Biomedical subjects

F Hofmann

Publications and source records attributed to F Hofmann.

At least 127 records · Page 7Linked to original sources

PubMed1998

Three amino acids in the C-linker are major determinants of gating in cyclic nucleotide-gated channels.

The activation of cyclic nucleotide-gated (CNG) channels is a complex process comprising the initial ligand binding and a consecutive allosteric transition from a closed to an open configuration. The cone and olfactory CNG channels differ considerably in cyclic nucleotide affinity and efficacy. In each channel, the cyclic nucleotide-binding site is connected to the last transmembrane segment of the channel by a linker peptide (C-linker) of approximately 90 amino acids. Here we report that replacement of three amino acids in the cone C-linker by the corresponding amino acids of the olfactory channel (I439V, D481A and D494S) profoundly enhanced the cAMP efficacy and increased the affinities for cAMP and cGMP. Unlike the wild-type cone channel, the mutated channel exhibited similar single-channel kinetics for both cGMP and cAMP, explaining the increase in cAMP efficacy. We thus conclude that the identified amino acids are major determinants of channel gating.

Amino Acid Sequence↗

PubMed1998

Clostridium difficile toxin B inhibits carbachol-induced force and myosin light chain phosphorylation in guinea-pig smooth muscle: role of Rho proteins.

1. Clostridium difficile toxin B glucosylates the Ras-related low molecular mass GTPases of the Rho subfamily thereby inactivating them. In the present report, toxin B was applied as a tool to test whether Rho proteins participate in the carbachol-induced increase in the Ca2+ sensitivity of force and myosin light chain (MLC) phosphorylation in intact intestinal smooth muscle. 2. Small strips of the longitudinal muscle of guinea-pig small intestine were incubated in toxin B (40 ng ml-1) overnight. Carbachol-induced force and intracellular [Ca2+], and, in a separate series, force and MLC phosphorylation, were determined. 3. Carbachol induced a biphasic contraction: an initial rapid increase in force (peak 1) followed by a partial relaxation and a second delayed increase in force (peak 2). The peak of the Ca2+ signal measured with fura-2 preceded peak 1 of force and then declined to a lower suprabasal steady-state level. Peak 2 was not associated with a significant increase in [Ca2+]. Toxin B nearly completely inhibited peak 2 while peak 1 was not significantly inhibited. Toxin B had no effect on the Ca2+ transient. 4. In control strips, MLC phosphorylation at peak 2 was 27.7% which was significantly higher than the resting value (18.6%). The inhibition of the second, delayed, rise in force induced by toxin B was associated with complete inhibition of the increase in MLC phosphorylation. The resting MLC phosphorylation was not significantly different from that of the control strips. 5. The initial increase in MLC phosphorylation determined 3 s after exposure to carbachol was 54% in the control strips. Toxin B also inhibited this initial phosphorylation peak despite the fact that the Ca2+ transient and the initial increase in force were not inhibited by toxin B. This suggests that Rho proteins play an important role in setting the balance between MLC phosphorylation and dephosphorylation reactions even at high levels of intracellular Ca2+. 6. These findings are consistent with the hypothesis that the delayed rise in force elicited by carbachol is due to an increase in the Ca2+ sensitivity of MLC phosphorylation mediated by Rho proteins.

Adenosine Diphosphate Ribose↗

PubMed1998

Inhibition of insulin-stimulated glucose transport in 3T3-L1 cells by Clostridium difficile toxin B, Clostridium sordellii lethal toxin, and Clostridium botulinum C2 toxin.

The role of the actin cytoskeleton and/or GTPases of the Rho/Rac-family in glucose transport regulation was investigated in 3T3-L1 cells with clostridial toxins which depolymerize actin by inactivation of Rho/Rac (Clostridium difficile toxin B and Clostiridium sordellii lethal toxin (LT)) or by direct ADP-ribosylation (Clostridium botulinum C2 toxin). Toxin B and C2 reduced insulin-stimulated, but not basal, 2-deoxyglucose (2-DOG) uptake rates in 3T3-L1 fibroblasts. In parallel, the toxins produced morphological alterations of the cells reflecting disruption of the actin cytoskeleton. Both toxins reduced the maximum response to insulin but failed to alter the half-maximally stimulating concentrations of insulin. In 3T3-L1 adipocytes, the lethal toxin reduced the effect of insulin on 2-DOG uptake, whereas toxin B and C2 failed to affect glucose transport or cell morphology. When cells were exposed to the toxins after treatment with insulin, both toxin B and the lethal toxin, in contrast to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, failed to reduce the 2-DOG uptake rates. Thus, both translocation to the plasma membrane and internalization of glucose transporters were inhibited by the toxins, whereas the PI 3-kinase inhibitor selectively affects translocation. The data suggest that the effects of the clostridial toxins on trafficking of glucose transporters are mediated by the depolymerization of the actin cytoskeleton and are an indirect consequence of Rho or Rac inactivation. It is suggested that pathways signalling through Rac or Rho may play a modulatory role in glucose transport regulation through their effects on the actin network.

