Molecular mode of action of the large clostridial cytotoxins.
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Amino Acid Sequence↗
Biomedical subjects
F Hofmann
Publications and source records attributed to F Hofmann.
[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↗
[European and North American regulations on employing HBV-, HCV- and HIV-infected persons in health care].
In the United States of America (1991), in Canada (1998), in the UK (1993, 1994) and in Germany (1999) recommendations have been published concerning performance of "exposure-prone procedures" (EPP) by persons infected with Hepatitis-B virus (HBV), Hepatitis-C virus (HCV) or HIV. This review summarises the definition of EPP, methods to reduce the transmission of blood-borne pathogens, immunisation and screening policies, constitution of an advisory panel and the question of trace-back investigations described in these regulations.
Cross-Cultural Comparison↗
Effects of the dihydropyridine receptor subunits gamma and alpha2delta on the kinetics of heterologously expressed L-type Ca2+ channels.
Ba2+ currents through L-type Ca2+ channels were measured in tsA201 cells transiently transfected with expression vectors encoding the dihydropyridine (DHP) receptor subunits alpha1C, beta1a-GFP, alpha2delta and gamma. The subunit effect on channel function was studied by omitting either alpha2delta or gamma from the transfection mixture and analyzing the voltage dependence and kinetics of activation, inactivation and recovery from inactivation. Activation could be described by a single exponential function while the time course of inactivation of the Ba2+ current followed a double exponential function. Progressively longer depolarization led to increasingly slower recovery, indicating the successive occupancy of several inactive states. Activation parameters remained largely unaffected in y-deficient cells whereas the voltage dependence of inactivation was shifted by 16 mV to more positive potentials and the larger one of the two inactivation time constants was increased by one-third. On the other hand, alpha2delta-deficient cells showed decreased current density and slowed activation and inactivation. Recovery from inactivation was significantly slowed by gamma coexpression. This and the effect of the gamma subunit on steady-state inactivation were independent of the presence of alpha2delta. We conclude that y stabilizes L-type Ca2+ channel inactivation in a way similar to certain Ca(2+)-antagonistic drugs. Alpha2delta is not needed for this effect.
Barium↗
State- and isoform-dependent interaction of isradipine with the alpha1C L-type calcium channel.
We investigated the dihydropyridine (DHP) inhibition of barium current (I(Ba)) through the smooth muscle alpha1Ch and cardiac alpha1Ca splice variants of the L-type calcium channel using a whole-cell patch-clamp method. IC50 values for inhibition of current amplitude of the alpha1Cb channel were three to fivefold lower than for the alpha1Ca channel at holding potentials between -80 mV and -30 mV. No difference was found in either the transition of the channels into an inactivated state in the absence or presence of drug, or in the recovery from inactivation under control conditions. However, isradipine slowed the recovery from inactivation of alpha1Ca more effectively than alpha1Cb. To evaluate the interaction of isradipine with the open channel state of both splice variants, interactions with the inactivated state were selectively suppressed by the mutation of three amino acids in the IVS6 segment (Y1485I, M1486F, I1493L) in alpha1CbCh30 and alpha1CaCh30 channels. The extent of this interaction was seen by an acceleration of current decay. This was found to be identical for both splice variants. Our results suggest that the higher DHP selectivity of the alpha1Cb versus the alpha1Ca channel is caused by the structural difference in the binding site and not by different transitions between resting, open and inactivated states.
Alternative Splicing↗
Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization.
Retinal vasculogenesis and ischemic retinopathies provide good model systems for study of vascular development and neovascularization (NV), respectively. Vascular endothelial cell growth factor (VEGF) has been implicated in the pathogenesis of retinal vasculogenesis and in the development of retinal NV in ischemic retinopathies. However, insulin-like growth factor-I and possibly other growth factors also participate in the development of retinal NV and intraocular injections of VEGF antagonists only partially inhibit retinal NV. One possible conclusion from these studies is that it is necessary to block other growth factors in addition to VEGF to achieve complete inhibition of retinal NV. We recently demonstrated that a partially selective kinase inhibitor, PKC412, that blocks phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors and several isoforms of protein kinase C (PKC), completely inhibits retinal NV. In this study, we have used three additional selective kinase inhibitors with different selectivity profiles to explore the signaling pathways involved in retinal NV. PTK787, a drug that blocks phosphorylation by VEGF and PDGF receptors, but not PKC, completely inhibited retinal NV in murine oxygen-induced ischemic retinopathy and partially inhibited retinal vascularization during development. CGP 57148 and CGP 53716, two drugs that block phosphorylation by PDGF receptors, but not VEGF receptors, had no significant effect on retinal NV. These data and our previously published study suggest that regardless of contributions by other growth factors, VEGF signaling plays a critical role in the pathogenesis of retinal NV. Inhibition of VEGF receptor kinase activity completely blocks retinal NV and is an excellent target for treatment of proliferative diabetic retinopathy and other ischemic retinopathies.
