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F Hofmann

Publications and source records attributed to F Hofmann.

At least 181 records · Page 10Linked to original sources
PubMed1996

The ras-related protein Ral is monoglucosylated by Clostridium sordellii lethal toxin.

Clostridium sordellii lethal toxin (LT), a cytotoxin which causes preferential destruction of the actin cytoskeleton, has been recently identified as glucosyltransferase to modify the low molecular mass GTPases Rac, Ras and Rap. We report here on LT produced by C. sordellii strain 6018 which glucosylates in addition to Rac, Ras and Rap the Ral protein. LT from strain VPI9048 however does not glucosylate Ral. Besides recombinant Ral, cellular Ral is also substrate. In the GDP-bound form, Ral is a superior substrate to the GTP form. Acceptor amino acid for glucose is threonine-46 which is equivalent to threonine-35 in H-Ras located in the effector region. The Ral-glucosylating toxin is a novel isoform of Ras-modifying clostridial cytotoxins.

Animals↗
PubMed1996

Protein phosphatase 2A is essential for the activation of Ca2+-activated K+ currents by cGMP-dependent protein kinase in tracheal smooth muscle and Chinese hamster ovary cells.

The regulation of Ca2+-activated K+ channels (KCa channels) by cGMP-dependent protein kinase (cGMP kinase) and its molecular mechanism were investigated in Chinese hamster ovary (CHO) and tracheal smooth muscle cells. In CHO wild-type cells (CHO-WT cells) and in CHO cells stably transfected with cGMP kinase Ialpha (CHO-cGK cells), KCa channels with intermediate conductance (approximately 50 picosiemens) were identified. Due to the basal activity of cGMP kinase, Ca2+-activated K+ currents had a higher sensitivity toward the cytosolic Ca2+ concentration in CHO-cGK cells than in CHO-WT cells. Dialysis of the active fragment of cGMP kinase (300 n) into CHO-WT cells or of cGMP into CHO-cGK cells increased the Ca2+-activated K+ current, while the catalytic subunit of cAMP-dependent protein kinase (cAMP kinase) was without effect. In cell-attached patches obtained from freshly isolated bovine tracheal smooth muscle cells, the open state probability (NPo) of maxi-KCa channels (conductance of approximately 260 picosiemens) was enhanced by 300 microM 8-(4-chlorophenylthio)-cGMP, a specific and potent activator of cGMP kinase. In contrast, 1 microM isoprenaline, 20 microM forskolin, and 3 mM 8-bromo-cAMP failed to enhance KCa channel activity. In excised inside-out patches, only the active fragment of cGMP kinase (but not that of cAMP kinase) increased NPo when applied to the cytosolic side of the patch. The enhancement of NPo by cGMP kinase was inhibited in CHO cells as well as in tracheal smooth muscle cells by the cGMP kinase inhibitor KT 5823 (1 microM) and the protein phosphatase (PP) inhibitors microcystin (5 microM) and okadaic acid (10 nM). The catalytic subunit of PP2A (but not that of PP1) mimicked the effect of cGMP kinase on NPo in excised inside-out patches. The results show that cGMP kinase regulates two different KCa channels in two unrelated cell types by the same indirect mechanism, which requires the activity of PP2A. The regulation of the KCa channel is specific for cGMP kinase and is not mimicked by cAMP kinase.

Animals↗
PubMed1996

Primary structure of a novel ABC transporter with a chromosomal localization on the band encoding the multidrug resistance-associated protein.