3T3 Cells↗

PubMed1998

Cyclic nucleotide-gated channels--mediators of NO:cGMP-regulated processes.

Cyclic nucleotide-gated (CNG) channels represent one of the three known cellular receptor classes for cGMP. Activation of CNG channels by binding of cyclic nucleotides to a site in the C-terminus results in opening of the channel pore, entry of Ca2+ into the cell and subsequent induction of Ca2+-dependent processes. In this review we will summarize new data on the complex molecular structure and the activation mechanism of CNG channels. In addition, we will discuss the role of CNG channels as mediators of NO:cGMP-dependent cellular processes.

Animals↗

PubMed1998

Regulation of stably expressed and native BK channels from human myometrium by cGMP- and cAMP-dependent protein kinase.

The cloned BK channel alpha subunit from human myometrium was stably expressed in Chinese hamster ovary cells, either alone (CHOalpha cells) or in combination with the auxiliary beta subunit (CHOalpha+beta cells). We studied basic channel properties and the effects of cGMP- and cAMP-dependent protein kinases on the BK channel activity. Coexpression of alpha and beta subunits enhanced the Ca2+ and voltage sensitivity of the BK channel, and decreased the inhibitory potency of iberiotoxin. Blocking and stimulating effects on BK channel activity by charybdotoxin and nitric oxide, respectively, were independent of the beta subunit. The cGMP kinase Ialpha and cAMP kinase failed to affect BK channel activity in CHOalpha and CHOalpha+beta cells at different [Ca2+]i and voltages. In contrast, BK channels in freshly isolated myometrial cells from postmenopausal women responded to cAMP kinase and cGMP kinase with a fourfold and twofold decrease in their open probability (NPo), respectively. These effects could be reversed by alkaline phosphatase and remained unaffected by the phosphatase inhibitor okadaic acid (100 nM). In 28% of myometrial cells, however, cAMP and cGMP kinases increased NPo 2-fold and 3.5-fold, respectively. This stimulation was enhanced rather than reversed by alkaline phosphatase and was abolished by 100 nM okadaic acid. The results suggest that in stably transfected CHO cells the expressed BK channel is not regulated by cAMP kinase and cGMP kinase. However, in native myometrial cells stimulatory and inhibitory regulation of BK channels by cAMP kinase and cGMP kinase was observed, suggesting that channel regulation by the protein kinases requires factors that are not provided by CHO cells. Alternatively, failure of regulation may have been due to the primary structure of the myometrial BK channel protein used in this study.

Animals↗

PubMed1998

Factors influencing immunity against diphtheria in adults.

In about 50% of all adult Germans, serological immunity to diphtheria is lacking even though a vaccination history exists in most cases. In this study, the factors contributing to diphtheria immunity, up to 43 years after the last diphtheria vaccination, were analysed. Serum samples were taken from 287 adults whose complete vaccination history was available, and who had received a standard primary vaccination. The sera were tested by tissue culture neutralization assay for diphtheria antitoxin levels. The time lapse since the last diphtheria vaccination proved to be the most important factor influencing the protection rate. Secondly, the factor last vaccination within the first 3 years of life and, finally, female gender were found to correlate significantly with a low protection rate. No direct effect could be established for age, number of previous vaccinations, occupational contact with patients or travel within any endemic region during the last 10 years. Since diphtheritic infection can occur among previously vaccinated persons, the immunity gap observed among adults should be closed by regular diphtheria boosters. Special attention should be paid to the three groups mentioned. Further investigation is required to determine whether or not a single booster vaccination is sufficient to provide long-term protection for those in the risk groups indicated above.

Adolescent↗

PubMed1998

Activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor 1 increases intestinal permeability in Caco-2 cells.