Aging↗
Differential role of cyclic GMP-dependent protein kinase II in ion transport in murine small intestine and colon.
BACKGROUND & AIMS: The aim of this study was to determine the role of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (cGK) type II in intestinal fluid homeostasis under basal conditions and following exposure to cGMP-linked secretagogues, e.g., Escherichia coli heat-stable enterotoxin (STa) and guanylin. METHODS: Fluid and ion transport was determined in different segments of the intestine of wild-type and cGK II-deficient mice by ligated loop assays in vivo, and by short-circuit current and isotope flux measurements in vitro. RESULTS: Small intestinal fluid absorption in vivo was enhanced in cGK II-deficient mice under basal conditions and in the presence of STa. Furthermore, STa, guanylin, and 8-pCPT-cGMP stimulation of electrogenic anion secretion and inhibition of Na(+) absorption in vitro were markedly reduced in the small intestine from cGK II -/- mice but not in proximal colon. The type III phosphodiesterase inhibitor amrinone mimicked STa action in cGK II -/- mice, and also stimulated ion secretion in humans. CONCLUSIONS: This study shows that the cGMP/cGK II pathway regulates fluid homeostasis in the small intestine under basal conditions and mediates STa effects by both increasing anion secretion and inhibiting Na(+) absorption. It also demonstrates the presence of a cGK II-independent pathway for STa/cGMP-provoked secretion predominantly in the colon, which possibly involves a cGMP-inhibitable phosphodiesterase and/or activation of the cAMP-dependent protein kinase pathway.
Animals↗
Nomenclature of voltage-gated sodium channels.
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Animals↗
Nomenclature of voltage-gated calcium channels.
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Animals↗
Neuronal distribution and functional characterization of the calcium channel alpha2delta-2 subunit.
The auxiliary calcium channel alpha2delta subunit comprises a family of three genes, alpha2delta-1 to 3, which are expressed in a tissue-specific manner. alpha2delta-2 mRNA is found in the heart, skeletal muscle, brain, kidney, liver and pancreas. We report here for the first time the identification and functional characterization of alpha2delta-2 splice variants and their mRNA distribution in the mouse brain. The splice variants differ in the alpha2 and delta protein by eight and three amino acid residues, respectively, and are differentially expressed in cardiac tissue and human medullary thyroid carcinoma (hMTC) cells. In situ hybridization of mouse brain sections revealed the highest expression of alpha2delta-2 mRNA in the Purkinje cell layer of the cerebellum, habenulae and septal nuclei, and a lower expression in the cerebral cortex, olfactory bulb, thalamic and hypothalamic nuclei, as well as the inferior and superior colliculus. As the in situ data did not suggest a specific colocalization with any alpha1 subunit, coexpression studies of alpha2delta-2 were carried out either with the high-voltage-gated calcium channels, alpha1C, alpha1E or alpha1A, or with the low-voltage-gated calcium channel, alpha1G. Coexpression of alpha2delta-2 increased the current density, shifted the voltage dependence of channel activation and inactivation of alpha1C, alpha1E and alpha1A subunits in a hyperpolarizing direction, and accelerated the decay and shifted the steady-state inactivation of the alpha1G current.
Alternative Splicing↗
A role for the small molecular weight GTPases, Rho and Cdc42, in muscarinic receptor signaling to focal adhesion kinase.
An enhanced tyrosine phosphorylation of focal adhesion kinase (FAK) is elicited during neuronal growth cone remodeling and requires the maintenance of agonist-sensitive pools of phosphatidylinositol 4,5-bisphosphate (PIP2). Rho family GTPases are putative regulators of both PIP2 synthesis and growth cone remodeling, including neurite outgrowth elicited by muscarinic cholinergic receptor (mAChR) stimulation. In this study, we investigated the interrelationships among Rho family GTPases, PIP2 synthesis, and mAChR signaling to FAK in SH-SY5Y neuroblastoma cells. Preincubation with Clostridium difficile toxin B (Tox B), an inhibitor of Rho, Rac, and Cdc42, attenuated mAChR-stimulated FAK and paxillin tyrosine phosphorylation and lysophosphatidic acid (LPA)-induced FAK phosphorylation to a similar extent (75% decreases at 200 pg/ml Tox B) but did not affect mitogen-activated protein kinase activation elicited by either phorbol ester or an mAChR agonist. In contrast, preincubation with selective inhibitors of either Rho (C3 exoenzyme) or Rho kinase (HA-1 077) resulted in 80-90% reductions in LPA-induced FAK phosphorylation but only 40-50% decreases in mAChR-stimulated phosphorylation. Moreover, mAChR-mediated FAK phosphorylation was significantly attenuated in cells scrape-loaded with dominant-negative N17Cdc42 but not N17Rac1. Tox B had little or no effect on agonist-sensitive pools of PIP2 but inhibited mAChR-driven actin cytoskeletal remodeling. The results suggest that the Rho family GTPases, Rho and Cdc42, link mAChR stimulation to increases in FAK phosphorylation independently of effects on PIP2 synthesis.