Complementary DNA clones encoding a novel protein, ABC-C, with the typical structural features of the ABC transporter family were identified in a human medullary thyroid carcinoma cell line. The transporter consists of 1704 amino acid residues with two homologous repeats, each harboring six putative transmembrane helices and an ATP-binding cassette motif. The mRNA is expressed highest in normal lung, but also in varying amounts in other tissues and in C-cell carcinoma. The ABC-C gene is mapped on chromosome 16p13.3, in close physical proximity to another ABC transporter, the multidrug resistance-associated protein. This related protein is assumed to confer resistance to chemotherapeutic drugs in small cell lung carcinoma. The genomic clustering of both transporters, typical also for other members of the ABC family, supports the notion that ABC-C may be involved in development of resistance to xenobiotics.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

The IVS6 segment of the L-type calcium channel is critical for the action of dihydropyridines and phenylalkylamines.

The current through the L-type calcium channel is inhibited and stimulated by distinct dihydropyridines at very low concentrations. The molecular determinants for the high affinity block and stimulation were investigated using chimeras between the class C and E calcium channels. Mutation of three amino acids in the last putative transmembrane segment (IVS6) of the alpha1C subunit decreased the affinity for (+)isradipine 100-fold without significantly affecting the basic properties of the expressed channel. Mutation of two of these three amino acids completely abolished the stimulatory effect of the calcium channel agonist Bay K 8644. These mutations only slightly affected the blocking efficacy of mibefradil and the phenylalkylamine devapamil. Three distinct but adjacently located amino acids mediated the high affinity block by devapamil. These results suggest that the IVS6 segment of the alpha1C subunit is critical for the high affinity interaction between the L-type calcium channel and the calcium channel agonist Bay K 8644 and the two antagonists isradipine and devapamil.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Inactivation of Ras by Clostridium sordellii lethal toxin-catalyzed glucosylation.

The lethal toxin (LT) from Clostridium sordellii belongs to the family of large clostridial cytotoxins causing morphological alterations in cultured cell lines accompanied by destruction of the actin cytoskeleton. C. sordellii LT exhibits 90% homology to Clostridium difficile toxin B, which has been recently identified as a monoglucosyltransferase (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503). We report here that LT too is a glucosyltransferase, which uses UDP-glucose as cosubstrate to modify low molecular mass GTPases. LT selectively modifies Rac and Ras, whereas the substrate specificity of toxin B is confined to the Rho subfamily proteins Rho, Rac, and Cdc42, which participate in the regulation of the actin cytoskeleton. In Rac, both toxin B and LT share the same acceptor amino acid, threonine 35. Glucosylation of Ras by LT results in inhibition of the epidermal growth factor-stimulated p42/p44 MAP-kinase signal pathway. LT is the first bacterial toxin to inactivate Ras in intact cells.

3T3 Cells↗
PubMed1996

Differential effects of cdk2 and cdk3 on the control of pRb and E2F function during G1 exit.

The cyclin-dependent kinases cdk2 and cdk3 are required for the G1-S transition in mammalian cells. Here we show that G1 arrest induced by the corresponding dominant-negative mutants of these enzymes, cdk2dn or cdk3dn, is resistant to the action of SV40 T antigen (T). In the presence of cdk2dn, T released active E2F from negative control by pRb and its related family members (pocket proteins) but failed to induce S-phase. Therefore, among other targets, cdk2 also phosphorylates nonpocket protein substrates in promoting S-phase entry, and T does not mimic all cdk2 functions. In the presence of cdk3dn, however, T failed to induce cell cycle progression or stimulate E2F-dependent transcription activity. Dominant-negative cdk3 inhibited E2F-1, E2F-2, and, less significantly, E2F-3, but not E2F-4 transcription activity. The inhibition occurred in a pRb-independent manner and did not affect the DNA-binding capacity of the transcription factor. Cdk3 bound specifically to E2F-1/DP-1 complexes in vivo, most likely through DP-1. Thus, cdk3 function contributes to the activation of E2F-1, E2F-2, and partially E2F-3 and, thereby, participates in the process of S-phase entry.

Antigens, Viral, Tumor↗
PubMed1996

Molecular cloning and expression of the Modulatory subunit of the cyclic nucleotide-gated cation channel.