The cytotoxic necrotizing factor 1 (CNF1) activates Rho GTPases by deamidation of glutamine-63 and thereby induces redistribution of the actin cytoskeleton and formation of stress fibers. Here, we have studied the effects of CNF1 on the transepithelial resistance of Caco-2 cells, a human intestinal epithelial cell line, in comparison with the Rho-inactivating toxin B of Clostridium difficile. Whereas toxin B decreased the transepithelial resistance of Caco-2 cells by about 80% after 4 h, CNF1 reduced it by about 40%. Significant changes of the transepithelial resistance induced by CNF1 were detected after 3 h of incubation. Half-maximal effects were observed with 10 and 41 ng of CNF1 and toxin B per ml, respectively. Flux measurement revealed no CNF1-induced increase of fluorescein isothiocyanate-dextran permeation within the first 4 h of incubation and a 2.9-fold increase after 24 h of incubation. In contrast, toxin B induced a 28-fold increase of permeation after 24 h. As detected by rhodamine-phalloidin staining, CNF1 increased polymerization of F actin at focal contacts of adjacent cells and induced formation of stress fibers. The data indicate that not only depolymerization but also polymerization of actin and subsequent reorganization of the actin cytoskeleton alter the barrier function of intestinal tight junctions.

Bacterial Toxins↗

PubMed1998

Chimeric clostridial cytotoxins: identification of the N-terminal region involved in protein substrate recognition.

Clostridium sordellii lethal toxin is a member of the family of large clostridial cytotoxins that glucosylate small GTPases. In contrast to Clostridium difficile toxins A and B, which exclusively modify Rho subfamily proteins, C. sordellii lethal toxin also glucosylates Ras subfamily proteins. By deletion analysis and construction of chimeric fusion proteins of C. sordellii lethal toxin and C. difficile toxin B, we localized the enzyme activity of the lethal toxin to the N terminus of the holotoxin and identified the region involved in protein substrate specificity. The toxin fragment of the N-terminal 546 amino acid residues of C. sordellii lethal toxin glucosylated Rho and Ras subfamily proteins, as the holotoxin did. Deletion of a further 30 amino acid residues from the C terminus of this active fragment drastically reduced glucotransferase activity and blocked glucohydrolase activity. Exchange of amino acid residues 364 through 516 of lethal toxin for those in the active toxin B fragment (1 to 546) allowed glucosylation of Ras subfamily proteins. In contrast, the chimera with amino acids 1 to 364 from toxin B, 365 to 468 from lethal toxin, and 469 to 546 from toxin B exhibited markedly reduced modification of Ras subfamily proteins, whereas modification of Rac and Cdc42 was hardly changed. The data indicate that the region of amino acid residues 364 through 516 primarily defines the substrate specificity of C. sordellii lethal toxin.

Bacterial Proteins↗

PubMed1998

The N-terminal part of the enzyme component (C2I) of the binary Clostridium botulinum C2 toxin interacts with the binding component C2II and functions as a carrier system for a Rho ADP-ribosylating C3-like fusion toxin.

The binary actin-ADP-ribosylating Clostridium botulinum C2 toxin consists of the enzyme component C2I and the binding component C2II, which are separate proteins. The active component C2I enters cells through C2II by receptor-mediated endocytosis and membrane translocation. The N-terminal part of C2I (C2IN), which consists of 225 amino acid residues but lacks ADP-ribosyltransferase activity, was identified as the C2II contact site. A fusion protein (C2IN-C3) of C2IN and the full-length C3-like ADP-ribosyltransferase from Clostridium limosum was constructed. The fusion protein C2IN-C3 ADP-ribosylated Rho but not actin in CHO cell lysates. Together with C2II, C2IN-C3 induced complete rounding up of CHO and HeLa cells after incubation for 3 h. No cell rounding was observed without C2II or with the original C3-like transferase from C. limosum. The data indicate that the N-terminal 225 amino acid residues of C2I are sufficient to cause the cellular uptake of C. limosum transferase via the binding component of C2II, thereby increasing the cytotoxicity of the C3-like exoenzyme several hundred-fold.

ADP Ribose Transferases↗

PubMed1998

Agents that inhibit Rho, Rac, and Cdc42 do not block formation of actin pedestals in HeLa cells infected with enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including Clostridium difficile toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.

Actins↗

PubMed1998

The structure of the murine calcium channel gamma-subunit gene and protein.

The gamma-subunit of voltage-gated calcium channels is a membrane protein that is associated with the skeletal muscle type of voltage gated calcium channels. Using a subunit-specific polyclonal antibody, the gamma-protein was detected in mouse skeletal muscle but not in brain, where at least five additional types of voltage-gated calcium channels are expressed. Murine genomic clones containing the full coding sequence of the gamma-subunit were isolated, the exons were mapped and sequenced. The murine gamma-subunit is encoded by a single copy gene containing 4 translated exons which are distributed over approximately 14 kilobases of DNA. The intron placement within the mouse gene correlates with the previously revealed organization of the human gamma-subunit gene, although the primary structures of the gamma-subunits are only moderately conserved between the murine, human, rat and rabbit proteins (75% identity).

