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W Nastainczyk

Publications and source records attributed to W Nastainczyk.

At least 37 records · Page 2Linked to original sources
PubMed1996

Isolation and characterization of six monoclonal antibodies raised against human RAD51 recombinant protein.

Monoclonal antibodies were produced against recombinant human RAD51 recombination protein. The antibodies of IgG subclasses were isolated from serum-free cell culture medium and purified by affinity chromatography on protein A-Sepharose. The antibodies can be used to detect specifically RAD51 protein on immunoblots of total cell lysates. Native RAD51 protein is specifically precipitated from lysates of human cells. In addition, these antibodies readily detect RAD51 in the cell nucleus by immunofluorescence staining. Epitope mapping on overlapping peptides spanning the complete primary amino acid sequence of human RAD51 revealed that three monoclonals recognize an epitope on RAD51 very close to the N-terminus of the protein (amino acids 16 to 20); the other three monoclonals interact with amino acids 85 to 95 of human RAD51.

Amino Acid Sequence↗
PubMed1995

Precise mapping of the tms1 binding site on p53.

Originally identified as multicopy suppressor of a lethal growth arrest caused by expression of a tumour mutant cDNA of p53 in fission yeast the tms1 gene product was found to form stable complexes with p53 in yeast. By using purified recombinant proteins multimeric complexes of tms1 and p53 could be demonstrated and recently the p53 binding site on the tms1 protein was established to the sequence YYITTEDFCT (aa 116-125) in the vicinity of a well conserved cell division motif. Here we report the precise mapping of the tms1 binding site on the p53 protein to the sequence LQIRGRERFE (aa 330-339) which defines a new functional domain on the p53 protein.

Amino Acid Sequence↗
PubMed1995

Full-length myotonin protein kinase (72 kDa) displays serine kinase activity.

We describe the full-length (72 kDa) myotonin protein kinase (Mt-PK) and demonstrate its kinase activity. The 72-kDa protein corresponds to the translation product from the first in-frame AUG codon. This protein was found in the cytoplasmic fraction, whereas the previously reported 55-kDa protein was observed in nuclear extracts. Only the 72-kDa protein was phosphorylated by [32P]phosphate in normal human fibroblasts. To investigate the putative kinase activity of Mt-PK, a construct containing the full-length open reading frame of Mt-PK was expressed in bacterial cells. The recombinant Mt-PK autophosphorylates a Ser residue and phosphorylates the synthetic peptide Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg, which contains a Ser residue in the phosphorylation site. We examined phosphorylation of the voltage-dependent Ca(2+)-release channel, or dihydropyridine receptor (DHPR), by recombinant Mt-PK. We observed that the beta subunit of DHPR was phosphorylated in vitro by Mt-PK. A beta-subunit DHPR peptide containing some of the Ser residues predicted to be phosphorylated was synthesized and found to be a substrate for Mt-PK in vitro. We conclude that the 72-kDa Mt-PK has a protein kinase activity specific for Ser residues.

Amino Acid Sequence↗
PubMed1995

Precise epitope mapping of three monoclonal antibodies raised against tms1 protein of fission yeast.

Recently we described the production of two monoclonal antibodies 10G2 and 10C4 of IgG3 subclass raised against the recombinant tms1 protein of fission yeast. Here we introduce a new monoclonal antibody 2E2 of IgG1 subclass and present the precise epitope mapping of these monoclonal antibodies using tms1 deletion mutants and synthetically produced oligopeptides spanning the tms1 protein by immunoblot analysis.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of a monoclonal anti-protein kinase CK2 beta-subunit antibody of the IgG class for the direct detection of CK2 beta-subunit in tissue cultures of various mammalian species and human tumors.

A murine monoclonal anti-protein kinase CK2 beta antibody was isolated and characterized. The antibody detects 1 pmol of purified recombinant CK2 beta-subunit after analysis on SDS-PAGE. Alternatively undenatured CK2 beta-subunit was detected by an ELISA assay either as recombinant CK2 beta-subunit or in the CK2 holoenzyme (alpha 2 beta 2). Here, concentrations of the first antibody of 1 ng/ml still allowed the detection of the subunit. Immunoblotting of crude cellular extracts from various tissue cultures (man, mouse, and hamster), from human tumors, and the nonneoplastic tissue allowed the detection of the CK2 beta-subunit. The detected epitope of this antibody was, as determined by the epitope analysis technique, 123GLSDI127.

Amino Acid Sequence↗
PubMed1994

Amyotrophic lateral sclerosis patient antibodies label Ca2+ channel alpha 1 subunit.

