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Biomedical subjects

F Hofmann

Publications and source records attributed to F Hofmann.

At least 73 records · Page 4Linked to original sources
PubMed2000

Structural consequences of mono-glucosylation of Ha-Ras by Clostridium sordellii lethal toxin.

Mono-glucosylation of Ha-Ras by Clostridium sordellii lethal toxin at effector region threonine 35 has diverse effects on the Ras GTPase cycle, the dominant one of which is the inhibition of Ras-Raf coupling, leading to complete blockade of Ras downstream signaling. To understand the structural basis of the functional consequences of glucosylation, the X-ray crystal structure of glucosylated Ras-GDP was compared with that of non-modified Ras. Glucosylated Ras exhibits a different crystal packing but the overall three-dimensional structure is not altered. The glucose group does not affect the conformation of the effector loop. Due to steric constraints, the glucose moiety prevents the formation of the GTP conformation of the effector loop, which is a prerequisite for binding to the Raf-kinase. The X-ray crystal data also revealed the alpha-anomeric configuration of the bound glucose, indicating that the glucose transfer proceeds under retention of the C-1 configuration of the d-alpha-glucose. Therefore, glucosylation preserves the inactive conformation of the effector loop independently of the nucleotide occupancy, leading to a complete inhibition of downstream signaling of Ras.

Bacterial Toxins↗
PubMed2000

Mutations in the S4 domain of a pacemaker channel alter its voltage dependence.

In an attempt to study the functional role of the positively charged amino acids present in the S4 segment of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, we have introduced single and sequential amino acid replacements throughout this domain in the mouse type 2 HCN channel (mHCN2). Sequential neutralization of the first three positively charged amino acids resulted in cumulative shifts of the midpoint voltage activation constant towards more hyperpolarizing potentials. The contribution of each amino acid substitution was approximately -20 mV. Amino acid replacements to neutralize either the first (K291Q) or fourth (R300Q) positively charged amino acid resulted in the same shift (about 20 mV) towards more hyperpolarized potentials. Replacing the first positively charged amino acid with the negatively charged glutamic acid (K291E) produced a shift of approximately -50 mV in the same direction. None of the above amino acid substitutions had any measurable effect on the time course of channel activation. This suggests that the S4 domain of HCN channels critically controls the voltage dependence of channel opening but is not involved in regulating activation kinetics. No channel activity was detected in mutants with neutralization of the last six positively charged amino acids from the S4 domain, suggesting that these amino acids cannot be altered without impairing channel function.

Amino Acid Sequence↗
PubMed2000

The alpha 1-subunit of smooth muscle Ca(2+) channel preserves multiple open states induced by depolarization.

The cloned alpha 1-subunits of the smooth muscle Ca(2+) channel (alpha (1C-b)) from rabbit lung were expressed in Chinese hamster ovary cells. The effect of large depolarizations was examined using cell-attached patch clamp techniques. After large, long-duration depolarizations (to +80 mV, 4 s), the cloned smooth muscle Ca(2+) channels were still open, and also showed slow channel closure upon repolarization. The sum of unitary channel currents revealed that the tail current seen after large conditioning depolarizations had a slower deactivation time constant compared to that seen when the cell membrane was depolarized briefly with a test step (to +40 mV), suggesting that large depolarizations transform the conformation of the Ca(2+) channels to a second open state. The decay time course of the tail current induced by large conditioning depolarizations was prolonged by reducing the negativity of the repolarization step, and vice versa. Using the slow deactivating characteristic, the current-voltage relationship was directly measured by applying a ramp pulse after a large depolarization. Its slope conductance was approximately 26 pS. Since the patch pipettes contained Ca(2+) agonists, the transition of the Ca(2+) channel conformation to the second, long open state during a large depolarization was distinct from that caused by Ca(2+) agonists, suggesting that the cloned alpha 1-subunits of smooth muscle Ca(2+) channels preserve the characteristic features seen in native smooth muscle Ca(2+) channels. In addition, when skeletal muscle beta-subunits were coexpressed with the alpha 1-subunits, the long channel openings after large, long-duration depolarizations were frequently suppressed. This phenomenon could be explained if the skeletal muscle beta-subunits increased the inactivation rate during the preconditioning depolarization.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2000

New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis.

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or PTK787 / ZK222584), reversibly inhibit Flt-1 and KDR with IC(50) values < 0.1 microM. CGP 79787D also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing the KDR receptor (ED(50) = 34 nM). Modification of the 1-anilino moiety afforded derivatives with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmethyl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as candidates for clinical evaluation.

