.

Adult↗
PubMed1993

Nuclear p53 protein accumulates preferentially in medullary and high-grade ductal but rarely in lobular breast carcinomas.

Striking differences were found between different histological types of breast cancer when 263 invasive breast carcinomas were tested for nuclear p53 accumulation in formaldehyde-fixed paraffin sections. Nuclear p53 accumulation was found in > 10% of tumor cells in 61% of medullary carcinomas (22/36), 37% of grade 3 ductal not otherwise specified carcinomas (32/86), 4% of lobular carcinomas (2/47), and 0% (0/7) of mucinous carcinomas. Strong cytoplasmic p53 staining was noted in 32% of lobular carcinomas. High percentages of medullary and high-grade ductal breast carcinomas accumulate nuclear p53, but these tumors have favorable and poor prognoses, respectively. Thus, whereas nuclear p53 accumulation can be associated in these tumors with high morphological malignancy grades in general and with tumor cell proliferation in particular, p53 accumulation is not necessarily correlated with biological aggressiveness. Overall incidence of p53-positive tumors in a particular series of breast carcinomas (in our study 28%) will depend on the ratio of ductal not otherwise specified, medullary, and lobular carcinomas.

Breast Neoplasms↗
PubMed1993

[Indications and application of BiPaP therapy].

BiPAP allows the independent adjustment of in- and expiratory pressure. BiPAP may thus be used as a "physiologic" CPAP if high expiratory pressure values are not tolerated or as a pressure limited ventilator in pts. suffering from central sleep related breathing disorders (SRBD). 12 pts with SRBD were treated with nBiPAP: 7 did not tolerate the expiration against the elevated pressure, 4 pts suffering from MSA who still presented relevant CSA or central Hypoventilation during nCPAP and 1 pt. with CSA who did not respond to nCPAP. Mean age was 52.3 y (range 33-70) and mean broca index was 149.6 (range 115-254). RDI before therapy ranged between 33-152/h (mean value 79/h); in the five pts. who tolerated nCPAP, the reduction of the RDI obtained was considered insufficient (RDI during nCPAP: 16-71/h). During BiPAP mean RDI was 6.8/h (range 1-17) at pressure levels of 10/7 to 16/13 cm H2O (inspiratory/expiratory pressure) in those pts. that had not tolerated CPAP and 7.9/h (range 2-16.5) in the pts. suffering from central SRBD: in one pt. BiPAP could successfully replace a combined treatment with CPAP and IPPV. 11 pts. are using BiPAP for 10 to 25 months (mean value 13.5) now at home. If CPAP proves ineffective (central SRBD, high expiratory pressure) BiPAP constitutes an effective alternative therapy.

Adult↗