Sporadic amyotrophic lateral sclerosis is an idiopathic human degenerative disease of spinal cord and brain motor neurons. Prior studies demonstrated that most patients with amyotrophic lateral sclerosis possess immunoglobulins that bind to purified L-type voltage-gated calcium channels, that titers of anti-voltage-gated calcium channel antibodies correlate with disease progression rates, and that amyotrophic lateral sclerosis patient-derived antibodies (ALS IgG) produce electrophysiological changes in the function of voltage-gated calcium channels. Using Western transfer immunoblots and enzyme-linked immunosorbent assays, the calcium ionophore-forming alpha 1 subunit of the voltage-gated calcium channel is now identified as the major voltage-gated calcium channel antigen to which ALS IgG binds. Additionally, the binding of an L-type voltage-gated calcium channel alpha 1 subunit-directed monoclonal antibody, which itself mimics the effects of ALS IgG on skeletal muscle voltage-gated calcium channel currents, is selectively prevented by preaddition of ALS IgG. Voltage-gated calcium channel-binding IgG from patients with Lambert-Eaton myasthenic syndrome appears to be differentiated from ALS IgG by the reactivity of the former to both alpha 1 and beta subunits of the calcium channel. These assays provide further evidence linking amyotrophic lateral sclerosis to an autoimmune process, and suggest one means to differentiate immunoglobulins from patients with amyotrophic lateral sclerosis from those of patients with another autoimmune disease expressing calcium channel antibodies.

Amyotrophic Lateral Sclerosis↗
PubMed1994

Isolation and characterization of two monoclonal antibodies raised against tms1 protein of fission yeast.

Monoclonal antibodies were produced against recombinant tms1 protein of fission yeast. The antibodies of IgG3 subclass were isolated from serum-free cell culture medium and purified by affinity chromatography on protein A-Sepharose. The antibodies can be used to detect specifically the tms1 protein on immunoblots of total yeast lysates. In addition, native tms1 protein is specifically precipitated by these antibodies from yeast lysates.

Animals↗
PubMed1994

Detection of skeletal muscle calcium channel subunits in cultured neonatal rat cardiac myocytes.

A monoclonal antibody (mAb) 8B7, that recognizes specifically the alpha 1-subunit of dihydropyridine-sensitive calcium channels in skeletal muscle, exerted a dose-dependent positive chronotropic effect on cultured spontaneously beating neonatal rat cardiac myocytes. The antibody-induced increase in beating frequency was similar to that elicited by 1 microM of (-)-isoprenaline. The mAb 8B7 recognized a polypeptide that is analogous to the 170-kDa calcium channel alpha 1-subunit of rabbit skeletal muscle in wheat germ lectin purified proteins solubilized from cultured rat heart myocytes by Western blot analysis. Furthermore, the mAb 7C3, previously shown to recognize specifically the skeletal muscle calcium channel beta-subunit, reacted with a 55-kDa polypeptide in these cultured myocytes. No immunoreactivity, however, was obtained when proteins solubilized from both neonatal or adult rat heart tissue were employed in Western blotting or immunoprecipitation experiments. RNA-hybridization analysis revealed that primary cultures derived from neonatal rat hearts expressed two types of mRNA encoding the skeletal muscle as well as the cardiac calcium channel alpha 1-subunit, while neonatal rat heart tissue expressed only the message for cardiac calcium channel. These results provide evidence for a functional expression of skeletal muscle-type calcium channel subunits during the cultivation of neonatal rat heart cells.

Animals↗
PubMed1993

Stable co-expression of calcium channel alpha 1, beta and alpha 2/delta subunits in a somatic cell line.

1. The high-voltage-activated L-type calcium channel is a multi-protein complex of alpha 1, alpha 2/delta, beta and gamma subunits. The alpha 1 subunit contains the voltage-dependent calcium-conducting pore. Chinese hamster ovary (CHO) cells were stably transfected with the complementary DNA of the alpha 1, beta and alpha 2/delta subunits. These subunits were not detected in wild-type CHO cells. 2. The alpha 1 (CaCh2b) subunit itself directed the expression of functional calcium channels which bound calcium channel blockers and showed voltage-dependent activation and inactivation. 3. The co-expression of the alpha 1 subunit with the beta subunit (CaB1 gene) enhanced the density of the dihydropyridine binding sites 2- to 3-fold and increased dihydropyridine-sensitive barium inward currents (IBa) up to 3.5-fold from -13.3 microA/cm2 (alpha 1 subunit) to -46.7 microA/cm2 (alpha 1 and beta subunits). 4. Co-expression of the beta subunit did not change the sensitivity of IBa towards dihydropyridines, but accelerated current activation and inactivation and shifted the half-maximal steady-state activation and inactivation to slightly more hyperpolarizing potentials. 5. The co-expression of the alpha 2/delta subunit together with alpha 1 and beta subunits accelerated the inactivation kinetics of the channel without a major effect on the other parameters. 6. These results indicate that the beta and alpha 2/delta subunit interact with the alpha 1 subunit and modulate thereby the properties of the alpha 1 subunit-dependent inward current.