Administration, Oral↗
PubMed2000

Modulation of the smooth-muscle L-type Ca2+ channel alpha1 subunit (alpha1C-b) by the beta2a subunit: a peptide which inhibits binding of beta to the I-II linker of alpha1 induces functional uncoupling.

Modulation of the smooth-muscle Ca(2+) channel alpha1C-b subunit by the auxiliary beta2a subunit was studied in the HEK 293 (cell line from human embryonic kidney cells) expression system. In addition, we tested whether the alpha1-beta interaction in functional channels is sensitive to an 18-amino-acid synthetic peptide that corresponds to the sequence of the defined major interaction domain in the cytoplasmic I-II linker of alpha1C (AID-peptide). Ca(2+) channels derived by co-expression of alpha1C-b and beta2a subunits exhibited an about 3-fold higher open probability (P(o)) than alpha1C-b channels. High-P(o) gating of alpha1C-b.beta2a channels was associated with the occurrence of long-lasting channel openings [mean open time (tau)>10 ms] which were rarely observed in alpha1C-b channels. Modulation of fast gating by the beta2a subunit persisted in the cell-free, inside-out recording configuration. Biochemical experiments showed that the AID-peptide binds with appreciable affinity to beta2 subunits of native Ca(2+) channels. Binding of the beta2 protein to immobilized AID-peptide was specifically inhibited (K(i) of 100 nM) by preincubation with free (uncoupled) AID-peptide, but not by a corresponding scrambled peptide. Administration of the AID-peptide (10 microM) to the cytoplasmic side of inside-out patches induced a substantial reduction of P(o) of alpha1C-b.beta2a channels. The scrambled control peptide failed to affect alpha1C-b. beta2a channels, and the AID-peptide (10 microM) did not modify alpha1C-b channel function in the absence of expressed beta2a subunit. Our results demonstrate that the beta2a subunit controls fast gating of alpha1C-b channels, and suggest the alpha1-beta interaction domain in the cytoplasmic I-II linker of alpha1C (AID) as a possible target of modulation of the channel. Moreover, our data are consistent with a model of alpha1-beta interaction that is based on multiple interaction sites, including AID as a determinant of the affinity of the alpha1-beta interaction.

Animals↗
PubMed2000

Absence of the gamma subunit of the skeletal muscle dihydropyridine receptor increases L-type Ca2+ currents and alters channel inactivation properties.

In skeletal muscle the oligomeric alpha(1S), alpha(2)/delta-1 or alpha(2)/delta-2, beta1, and gamma1 L-type Ca(2+) channel or dihydropyridine receptor functions as a voltage sensor for excitation contraction coupling and is responsible for the L-type Ca(2+) current. The gamma1 subunit, which is tightly associated with this Ca(2+) channel, is a membrane-spanning protein exclusively expressed in skeletal muscle. Previously, heterologous expression studies revealed that gamma1 might modulate Ca(2+) currents expressed by the pore subunit found in heart, alpha(1C), shifting steady state inactivation, and increasing current amplitude. To determine the role of gamma1 assembled with the skeletal subunit composition in vivo, we used gene targeting to establish a mouse model, in which gamma1 expression is eliminated. Comparing litter-matched mice with control mice, we found that, in contrast to heterologous expression studies, the loss of gamma1 significantly increased the amplitude of peak dihydropyridine-sensitive I(Ca) in isolated myotubes. Whereas the activation kinetics of the current remained unchanged, inactivation of the current was slowed in gamma1-deficient myotubes and, correspondingly, steady state inactivation of I(Ca) was shifted to more positive membrane potentials. These results indicate that gamma1 decreases the amount of Ca(2+) entry during stimulation of skeletal muscle.

Animals↗
PubMed2000

Involvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases.