The cDNA of three variants of a cyclic nucleotide-gated (CNG) channel modulatory subunit (CNG4c-CNG4e) has been cloned. CNG4c, CNG4d, and CNG4e differ slightly from each other within an amino-terminal sequence that was originally reported as part of the bovine retinal glutamic acid-rich protein (GARP). The core region of CNG4 is homologous to the second subunit of the human rod photoreceptor channel (hRCNC2b), suggesting that both proteins are alternatively spliced products of the bovine and human homologue of the same gene. CNG4 transcripts are present in retina, testis, kidney, heart, and brain. Expression of CNG4 in HEK293 cells did not lead to detectable currents. Coexpression of CNG4 with the principal subunit of the bovine testis CNG channel (CNG3) resulted in currents which differed in several aspects from that induced by CNG3 alone. The heterooligomeric CNG3/CNG4 and the homooligomeric CNG3 channels were modified by Ca2+-calmodulin and some calmodulin antagonists. The results suggest that CNG4 forms functional heterooligomeric channels with CNG3 in vitro and probably also in intact tissues.

Amino Acid Sequence↗
PubMed1996

Analysis of the catalytic site of the actin ADP-ribosylating Clostridium perfringens iota toxin.

The enzyme component of actin ADP-ribosylating Clostridium perfringens iota toxin was affinity labelled by UV irradiation in the presence of [carbonyl-14C]NAD. A peptide containing the radiolabel was generated by CNBr cleavage and subsequent proteolysis with trypsin. Its amino acid sequence is Gly-Ser-Pro-Gly-Ala-Tyr-Leu-Ser-Ala-Ile-Pro-Gly-Tyr-Ala-Gly-X-Tyr-Glu-Va l-Leu-Leu-Asn-His-Gly-Ser-Lys corresponding with the region Gly-363 through Lys-388 in the C. perfringens iota toxin. Mass spectrometric data as well as results of the PTH-amino acid analysis are in line with a modification of a glutamic acid side chain located at position 378. Therefore, in addition to Glu-380, as could be concluded by analogy with other ADP-ribosyltransferases, Glu-378 may play a pivotal role in the active site of C. perfringens iota toxin.

ADP Ribose Transferases↗
PubMed1996

Ca(2+)-dependent inactivation of the class C L-type Ca2+ channel is a property of the alpha 1 subunit.

The stably expressed Ca2+ channel alpha 1C-a and alpha 1C-b subunit were used to investigate the molecular basis for Ca(2+)-dependent inactivation of the L-type current. The Ba2+ current (IBa) of both channels had similar kinetics and inactivated with one time constant of about 400 ms at +20 mV, whereas the Ca2+ current (ICa) could be fitted only with a bi-exponential function. The fast (tau f) and the slow (tau s) time constant were about 20 ms and 400 ms, respectively. The inactivation of ICa strongly depended on the entry of Ca2+ as shown by prepulses and variation of the intracellular Ca2+ chelator. Coexpression of the alpha 1C subunits with the auxiliary alpha 2/delta and beta subunits accelerated the voltage-dependent but not the Ca(2+)-dependent inactivation of the channels. These results suggest that the alpha 1C subunit of L-type Ca2+ channels itself mediates the Ca(2+)-dependent inactivation of the current.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Expression and functional characterization of the cardiac L-type calcium channel carrying a skeletal muscle DHP-receptor mutation causing hypokalaemic periodic paralysis.

