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Biomedical subjects

F Hofmann

Publications and source records attributed to F Hofmann.

At least 145 records · Page 8Linked to original sources
PubMed1997

Localization of the glucosyltransferase activity of Clostridium difficile toxin B to the N-terminal part of the holotoxin.

Clostridium difficile toxin B that is one of the largest cytotoxins (270 kDa) known acts on Rho subfamily proteins by monoglucosylation (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503). By deletion analysis we identified the enzyme and cytotoxic activity of the toxin to be located at the N terminus of the holotoxin. A 63-kDa fragment of toxin B covering the first 546 amino acid residues glucosylated Rho, Rac, and Cdc42, but not Ras, by using UDP-glucose as a cosubstrate. As known for the holotoxin, glucosylation by the toxin fragment was favored with the GDP-bound form of the low molecular mass GTPases. Microinjection of the toxin fragment into NIH-3T3 cells induced rounding up of cells and redistribution of the actin cytoskeleton. In contrast, a toxin fragment encompassing the first 516 amino acid residues was at least 1000-fold less active than toxin fragment 1-546 and cytotoxically inactive. The data give direct evidence for location of the enzyme activity of C. difficile toxin B at the N-terminal 546 amino acids residues and indicate a functionally and/or structurally important role of the region from amino acid residues 516 through 546 for enzyme and cytotoxic activities.

3T3 Cells↗
PubMed1997

Identification of the amino acid sequences responsible for high affinity activation of cGMP kinase Ialpha.

The cGMP-dependent protein kinases (cGK) Ialpha and Ibeta have identical cGMP binding sites and catalytic domains. However, differences in their first 100 amino acids result in 15-fold different activation constants for cGMP. We constructed chimeras to identify those amino acid sequences that contribute to the high affinity cGK Ialpha and low affinity cGK Ibeta phenotype. The cGK Ialpha/Ibeta chimeras contained permutations of six amino-terminal regions (S1-S6) including the leucine zipper (S2), the autoinhibitory domain (S4), and the hinge domain (S5, S6). The exchange of S2 along with S4 switched the phenotype from cGK Ialpha to cGK Ibeta and vice versa, suggesting that the domains with the highest homology between the two isozymes determine their affinity for cGMP. The high affinity cGK Ialpha phenotype was also obtained by a specific substitution within the hinge domain. Chimeras with the sequence of cGK Ialpha in S5 and cGK Ibeta in S6 were activated at up to 6-fold lower cGMP concentrations than cGK Ialpha. Based on the activation constants of all chimeras constructed, empirical weighting factors have been calculated that quantitatively describe the contribution of the individual amino-terminal domains S1-S6 to the high affinity cGK Ialpha phenotype.

Amino Acid Sequence↗
PubMed1997

[Diphtheria booster immunization for adults].

BACKGROUND AND OBJECTIVE: Nearly 50% of adults in Germany have no measurable antibody protection against diphtheria, even though most of them will have been vaccinated against it in their childhood. We investigated how vaccination should be performed in this situation to provide lasting protection. MATERIAL AND METHODS: 100 adults (53 men, 47 women; mean age 27.7 [19-54] years) whose last diphtheria vaccination had been at least 10 years previously, were injected up to three times with customary commercial monovalent diphtheria vaccine for adults (5 IU toxoid/0.5 ml), at intervals of 4-8 weeks. Each time, before and 4-8 weeks after the latest injection, the antitoxin level was determined by cell-culture neutralization test. RESULTS: Before the vaccination 30 of the 100 subjects had protection against diphtheria (antitoxin level > 0.1 IU/ml), 24 had "basic immunity" (0.01 to < 0.1 IU/ml), and 46 no measurable protection (< 0.01 IU/ml). After the first booster the protection threshold had not been reached in seven of the 100, all of them women. The main determinant for the pre- and post-vaccination antitoxin level was the interval since the last vaccination. In addition, women had significantly lower pre- and postvaccination levels than men. But after the second and third booster the antitoxin level of the seven women had risen to above the protection threshold, while the geometric mean of the whole collective had fallen, despite further boosters. It was 0.015 IU/ml before vaccination, 1.156 IU/ml after the first booster, 0.924 IU/ml after the second, and 0.952 after the third. CONCLUSION: Those adults who were last vaccinated against diphtheria more than 20 years ago should have two booster shots, more than 8 weeks apart, so that the highest possible and most lasting antitoxin level can be achieved. Women in particular should have at least two booster shots, as their antitoxin response tends to be less than that of men.