Actins↗
Characterization of the catalytic domain of Clostridium novyi alpha-toxin.
Clostridium novyi alpha-toxin belongs to the family of large clostridial cytotoxins which act on cells through the modification of small GTP-binding proteins. We present here an analysis of the catalytic domain of alpha-toxin. A NH(2)-terminal 551-amino-acid fragment, alpha 551, was found to contain the full enzyme activity of the holotoxin, whereas a slightly shortened fragment encompassing 509 amino acids showed no detectable enzyme activity. Further characterization of the enzymatically active fragment alpha 551 revealed a substrate specificity for both UDP-N-acetylglucosamine and UDP-glucose. A Michaelis-Menten constant of 17 microM was determined for the substrate UDP-N-acetylglucosamine, while that for UDP-glucose was about 20 times higher, indicating a weaker affinity of the toxin for the latter substrate. Mutation of the aspartic acids of a conserved motif DXD within alpha 551 reduced enzyme activity >700-fold and inhibited cytotoxicity after microinjection in cells. Inhibition of enzyme activity of the DXD mutant could be partially overcome by increased concentrations of manganese ions, suggesting the involvement of these aspartic acids in Mn(2+) binding. By construction of chimeras of enzymatically active fragments of C. sordellii lethal toxin and C. novyi alpha-toxin, we located the region involved in nucleotide-sugar specificity to amino acids 133 through 517.
Bacterial Toxins↗
New method to generate enzymatically deficient Clostridium difficile toxin B as an antigen for immunization.
The family of the large clostridial cytotoxins, encompassing Clostridium difficile toxins A and B as well as the lethal and hemorrhagic toxins from Clostridium sordellii, monoglucosylate the Rho GTPases by transferring a glucose moiety from the cosubstrate UDP-glucose. Here we present a new detoxification procedure to block the enzyme activity by treatment with the reactive UDP-2', 3'-dialdehyde to result in alkylation of toxin A and B. Alkylation is likely to occur in the catalytic domain, because the native cosubstrate UDP-glucose completely protected the toxins from inactivation and the alkylated toxin competes with the native toxin at the cell receptor. Alkylated toxins are good antigens resulting in antibodies recognizing only the C-terminally located receptor binding domain, whereas formaldehyde treatment resulted in antibodies recognizing both the receptor binding domain and the catalytic domain, indicating that the catalytic domain is concealed under native conditions. Antibodies against the native catalytic domain (amino acids 1 through 546) and those holotoxin antibodies recognizing the catalytic domain inhibited enzyme activity. However, only antibodies against the receptor binding domain protected intact cells from the cytotoxic activity of toxin B, whereas antibodies against the catalytic domain were protective only when inside the cell.
Animals↗
Rising behind NO: cGMP-dependent protein kinases.
Over the past few years, a wealth of biochemical and functional data has been gathered on mammalian cGMP-dependent protein kinases (cGKs). In mammals, three different kinases are encoded by two genes. Mutant and chimeric cGMP kinase proteins generated by molecular biology techniques have yielded important biochemical knowledge, such as the function of the N-terminal domains of cGKI and cGKII, the identity of the cGMP-binding sites of cGKI, the substrate specificity of the enzymes and structural details of the catalytic center. Genetic approaches have proved to be especially useful for the analysis of the biological function of cGKs. Recently, some of the in vivo targets and mechanisms leading to smooth muscle relaxation have been identified. In vivo targets are the myosin-binding subunit of myosin phosphatase (PP1M), a member of the protein phosphatase 1, the calcium-activated maxi K(+) channel and a new protein named IRAG that forms a complex with the inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) receptor and cGKI. Phosphorylation of PP1M by cGKI(alpha) activates myosin phosphatase, whereas phosphorylation of IRAG by cGKI(beta) decreases Ins(1,4, 5)P(3)-induced calcium release. cGKII regulates in vivo intestinal fluid secretion by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), bone growth and renal renin secretion by phosphorylation of unknown proteins.