A histidine substitution for the outermost arginine in II/S4 of the alpha1 subunit of the human skeletal muscle dihydropyridine (DHP) receptor has been reported to cause hypokalaemic periodic paralysis (HypoPP). This mutation shifts the voltage dependence of L-type Ca curent inactivation in myotubes from HypoPP patients by -40 mV without affecting activation. Based on the strong homology of II/S4 in cardiac and skeletal muscle alpha1, we introduced the corresponding mutation into the rabbit cardiac alpha1 subunit (R650H). Wild type (WT) and mutant constructs were transiently transfected in HEK cells together with beta and alpha2delta subunits and Ca and Ba currents were studied using the whole-cell patch-clamp technique. In contrast to the results obtained from human myotubes, R650H produced a small (-5 mV) but significant shift of both the steady-state activation and inactivation curves. When external pH was increased from 7.4 to 8.4 in order to favour deprotonization of H650, the only difference between WT and mutant channels was a slightly reduced steepness of the inactivation curve. Additional cotransfection of the gamma subunit which is only found in skeletal but not in heart muscle, shifted the inactivation curves of both WT and R650H by -20 mV. We conclude that R650 plays a different role in voltage-dependent gating of the cardiac L-type Ca channel than the corresponding residue in the human skeletal muscle L-type channel, since a distinct and selective effect on the midpoint voltage of steady-state inactivation could not be found for R650H.

Animals↗
PubMed1996

Immunogenicity of an inactivated hepatitis A vaccine administered according to two different schedules and the interference of other "travellers" vaccines with the immune response.

A total of 2036 persons consulting vaccination centers in Germany were vaccinated with an inactivated hepatitis A vaccine (containing 720 ELISA units of antigen) either according to the standard schedule (two vaccinations given 4 weeks apart) or to an abbreviated schedule (two vaccinations given 2 weeks apart) in a controlled clinical study. The abbreviated schedule induced a similar rate of seroconversion and geometric mean antibody titre as compared to the standard schedule. The incidence of reactions reported after vaccination was similar in both groups. When other "travellers" vaccines were given simultaneously neither the immunogenicity nor the reactogenicity of the hepatitis A vaccine were influenced. These findings have considerable practical importance in the prevention of hepatitis A in travellers.

Adolescent↗
PubMed1996

High permeation of L-type Ca2+ channels at physiological [Ca2+]: homogeneity and dependence on the alpha 1-subunit.

Molecular cloning has identified multiple isoforms of dihydropyridine-sensitive C-class L-type Ca2+ channels. We tested the hypotheses that L-type (C-class) channels exhibit homogeneous high permeation properties at physiological Ca2+ concentrations and membrane potentials. We measured unitary currents through single dihydropyridine-sensitive omega-conotoxin-insensitive endocrine and smooth muscle L-type Ca2+ channels in rat pituitary GH3 and rat aortic A7r5 cell lines. We also measured unitary currents through smooth muscle (Cb) Ca2+ channel alpha 1-subunits in Chinese hamster ovary (CHO) cells. Our results show that single channel conductances of all three L-type (C-class) channels are uniform with high Ba2+ concentrations, e.g., approximately 23 pS with 110 mM Ba2+. The single channel conductances were reduced to similar values when the Ba2+ concentration was lowered to near-physiological values: 11.1, 9.3, and 8.4 pS in GH3, A7r5, and CHO cells at 2 mM Ba2+, respectively. The single channel conductances were not significantly different with near-physiological Ca2+ concentrations: 5.5, 5.9, and 4.9 pS in GH3, A7r5, and CHO cells at 2 mM Ca2+, respectively. The data suggest that L-type (C-class) channels are homogeneous in terms of Ca2+ permeation at physiological charge carrier concentrations and membrane potentials. Furthermore, the data indicate that the relatively high Ca2+ permeation under physiological conditions is determined by the intrinsic properties of the pore-forming Ca2+ channel alpha 1-subunit.

Animals↗
PubMed1996

Influence of L-type Ca channel alpha 2/delta-subunit on ionic and gating current in transiently transfected HEK 293 cells.