Adult↗
PubMed1997

Regional expression and cellular localization of the alpha1 and beta subunit of high voltage-activated calcium channels in rat brain.

The neuronal high voltage-activated calcium channels are a family of ion channels composed from up to five different alpha1 and four different beta subunits. The neuronal distribution and subunit composition of calcium channels were investigated using subunit-specific antibodies and riboprobes. The beta subunit-specific antibodies identified the presence of beta1a in skeletal muscle; beta2 in heart; and beta2, beta3, and beta4 in brain. The beta3 protein was widely distributed in rat brain, with prominent labeling of olfactory bulb, cortex, hippocampus, and habenula. The beta4 protein was also widely expressed, most prominently in the cerebellum. beta2 protein was expressed at only low levels. In situ hybridization with beta subunit-specific riboprobes confirmed the differential expression pattern of the individual subunits. Hybridization with riboprobes specific for the alpha1A, alpha1B, alpha1C, and alpha1D subunits showed a broad distribution of alpha1A and alpha1B transcripts, whereas the expression level of alpha1C and alpha1D mRNA was lower and more spatially restricted. The overall expression pattern and cellular localization suggested that beta4 may associate predominantly, but probably not exclusively, with the alpha1A subunit, and beta3 with the alpha1B subunit. In certain brain areas such as the habenula, the beta3 subunit may associate with other alpha1 subunits too. Furthermore, the beta2 subunit may form complexes with different alpha1 subunits in brain and cardiac muscle. These results demonstrate that a given beta subunit may associate with different alpha1 subunits in a cell type-dependent manner, contributing to the diversity of the neuronal calcium channels.

Animals↗
PubMed1997

ADP-ribosylating and glucosylating toxins as tools to study secretion in RBL cells.

The influence of different ADP-ribosylating and glucosylating cytotoxins on stimulated protein tyrosine phosphorylation and secretion in rat basophilic leukemia (RBL) cells was studied. Treatment of RBL cells with Clostridium botulinum C2 toxin, which specifically ADP-ribosylated monomeric G-actin and caused complete depolymerization of the actin cytoskeleton in intact cells, inhibited Fc epsilon RI receptor-mediated tyrosine phosphorylation of various proteins in a time- and concentration-dependent manner with maximal effects at 100 ng/ml C2I and 200 ng/ml C2I. C2 toxin (10 ng/ml C2I and 20 ng/ml C2II) increased antigen- or calcium ionophore (A23187)-stimulated [3H]serotonin release maximally by about 3 fold. Clostridium botulinum C3, which ADP-ribosylated Rho in intact RBL cells, had no effect on protein tyrosine phosphorylation and stimulated secretion. In contrast, the cytotoxic Clostridium difficile toxin B (ToxB), which glucosylated the Rho-subtype family members RhoA and Cdc42, blocked or reduced antigen- or calcium ionophore-mediated [3H]serotonin release, respectively, and decreased tyrosine phosphorylation of a 110 kDa protein. The data indicate that different actin pools control tyrosine phosphorylation and secretion in RBL cells and suggest that Rho subfamily proteins regulate secretion independently of the actin cytoskeleton.

ADP Ribose Transferases↗
PubMed1997

Identification and functional characterization of a calcium channel gamma subunit.

A positive selection technique was used to identify novel auxiliary calcium channel subunits that are similar to the skeletal muscle gamma subunit. A new rat gamma subunit cDNA was found, which was highly expressed in skeletal muscle tissue and was detected by RT-PCR in cardiac tissue. The 223-amino-acid-protein shares 84% and 79% identity, respectively, with the human and rabbit skeletal muscle subunits. Northern blot analysis revealed a single transcript of 1.5 kb in rat skeletal muscle, but not in cardiac tissue. Transient coexpression with the cardiac calcium channel complex demonstrated that the gamma subunit shifted the inactivation curve to negative potentials and accelerated current inactivation without changing other voltage-dependent properties of the channel.