Alternative Splicing↗

PubMed1998

Isradipine interacts with the open state of the L-type calcium channel at high concentrations.

Triple mutation of Tyr1485, Met1486 and Ile1493 in the IVS6 segment of alpha 1C-b subunit of the L-type calcium channel results in a loss of the high affinity inhibition by isradipine. The mutant channel (Ch30) yet exhibits a concentration-dependent inhibition by isradipine with a 110-fold lower affinity. The mechanisms underlying the remaining low affinity block were investigated. Isradipine accelerated the current decay in Ch30 but not in wild type channel in a concentration dependent manner. Dependence of the current amplitude inhibition on holding potential was parallel in Ch30 and in wild type channels, while the acceleration of current decay in Ch30 was independent of the membrane potential. The recovery from voltage-dependent inactivation was biphasic in both channels and was slowed down by isradipine in the wild type but not in the Ch30 channel. The change of the charge carrier (Ba2+ or Ca2+) and calcium chelator (EGTA or BAPTA) did not affect the acceleration of current decay indicating that isradipine did not interact with the Ca(2+)-inactivated state of the channel. These results demonstrate that the mutations of Ch30 affect selectively the high affinity inhibition of an inactivated channel and unmask a low affinity interaction of isradipine with an open state of the channel.

Barium↗

PubMed1998

[Tick-borne encephalitis transmitted by raw milk--what is the significance of this route of infection? Studies in the epidemic region of South-West Germany].

Despite the observation of TBE cases after consumption of raw milk from cows or goats, so far the proof of the alimentary route of human infection has not been possible. In the regions of southwestern Germany, where TBE is known to be endemic, milk-borne TBE infections have not yet been observed. To assess the significance of raw milk consumption for viral transmission, a cross-sectional study (114 forestry workers, 177 individuals exposed during their leisure-time activities, 170 non-exposed individuals) and a case-control study (50 TBE patients, 150 controls) were carried out. The results of the study show that both the time spent in the endemic region and also the professional exposure to ticks do influence TBE seroprevalence, whereas the consumption of raw milk (milk directly from the farmer) is no major risk factor for TBE infection or disease. Among leisure-time activities, only hunting has an effect comparable to the one of the professional exposure to ticks. Besides epidemiological data obtained in humans, serological investigations of cows were performed. Here it is shown that milk-producing animals are involved in the natural transmission of TBE virus. Since former studies had shown that TBE-viremic animals excrete the virus with the milk--although over a short period and in low concentrations--the occurrence of milk-borne TBE infections cannot be excluded in the endemic regions of south-western Germany. However, from the epidemiological point of view, their significance may be neglected. Vaccination has proven the most reliable means of TBE prevention, irrespective of the route of infection.

Animals↗

PubMed1998

[Current hepatitis B epidemiology in Germany].

Hepatitis B (HB) is the most common occupational hazard for health care workers. On the other hand 90% of the notified HB cases in Germany are observed among the general population (including other high-risk groups like i.v. drug abusers etc.). About 350 million persons all over the world are carriers of the hepatitis B virus (HBV). The availability of effective HB vaccines since 1982 made a control of HB possible. In this communication we report on epidemiological changes due to hepatitis B vaccination in a large university hospital in Germany. Health care workers and non-health care workers occupied at Freiburg university hospital were tested for HBV markers in 1984/85 (n = 4218), 1989/90 (n = 4081) and 1994/95 (n = 4022) during routine occupational health check-ups. Vaccinations were performed using a plasma-derived vaccine (1984/85) or a vaccine obtained by genetic engineering, respectively (1989/90, 1994/95). In 1984/85 prevalence of anti-HBs/HBc in German health care workers (12.4%) was 2.5 times higher than the one in non-health care workers (4.9%), in 1994/95 anti-HBs/HBc prevalence in both groups (4.4 vs. 4.5%) was comparable. On the other hand HBV carriage in persons occupied in professions without blood contact increased from 1984/85 (0.5%) to 1994/95 (1.1%). Therefore, the number of HBV carriers (ca. 1.1 millions) in Germany can be roughly estimated. Our data indicate a high degree of effectiveness of hepatitis B vaccines. Vaccination programme for the general population in accordance with German (STIKO) and WHO recommendations are necessary for the control of HBV infection.