Animals↗
Low voltage activated calcium channels: from genes to function.
Cloning of three members of low-voltage-activated (LVA) calcium channel family, predominantly neuronal alpha1G and alpha1I, and ubiquitous alpha1H, enabled to investigate directly their electrophysiological and pharmacological profile as well as their putative subunit composition. All the three channels are half-activated at membrane potential about -40 mV and half-inactivated at about -70 mV. Kinetics of alpha1G and alpha1H channels activation and inactivation are similar and faster than that of alpha1I channel. All the three channels are blocked with high affinity by the organic blocker mibefradil. Another high affinity blocker is kurtoxin. Cloned LVA channels are relatively insensitive to antiepileptics, dihydropyridines and omega-conotoxins. Ni2+ is high affinity blocker of alpha1H channel only. Amiloride inhibits the alpha1H channel. The subunit composition of LVA channel remains unclear. Out of known high-voltage-activated calcium channel subunits, alpha2delta-2 and gamma-5 subunits significantly and systematically modified activation and/or inactivation of the current. In contrast, alpha2delta-1, alpha2delta-3, gamma-2 and gamma-4 subunits failed to modulate the current or had only minor effects.
Alternative Splicing↗
[Instructions after vaccination].
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Humans↗
Dihydropyridine enantiomers block recombinant L-type Ca2+ channels by two different mechanisms.
1. The molecular basis of the state-dependent block of L-type Ca2+ channels by dihydropyridines is still poorly understood. Therefore, we studied the enantioselectivity of Ca2+ channel block by isradipine enantiomers at three holding potentials (-80, -60 and -40 mV) in Chinese hamster ovary (CHO) cells stably expressing the rabbit lung alpha1C-b-subunit. 2. The extent of enantioselectivity did not markedly change with the holding potential (IC50 ratios of 104-138), whereas the potency of both isradipine enantiomers increased with depolarisation of the holding potential. 3. In addition to its block of the peak Ca2+ channel current, Ipeak, (-)-isradipine inhibited the relative current at the end of the test pulse, the so-called Ilate, normalised to Ipeak (Ilate/Ipeak). This effect was unaffected by the holding potential and revealed distinct kinetics compared to the development of conventional block of Ipeak. 4. When these effects were studied using an alpha1C-b-mutant lacking the high-affinity dihydropyridine binding site, expressed in human embryonic kidney (HEK 293) cells, both enantiomers blocked Ilate/Ipeak to a similar degree. 5. Our data are discussed within the framework of the 'guarded receptor' and the 'modulated receptor' hypotheses. The very different properties of the block of Ilate/Ipeak compared to those of the conventional high-affinity block of Ipeak suggest the existence of an additional mechanism possibly mediated via a second, distinct binding site.
Animals↗
Mechanisms and kinetics of procathepsin D activation.
In vitro, procathepsin D is activated to pseudocathepsin D by incubation at low pH. To investigate the mechanism of this activation, recombinant human procathepsin D and two mutants were generated in a baculovirus expression system. One mutant carried a point mutation within the catalytic domain, which resulted in a catalytically inactive enzyme form (D77A). The other carried a point mutation within the propeptide, which prevented activation by processing at the 'autoproteolysis-site' (L26P). Neither mutant is capable of processing itself to form pseudocathepsin D, and L26P is not able to process D77A. Despite the inability of L26P to cleave either its own or a wild-type prosequence, it did exhibit activity against a synthetic peptide substrate. The ability of intact precursor (zymogen) to cleave a peptide, but not a protein substrate, offers new insights into the mechanism of inhibition by the propeptide. Mature cathepsin D can process the inactive D77A mutant to the pseudoform, demonstrating that processed species are capable of cleaving zymogen molecules in an intermolecular interaction. In addition, kinetic studies provide evidence for a two-phase mechanism for the conversion of procathepsin D to pseudocathepsin D, one phase where the first molecules of pseudocathepsin D are formed at a low rate and a second phase where the process is autocatalytically accelerated by newly formed pseudocathepsin D molecules. Finally, with the help of the mutants L26P and D77A it was observed that at least two additional proteinase activities, found in conditioned media from insect cell culture, are capable of activating procathepsin D by cleaving it within the proregion. This observation suggests that there are likely to be multiple proteinases in the extracellular matrix that are capable of activating procathepsin D, thereby triggering the second autocatalytic phase. This may also be important for solid tumors, where the presence of cathepsin D has been correlated with tumor growth and invasion.
Amino Acid Sequence↗