We have measured ionic and gating currents in human embryonic kidney (HEK 293) cells transiently transfected with cDNAs encoding subunits of the cardiac voltage-gated L-type Ca2+ channel. Robust recombinant ionic current and associated nonlinear charge movement could be measured over a broad voltage range without contamination by endogenous channel activity. Coexpression of the alpha 2/delta-subunit along with alpha 1- and beta 2-subunits speeded activation and deactivation kinetics and significantly increased the maximal conductance of ionic current. Charge movement was measured at voltages negative to the threshold for activation of ionic current, and gating charge could be immobilized at positive holding potentials that did not inactivate ionic current. The ratio of maximal ionic conductance to maximal charge moved remained the same in the absence or presence of the alpha 2/delta-subunit. However, the maximal amount of charge moved was increased about twofold in the presence of the alpha 2/delta-subunit. These results suggest that coexpression of the alpha 2/delta-subunit enhances the expression of functional L-type channels and, in addition, provide evidence that most of the L-type channel-associated nonlinear charge movement is caused by transitions between nonconducting states of the channel protein that precede the open and inactivated states.

Calcium Channels↗
PubMed1996

[Occupational medicine in public health service--evaluation results of an continuing education symposium].

The demand for a quality controlled and quality proved continuing medical education (CME) has grown in the past. Especially the German "Bundesärztekammer" (society of physicians of Germany) has developed new models for CME and it's evaluation. This paper presents the background, methodology and results of two evaluations of CME for occupational physicians in health care institutions. The two symposia in 1994 and 1995 were evaluated using the same questionnaire. The comparative analysis of the results indicates the feasibility of the instrument and the validity of the results when used together with the results on an open discussion at the end of the symposium. It also demonstrates the value of the evaluation for future planning of comparable programs.

Adult↗
PubMed1996

Reconstitution of the skeletal muscle dihydropyridine receptor. Functional interaction among alpha 1, beta, gamma and alpha 2 delta subunits.

The L-type voltage-dependent Ca2+ channel purified from skeletal muscle by virtue of its dihydropyridine (DHP) binding activity, is composed of alpha 1, alpha 2 delta, beta and gamma subunits. The alpha 1 subunit has the ability to function alone as a Ca2+ channel and a receptor for DHP and other Ca2+ channel antagonists. In this study, the non-alpha 1 components coexpressed with alpha 1 in COS cells were investigated for their effects on DHP binding and suppression of an anomalous allosteric regulation of the phenylalkylamine (-)D600 in complexes lacking one or more subunits. (-)D600 increased DHP binding to membranes of COS cells expressing alpha 1 beta while it did not affect DHP binding to skeletal muscle membranes. Coexpression of gamma or alpha 2 delta with alpha 1 beta partially suppressed this effect. Coexpression of all the subunits completely eliminated the stimulatory effect of (-)D600, while at the same time increasing the affinity of the complex for DHP to that stabilized in partial complexes by the phenylalkylamine. These results demonstrate that all of the components that co-purify are required for the formation of a functional DHP receptor having the properties of the native skeletal muscle DHP receptor.

Animals↗
PubMed1995

Expression of a chimeric, cGMP-sensitive regulatory subunit of the cAMP-dependent protein kinase type I alpha.

To study the fluctuations of cGMP in living cells through changes of energy transfer of dissociable fluorescence labeled subunits, we constructed a cGMP-sensitive probe by combining the N-terminus of the type I regulatory subunit of cAMP-dependent protein kinase (PKA) with the cGMP binding sites of cGMP-dependent protein kinase I alpha (PKG). This chimeric regulatory subunit retained PKA-like dimerization and PKG-compatible cGMP binding constants (Kd = 53 nM) for both binding sites. High affinity interaction with the PKA catalytic subunit was verified by Surface Plasmon Resonance (Kd = 3.15 nM). Additionally, the chimera inhibits the formation of wild-type holoenzyme with an apparent Ki of 1.05 nM. Furthermore, cGMP dissociated the mutant holoenzyme with an apparent activation constant of 146 nM. Thus, our construct provides all the requirements needed to investigate changes in intracellular cGMP concentrations.

Base Sequence↗