Large clostridial cytotoxins catalyze the glucosylation of Rho/Ras GTPases using UDP-glucose as a cosubstrate. By site-directed mutagenesis of Clostridium sordellii lethal toxin and Clostridium difficile toxin B fragments, we identified tryptophan 102, which is located in a conserved region within the catalytic domain of all clostridial cytotoxins, to be crucial for UDP-glucose binding. Exchange of Trp-102 with alanine decreased the glucosyltransferase activity by about 1,000-fold and blocked cytotoxic activity after microinjection. Replacement of Trp-102 by tyrosine caused a 100-fold reduction in enzyme activity, indicating a partial compensation of the tryptophan function by tyrosine. Decrease in glucosyltransferase and glycohydrolase activity was caused predominantly by an increase in the K(m) for UDP-glucose of these mutants. The data indicate that the conserved tryptophan residue is implicated in the binding of the cosubstrate UDP-glucose by large clostridial cytotoxins. Data bank searches revealed different groups of proteins sharing the recently identified DXD motif (Busch, C., Hofmann, F., Selzer, J., Munro, J., Jeckel, D., and Aktories, K. (1998) J. Biol. Chem. 273, 19566-19572) and a conserved region defined by a tryptophan residue equivalent to Trp-102 of C. sordellii lethal toxin. From our findings, we propose a novel family of glycosyltransferases which includes both prokaryotic and eukaryotic proteins.

Amino Acid Sequence↗
PubMed2000

Regulation of the calcium channel alpha(1G) subunit by divalent cations and organic blockers.

The pharmacological properties of the expressed murine T-type alpha(1G) channel were characterized using the whole cell patch clamp configuration. Ba(2+) or Ca(2+) were used as charge carriers. Both I(Ba) and I(Ca) were blocked by Ni(2+) and Cd(2+) with IC(50) values of 0.47+/-0.04 and 1.13+/-0.06 mM (Ni(2+)) and 162+/-13 and 658+/-23 microM (Cd(2+)), respectively. Ni(2+), but not Cd(2+), modified the gating of channel activation. Ni(2+) consistently accelerated channel deactivation while Cd(2+) had a similar effect only on I(Ca). The alpha(1G) channel was potently blocked by mibefradil in a dose- and voltage-dependent manner. I(Ba) was moderately blocked by phenytoin (IC(50) 73.9+/-1.9 microM) and was resistant to the block by valproate. Also 3 mM ethosuximide blocked 20 and 35% of the I(Ba) at a HP of -100 and -60 mV, respectively, while 5 mM amiloride inhibited I(Ba) by 38% and significantly slowed current activation. The alpha(1G) channel was not affected by 10 microM tetrodotoxin. Both 1 microM (+)isradipine and 10 microM nifedipine inhibited 18 and 14% of I(Ba) amplitude at a HP of -100 mV, and 23% and 29% of I(Ba) amplitude at a HP of -60 mV, respectively. The alpha(1G) current was minimally activated by 1 microM Bay K 8644.

Cadmium↗
PubMed2000

PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Angiogenesis Inhibitors↗
PubMed2000

Steady-state fully noninductive current driven by electron cyclotron waves in a magnetically confined plasma

A steady-state, fully noninductive plasma current has been sustained for the first time in a tokamak using electron cyclotron current drive only. In this discharge, 123 kA of current have been sustained for the entire gyrotron pulse duration of 2 s. Careful distribution across the plasma minor radius of the power deposited from three 0. 5-MW gyrotrons was essential for reaching steady-state conditions. With central current drive, up to 153 kA of current have been fully replaced transiently for 100 ms. The noninductive scenario is confirmed by the ability to recharge the Ohmic transformer. The dependence of the current drive efficiency on the minor radius is also demonstrated.

Journal Article↗
PubMed2000

A family of gamma-like calcium channel subunits.

The gamma subunit was initially identified as an auxiliary subunit of the skeletal muscle calcium channel complex. Evidence for the existence of further gamma subunits arose following the characterization of a genetic defect that induces epileptic seizures in stargazer mice. We present here the first account of a family of at least five putative gamma subunits that are predominantly expressed in brain. The gamma-2 and gamma-4 subunits shift the steady-state inactivation curve to more hyperpolarized potentials upon coexpression with the P/Q type alpha(1A) subunit. The coexpression of the gamma-5 subunit accelerates the time course of current activation and inactivation of the alpha(1G) T-type calcium channel.

Amino Acid Sequence↗
PubMed2000

Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta.

Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIbeta phosphorylated the IP3R and cGKIbeta, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIbeta, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIbeta in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.

Amino Acid Sequence↗
PubMed2000

Erectile dysfunction in cyclic GMP-dependent kinase I-deficient mice.