Amino Acid Sequence↗
PubMed1997

Intracellular Ca2+ inactivates L-type Ca2+ channels with a Hill coefficient of approximately 1 and an inhibition constant of approximately 4 microM by reducing channel's open probability.

The patch-clamp technique was used to characterize the mechanism of Ca2+-induced inactivation of cardiac L-type Ca2+ channel alpha(1C-a) + beta3 subunits stably expressed in CHO cells. Single Ca2+ channel activity was monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)BAYK 8644 and calpastatin plus ATP. This enabled stabilization of channel activity in the inside-out patch and allowed for application of steady-state Ca2+ concentrations to the intracellular face of excised membrane patches in an attempt to provoke Ca2+-induced inactivation. Inactivation was found to occur specifically with Ca2+ since it was not observed upon application of Ba2+. Ca2+-dependent inhibition of mean Ca2+ channel activity was characterized by a Hill coefficient close to 1. Ca2+ binding to open and closed states of the channel obtained during depolarization apparently occurred with similar affinity yielding half-maximal inhibition of Ca2+ channel activity at approximately 4 microM. This inhibition manifested predominantly in a reduction of the channel's open probability whereas availability remained almost unchanged. The reduction in open probability was achieved by an increase in first latencies and a decrease in channel opening frequency as well as channel open times. At high (12-28 microM) Ca2+ concentrations, 72% of inhibition occurred due to a stabilization of the closed state and the remaining 28% by a destabilization of the open state. Our results suggest that binding of one calcium ion to a regulatory domain induces a complex alteration in the kinetic properties of the Ca2+ channel and support the idea of a single EF hand motif as the relevant Ca2+ binding site on the alpha1 subunit.

Animals↗
PubMed1997

Molecular cloning of cyclic nucleotide-gated cation channel subunits from rat pineal gland.

We have investigated the subunit composition and the molecular structure of the CNG channel expressed in rat pineal gland. Three types of subunits have been cloned: an alpha-subunit (CNG1), two beta-subunit splice variants (rCNG4.1 and rCNG4.2) and a hydrophilic glutamic acid-rich protein (rGARP). In situ hybridization with sections of rat brain revealed the co-expression of CNG1, CNG4 and GARP in pinealocytes. In addition, CNG4-specific transcripts were detected in the arcuate, periventricular and supraoptic nuclei of the hypothalamus.

Alternative Splicing↗
PubMed1997

Transfer of the high affinity dihydropyridine sensitivity from L-type To non-L-type calcium channel.

To elucidate the mechanism underlying the interaction between the L-type Ca2+ channel and the dihydropyridines (DHPs), contribution of the repeat III was studied by constructing chimeras between the DHP-sensitive alpha1C and DHP-insensitive alpha1E subunits. The chimeras were transiently expressed in human embryonic kidney 293 cells and the whole-cell Ba2+ current (IBa) was recorded. Mutating Thr1061 to Tyr in IIIS5 of the alpha1C sequence completely abolished the inhibition and stimulation of IBa by the antagonist (+)-isradipine and agonist (-)-Bay K 8644, whereas mutating Gln1065 to Met in IIIS5 decreased the affinity for isradipine 100-fold without affecting the stimulating effect of Bay K 8644. The conserved amino acid residue Tyr1174 in IIIS6 of the alpha1C subunit was necessary for the high affinity DHP block. The DHP-dependent block and stimulation of IBa were transferred to the alpha1E channel by the mutation of two amino acid residues in IIIS5 (Y1295T, M1299Q), three residues in IIIS6 (F1406I, F1409I, V1414M) and three residues in IVS6 (I1706Y, F1707M, L1714I). The mutated alpha1E channel was stimulated 2.8-fold by 1 microM Bay K 8644 and blocked by isradipine with an IC50 value of 60 nM. These results show that mutation of Thr1061 in the alpha1C sequence results in a DHP-insensitive L-type channel and that transfer of the high affinity DHP sensitivity requires mutation of eight amino acid residues in the alpha1E sequence.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1997

Alternatively spliced IS6 segments of the alpha 1C gene determine the tissue-specific dihydropyridine sensitivity of cardiac and vascular smooth muscle L-type Ca2+ channels.