Animals↗
PubMed1992

Serum antibodies to L-type calcium channels in patients with amyotrophic lateral sclerosis.

BACKGROUND AND METHODS: Sporadic amyotrophic lateral sclerosis is a chronic, progressive degenerative disease of the motor neurons of the spinal cord and motor cortex. The cause is unknown. Recent electrophysiologic studies in animals indicate that immunoglobulins from patients with this disease alter presynaptic voltage-dependent calcium currents and calcium-dependent release of neurotransmitters. To determine whether similar interactions might be identified biochemically, we used an enzyme-linked immunosorbent assay (ELISA) to detect the reaction of serum IgG with purified complexes of L-type voltage-gated calcium channels from rabbit skeletal muscle. The results from patients with amyotrophic lateral sclerosis were compared with those obtained from patients with other types of motor neuron disease, patients with autoimmune and non-autoimmune neurologic diseases, and normal subjects. RESULTS: Serum samples from 36 of 48 patients with sporadic amyotrophic lateral sclerosis (75 percent) contained IgG that reacted with L-type calcium-channel protein, and serum reactivity on ELISA correlated with the rate of disease progression (Spearman rank-correlation coefficient, 0.62). Reactive serum was present in only 1 of 25 normal subjects and 1 of 35 control patients with no motor neuron disease. Antibodies to L-type voltage-gated calcium channels were identified in 6 of 9 patients with Lambert-Eaton syndrome, and in 3 of 15 patients with Guillain-Barré syndrome. CONCLUSIONS: Antibodies to L-type voltage-gated calcium channels are present in the serum of patients with amyotrophic lateral sclerosis, and antibody titers correlate with the rate of disease progression. Together with previous data, these results suggest a role for autoimmune mechanisms in the pathogenesis of sporadic amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis↗
PubMed1992

Target size analysis of prostaglandin endoperoxide synthase. Radiation inactivation of both cyclooxygenase and peroxidase correlated with the monomer of 72 kDa.

To determine the size of the functional catalytic unit of prostaglandin endoperoxide (prostaglandin H) synthase, radiation inactivation experiments were performed. Both microsomes from ovine seminal vesicles and purified enzyme were irradiated with 10 MeV electrons. The enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, showed mono-exponential inactivation curves dependent on radiation dose, indicating molecular masses of approximately 72 kDa. The enzyme in microsomes, in its native environment, as well as in its purified state after solubilisation with nonionic detergent showed identical molecular masses. The results clearly demonstrate that the monomer of the enzyme with an apparent molecular mass of 72 kDa (SDS/PAGE) is the functional unit for catalysis of both activities. Hence the two active sites of cyclooxygenase and peroxidase reside on the same polypeptide chain.

Animals↗
PubMed1991

Identification of the site of interaction of the dihydropyridine channel blockers nitrendipine and azidopine with the calcium-channel alpha 1 subunit.

The dihydropyridine binding site of the rabbit skeletal muscle calcium channel alpha 1 subunit was identified using tritiated azidopine and nitrendipine as ligands. The purified receptor complex was incubated either with azidopine or nitrenidpine at an alpha 1 subunit to ligand ratio of 1:1. The samples were then irradiated by a 200 W UV lamp. The ligands were only incorporated into the alpha 1 subunit, which was isolated by size exclusion chromatography and digested either by trypsin (azidopine) or endoproteinase Asp-N (nitrendipine). Each digest contained two radioactive peptides, which were isolated and sequenced. The azidopine peptides were identical with amino acids 13-18 (minor peak) and 1428-1437 (major peak) of the primary sequence of the skeletal muscle alpha 1 subunit. The nitrendipine peptides were identical with amino acids 1390-1399 (major peak) and 1410-1420 (minor peak). The sequence from amino acids 1390 to 1437 is identical in the alpha 1 subunits of skeletal, cardiac and smooth muscle and follows directly repeat IVS6. These results indicate that dihydropyridines bind to an area that is located at the putative cytosolic domain of the calcium channel.