The generation of nitric oxide (NO) in penile erectile tissue and the subsequent elevation of cyclic GMP (cGMP) levels are important for normal penile erection. Current treatments of erectile dysfunction elevate either cGMP levels by blocking cGMP degrading phosphodiesterase 5 or cyclic AMP (cAMP) levels by intrapenile injection of prostaglandin E1. The molecular target or targets of cGMP in erectile tissue and the role of cAMP for normal penile erection are not known. Herein, we report that mice lacking cGMP-dependent kinase I (cGKI) have a very low ability to reproduce and that their corpora cavernosa fail to relax on activation of the NO/cGMP signaling cascade. Elevation of cAMP by forskolin, however, induces similar relaxation in normal and cGKI-null corpus cavernosum. In addition, sperm derived from cGKI-null mice is normal, can undergo acrosomal reactions, and can efficiently fertilize eggs. Altogether, these data identify cGKI as the downstream target of cGMP in erectile tissue and provide evidence that cAMP signaling cannot compensate for the absence of the cGMP/cGKI signaling cascade in vivo.

Animals↗
PubMed2000

Molecular cloning and functional characterization of a new modulatory cyclic nucleotide-gated channel subunit from mouse retina.

Cyclic nucleotide-gated (CNG) channels play a key role in olfactory and visual transduction. Native CNG channels are heteromeric complexes consisting of the principal alpha subunits (CNG1-3), which can form functional channels by themselves, and the modulatory beta subunits (CNG4-5). The individual alpha and beta subunits that combine to form the CNG channels in rod photoreceptors (CNG1 + CNG4) and olfactory neurons (CNG2 + CNG4 + CNG5) have been characterized. In contrast, only an alpha subunit (CNG3) has been identified so far in cone photoreceptors. Here we report the molecular cloning of a new CNG channel subunit (CNG6) from mouse retina. The cDNA of CNG6 encodes a peptide of 694 amino acids with a predicted molecular weight of 80 kDa. Among the CNG channel subunits, CNG6 has the highest overall similarity to the CNG4 beta subunit (47% sequence identity). CNG6 transcripts are present in a small subset of retinal photoreceptor cells and also in testis. Heterologous expression of CNG6 in human embryonic kidney 293 cells did not lead to detectable currents. However, when coexpressed with the cone photoreceptor alpha subunit, CNG6 induced a flickering channel gating, weakened the outward rectification in the presence of extracellular Ca(2+), increased the sensitivity for L-cis diltiazem, and enhanced the cAMP efficacy of the channel. Taken together, the data indicate that CNG6 represents a new CNG channel beta subunit that may associate with the CNG3 alpha subunit to form the native cone channel.

Amino Acid Sequence↗
PubMed2000

Temporally controlled somatic mutagenesis in smooth muscle.

Ligand-dependent site-specific recombinases are powerful tools to engineer the mouse genome in specific somatic cell types at selected times during pre- and postnatal development. Current efforts are primarily directed towards increasing the efficiency of this recombination system in mice. We have generated transgenic mouse lines expressing a tamoxifen-activated Cre recombinase, CreER(T2), under the control of the smooth muscle-specific SM22 promoter. Both a randomly integrated transgene [SM-CreER(T2)(tg)] and a transgene that has been "knocked in" into the endogenous SM22 locus [SM-CreER(T2)(ki)] were expressed in smooth muscle-containing tissues. The level of CreER(T2) expression and tamoxifen-induced recombination was lower in SM-CreER(T2)(tg) mice compared with SM-CreER(T2)(ki) mice. Whereas no recombinase activity could be detected in vehicle-treated SM-CreER(T2)(ki) mice, administration of tamoxifen induced the excision of a loxP-flanked reporter transgene in up to 100% of smooth muscle cells. The recombined genome persisted for at least four months after tamoxifen treatment. SM-CreER(T2)(ki) transgenic mice should be useful to study the effects of various somatic mutations in smooth muscle.

Animals↗
PubMed2000

[Transmission of HBV, HCV and HIV by infectious medical personnel--presentation of an overview].

This report analyzes 46 cases of personnel-to-patient transmissions of hepatitis B (HB), hepatitis C (HC) and HIV in health care settings. Similar circumstances were found for transmission of HB (40 cases, 404 infected patients), HC (4 cases, 224 infected patients) and HIV (2 cases, 7 infected patients). Cases with the highest number of transmissions (one anesthesiologist with 217 HC transmissions, and one EEG technologist with 75 HB transmissions) were attributed to poor infection control practices. As long as infected health care workers (HCW) adhere to general infection control measures, a risk for transmission to patients exists only from infected surgeons who perform 'exposure-prone invasive procedures'. Whether changes in duties of infected HCW are necessary should be decided on an individual basis. Often, the infected personnel were assumed to have acquired the disease occupationally. Medical practices and devices bearing a risk of infection should constantly be reviewed with regard to risk for patients and personnel.

Cross Infection↗