Carrier State↗

PubMed1998

[Nosocomial hepatitis B virus, hepatitis C virus and HIV infections by infectious medial personnel].

Transmission of Hepatitis-B virus (HBV), Hepatitis-C virus (HCV) and Human immunodeficiency virus (HIV) from medical personnel to patients has been observed by many authors. In Germany, however, neither this type of nosocomial infection nor preventive measures have been discussed to date. This review deals with 302 cases documented in national and international journals (HBV 289, HCV 6, HIV 7). Methods of prevention (especially in surgery) are discussed.

Cross Infection↗

PubMed1997

[Occupational diseases of the spine in health care professions-- epidemiologic and insurance aspects (I). 1. Review of internationally publicized studies].

Due to spine-loading activities nursing professionals have a relatively high incidence/prevalence of low back pain. As lumbar disc disorders can be regarded as occupational diseases (since January 1993, occupational disease BK 2108, normally more than 10 years of exposition, lumbar disc disorders with pain and reduced motility) international literature is reviewed. Own results of different prevalence-, case-control- and incidence studies (relative risk for lumbago sciatica/odds ratio for disc prolaps appr. 3-6 depending on age and occupational age) are presented. Finally the question of compensation and prevention of disc disorders is discussed.

Female↗

PubMed1997

Evidence for differential roles of the Rho subfamily of GTP-binding proteins in glucose- and calcium-induced insulin secretion from pancreatic beta cells.

We utilized clostridial toxins (with known specificities for inhibition of GTPases) to ascertain the contribution of candidate GTPases in physiologic insulin secretion from beta cells. Exposure of normal rat islets or isolated beta (HIT-T15) cells to Clostridium difficile toxins A and B catalyzed the glucosylation (and thereby the inactivation) of Rac, Cdc42, and Rho endogenous to beta cells; concomitantly, either toxin reduced glucose- or potassium-induced insulin secretion from rat islets and HIT cells. Treatment of beta cells with Clostridium sordellii lethal toxin (LT; which modified only Ras, Rap, and Rac) also reduced glucose- or potassium-induced secretion. However, clostridial toxin C3-exoenzyme (which ADP-ribosylates and inactivates only Rho) was without any effect on either glucose- or potassium-induced insulin secretion. These data suggest that Cdc42, Rac, Ras, and/or Rap (but not Rho) may be needed for glucose- or potassium-mediated secretion. The effects of these toxins appear to be specific on stimulus-secretion coupling, since no difference in metabolic viability (assessed colorimetrically by quantitating the conversion of the tetrazolium salt into a formazan in a reduction reaction driven by nutrient metabolism) was demonstrable between control and toxin (A or LT)-treated beta cells. Toxin (A or LT) treatment also did not alter glucose- or potassium-mediated rises in cytosolic free calcium concentrations ([Ca2+]i), suggesting that these GTPases are involved in steps distal to elevations in [Ca2+]i. Recent findings indicate that the carboxyl methylation of Cdc42 is stimulated by only glucose, whereas that of Rap (Kowluru et al., J Clin Invest 98: 540-555, 1996) and Rac (present study) are regulated by glucose or potassium. Together, these findings provide direct evidence, for the first time, that the Rho subfamily of GTPases plays a key regulatory role(s) in insulin secretion, and they suggest that Cdc42 may be required for early steps in glucose stimulation of insulin release, whereas Rap and/or Rac may be required for a later step(s) in the stimulus-secretion coupling cascade (i.e. Ca2+-induced exocytosis of insulin).

ADP Ribose Transferases↗

PubMed1997

Essential role of the beta subunit in modulation of C-class L-type Ca2+ channels by intracellular pH.

Elevation of intracellular pH (pHi) enhances the activity of native L-type Ca2+ channels in cardiac and smooth muscle. We studied the modulation by pHi of expressed L-type Ca2+ channels comprised of either the alpha1c subunits alone or of alpha1c plus beta2a subunits. Ca2+ channels were expressed in human embryonic kidney cells (HEK 293) and pHi was increased from a basal level of 7.3 to 8.3 by exposure of cells to NH4Cl (20 mM) or by elevation of extracellular pH to 8.5. Elevation of pHi enhanced the activity of Ca2+ channels derived by coexpression of alpah1c and beta2a subunits. This alkalosis-induced stimulation of channel activity was mainly due to an increase in channel availability. Channels derived by expression of alpha1c alone were not affected by intracellular alkalosis. Our results demonstrate that the pHi sensitivity of L-type Ca2+ channels is conferred by the beta subunit of the channel complex.

Ammonium Chloride↗