Dihydropyridines (DHPs) block the vascular smooth muscle L-type Ca2+ channel at lower concentrations than the cardiac Ca2+ channel, although their alpha 1 subunit, which binds the DHPs, is derived from the same gene. This alpha 1C gene gives rise to several splice variants, among which the alpha 1C-b variant is affected by lower concentrations of nisoldipine than the alpha 1C-a variant. Functional expression of chimeras of alpha 1C-a and alpha 1C-b subunits demonstrated that the transmembrane segment IS6 is responsible for the different dihydropyridine sensitivity. Northern blot analysis showed that transcripts coding for the IS6 segment of the alpha 1C-a subunit were expressed in heart but not in aorta, whereas the IS6 segment of the alpha 1C-b subunit was expressed predominantly in vascular smooth muscle. In situ hybridization of rat heart sections confirmed this expression pattern of IS6 alpha 1C-a and IS6 alpha 1C-b in ventricular and smooth muscle myocytes, respectively. These results suggest that the different dihydropyridine sensitivities of cardiac and vascular L-type Ca2+ channels are caused at least partially by the tissue-specific expression of alternatively spliced IS6 segments of the alpha 1C gene.

Alternative Splicing↗
PubMed1997

[Non- and low-response after preventive hepatitis B vaccination].

20 years after the introduction of the hepatitis-B vaccine the question still remains unsolved as to how the 2 to 5 per cent of the vaccinees who, after basic immunisation, respond with no anti-HBs levels (0-10 IU/l nonresponder, NR) or low levels (11-99 IU/l low-responder, LR) should be boostered to acquire sufficient and long-lasting protective anti-HBs levels. In a retrospective analysis of booster results we found in 75 NR and LR that the probability for long-lasting protection is very low for NR, but better for LR (60%). In a second part of this publication, the efficacy of intradermal and intramuscular hepatitis-B boosters was examined by retrospective analysis of booster results among 26 NR and LR. A slight advantage of the intradermal application was found concerning post-booster anti-HBs increase and the maximal HBs values reached. Long-lasting protective anti-HBs values could not be established for either way of application. Finally, a survey of the literature summing up true results of intradermal hepatitis-B basic immunisations with reduced doses (mostly 2 micrograms), shows that whereas the seroconversion rates achieved by this method are almost compatible with those after regular basic vaccination, the GMT values were far lower than those after regular basic vaccination. Prospective and randomised studies are needed to show which kind of booster is most efficient for nonresponders and low-responders.

Adult↗
PubMed1997

[Spinal diseases in the nursing profession--a cross-sectional study].

A cross-sectional study on the profession-specific prevalence rates of back pain symptoms was conducted including N = 3332 nurses as the exposed group and N = 1720 office clerks as a control group. Only participants who had never changed from an exposed to an unexposed profession or vice versa, were included for the results presented here. Severity of symptoms was assessed, dividing into low back pain on the one hand, and lumbago-sciatica/sciatica on the other; frequency of pain episodes was measured using lifetime- and point-prevalence, in all these four combinations of severity and frequency the prevalence rates among nurses were clearly and significantly higher. Multivariate analyses revealed that profession is the most important risk factor among many others that were tested. Estimation of the prevalence rate for the heaviest symptom, the point-prevalence of lumbago-sciatica/sciatica, showed that about 4 out of 5 cases among nursing staff can be attributed to their profession. On the background of this high attributable risk the acceptance rate for LBP diseases of suspected occupational origin among nursing staff by statutory occupational health insurance bodies appears much too low.

Adult↗
PubMed1997

[Occupational medicine significance of hepatitis C in health care employees].