Amino Acid Sequence↗
PubMed1991

[Beta blockers and corneal sensitivity].

We performed several placebo-controlled, randomized, prospective, parallel, double-masked studies in ophthalmologically healthy people to determine the local anesthetic effect and the topical subjective tolerability of beta blockers with similar potency of action that are available in the Federal Republic of Germany. There are marked differences in the local anesthetic effects of the different beta blockers. Depending on the type of beta blockers the local anesthetic effect ranges from practically none up to anesthesia allowing contact glass examinations and removal of foreign bodies after application of only one drop. The subjective tolerability becomes worse with increasing strength of local anesthetic effect of the beta-blocker. There is a statistically significant correlation between the subjective tolerability and local anesthetic effect and the octanol-water distribution coefficient. Because of the local anesthetic effect, we recommend repeated checks of corneal sensitivity during beta blocker therapy. This should be done more frequently in people with sensitive eyes, "responders," contact lens wearers, and people with reduced corneal sensitivity for other reasons, especially during therapy with beta-blockers with marked local anesthetic effects. In addition to systemic side effects, cardioselectivity, influence on tear film, intrinsic sympathomimetic activity and topical tolerance, etc., the local anesthetic effect of beta blockers is a further important aspect in the "differential indications" of beta-blockers.

Adrenergic beta-Antagonists↗
PubMed1990

The dihydropyridine-sensitive calcium channel of the skeletal muscle: biochemistry and structure.

The dihydropyridine-sensitive calcium channel of the rabbit skeletal muscle is the first voltage-gated calcium channel which has been purified and biochemically characterized. The alpha 1-subunit, a 165 kDa protein, of the purified dihydropyridine receptor contains all regulatory sites of a L-type calcium channel and the calcium conducting unit. The purpose of this review is to summarize and discuss recent findings on the structure and possible function of the skeletal muscle calcium channel subunits.

Animals↗
PubMed1988

Evidence for coordinate, selective regulation of eicosanoid synthesis in platelet-derived growth factor-stimulated 3T3 fibroblasts and in HL-60 cells induced to differentiate into macrophages or neutrophils.

We used Swiss 3T3 fibroblasts stimulated with platelet-derived growth factor and HL-60 cells induced to differentiate into macrophages or neutrophils to study the regulation of prostaglandin and leukotriene synthesis. Addition of platelet-derived growth factor to quiescent 3T3 fibroblasts led within 4 h to a dramatic and preferential increase in prostacyclin synthesis from endoperoxide prostaglandin H2, and microsomal assays showed a strong platelet-derived growth factor-dependent increase in the maximal velocities (Vmax) of both prostaglandin H synthase and prostacyclin synthase. In contrast, addition of phorbol ester to HL-60 cells to induce differentiation into macrophages led within 4 h to a strong and preferential increase in thromboxane synthesis from prostaglandin H2, and microsomal assays disclosed a major rise in Vmax for both prostaglandin H synthase and thromboxane synthase. No comparable changes occurred in HL-60 cells that were differentiating into neutrophils, though upregulation of 5-lipoxygenase pathway enzymes occurred in both differentiation systems. Actinomycin D and cycloheximide prevented the appearance of all of these enzymes of eicosanoid synthesis in all three model systems. Thus, the distinctive patterns of eicosanoid synthesis that are seen in replicating fibroblasts and in differentiating macrophages and neutrophils appear to depend on a coordinate, selective upregulation of several enzymes of eicosanoid biosynthesis that is specific for each cell system.

Animals↗
PubMed1988

Site-specific phosphorylation of the purified receptor for calcium-channel blockers by cAMP- and cGMP-dependent protein kinases, protein kinase C, calmodulin-dependent protein kinase II and casein kinase II.

Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium-channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase II and casein kinase II phosphorylated the 165-kDa and the 55-kDa proteins of the purified CaCB receptor. The 130/28-kDa and the 32-kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165-kDa subunit with 2-3-fold higher initial rate than the 55-kDa subunit. Casein kinase II phosphorylated the 165-kDa and the 55-kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase II phosphorylated preferentially the 55-kDa protein. The 55-kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase II or casein kinase II and about 10 times faster by these enzymes than by cAMP kinase. Two-dimensional peptide maps of the 165-kDa subunit yielded a total of 11 phosphopeptides. Four or five peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase II and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55-kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165-kDa and the 55-kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase II and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor.

Animals↗