Transmission of Hepatitis C virus (HCV) is similar to the one observed with hepatitis B virus. The most important route of infection in health care personnel is by needlestick injury. In the course of a literature review 44 publications on HCV prevalence or incidence among hospital employees were studied and an overall transmission incidence of 2.2% was found. As none of the publications dealt with the problem of chronic Hepatitis C, 245 persons with elevated transaminases occupied Freiburg University Hospital were tested for HCV antibodies. 3 out of 82 non-medical professionals and 19 out of 163 health care workers were anti HCV positive (RR = 3.22; p < 0.05). Charwomen in medical departments, nurses and dentists had a higher relative risk than physicians and technical assistants. As no HCV vaccine is available the only way of HCV-prevention is compliance with universal precautions.

Adult↗
PubMed1997

[The family physician and his "patient with family". Significance of the family system in treatment of patients in general practice].

From a systemic perspective the general practitioner and the patient form a "medical-therapeutic system", which has the same features as other social systems. As the patient is also a part of his family system there are (more or less detectable) interactions between these two relevant systems as well. Compliance-from a systemic point of view-is not only an indication of the functioning of the "medical-therapeutic system" but also a result of those interactions between two relevant systems. Clinical trials show that with his interventions the general practitioner helps in solving problems of difficult, blocked family relationships with the goal of an adequate coping strategy for all involved family members.

Adult↗
PubMed1996

Intestinal secretory defects and dwarfism in mice lacking cGMP-dependent protein kinase II.

Cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinases (cGKs) mediate cellular signaling induced by nitric oxide and cGMP. Mice deficient in the type II cGK were resistant to Escherichia coli STa, an enterotoxin that stimulates cGMP accumulation and intestinal fluid secretion. The cGKII-deficient mice also developed dwarfism that was caused by a severe defect in endochondral ossification at the growth plates. These results indicate that cGKII plays a central role in diverse physiological processes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Difference in protein substrate specificity between hemorrhagic toxin and lethal toxin from Clostridium sordellii.

The hemorrhagic toxin (HT) from Clostridium sordellii is pharmacologically related to Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin which have been recently identified as mono-glucosyl-transferases. Here we report that HT, which is coexpressed with lethal toxin, is also a glucosyltransferase. Whereas lethal toxin glucosylates the Rho subfamily proteins Rac and Cdc42 and the Ras subfamily proteins H-Ras and Rap, the substrate specificity of HT is strictly confined to the Rho subfamily proteins Rho, Rac and Cdc42. Comparable to lethal toxin, transferase activity of HT is stimulated by Mn2+. Acceptor amino acid in Rho was identified by mutagenesis as threonine-37. C. sordellii HT is a novel member of the family of clostridial mono-glucosyl-transferases, a family which modifies the Rho and Ras GTPases.

3T3 Cells↗
PubMed1996

The retinoblastoma gene product protects E2F-1 from degradation by the ubiquitin-proteasome pathway.

E2F-1 plays a crucial role in the regulation of cell-cycle progression at the G1-S transition. In keeping with the fact that, when overproduced, it is both an oncoprotein and a potent inducer of apoptosis, its transcriptional activity is subject to multiple controls. Among them are binding by the retinoblastoma gene product (pRb), activation by cdk3, and S-phase-dependent down-regulation of DNA-binding capacity by cyclin A-dependent kinase. Here we report that E2F-1 is actively degraded by the ubiquitin-proteasome pathway. Efficient degradation depends on the availability of selected E2F-1 sequences. Unphosphorylated pRb stabilized E2F-1, protecting it from in vivo degradation. pRb-mediated stabilization was not an indirect consequence of G1 arrest, but rather depended on the ability of pRb to interact physically with E2F-1. Thus, in addition to binding E2F-1 and transforming it into a transcriptional repressor, pRb has another function, protection of E2F-1 from efficient degradation during a period when pRb/E2F complex formation is essential to regulating the cell cycle. In addition, there may be a specific mechanism for limiting free E2F-1 levels, failure of which could compromise cell survival and/or homeostasis.

Carrier Proteins↗