High-field NMR in pulsed magnets.
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Copper↗
Biomedical subjects
K H Müller
Publications and source records attributed to K H Müller.
Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1.
We have shown previously that the heavy metal-responsive transcriptional activator MTF-1 regulates the basal and heavy metal-induced expression of metallothioneins. To investigate the physiological function of MTF-1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF-1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF-1(-/-) embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy-chain subunit of the gamma-glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF-1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF-1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF-1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c-Jun and NF-kappaB (p65/RelA).
Animals↗
Conservation of msp, the gene encoding the major outer membrane protein of oral Treponema spp.
The major surface protein (Msp) of Treponema denticola has been implicated as a mediator of the interaction between the spirochete and the gingival epithelium in periodontal diseases. Previous studies showed that the Msp of T. denticola ATCC 35405 had porin activity, depolarized epithelial cell membranes, bound to extracellular matrix components of epithelial cells, and formed a regular hexagonal surface array in the treponemal outer membrane. The gene encoding Msp in ATCC 35405 was recently cloned, sequenced, and expressed in Escherichia coli (J. C. Fenno, K.-H. Müller, and B. C. McBride, J. Bacteriol. 178:2489-2496, 1996). In the present study, we identified genes encoding Msp-like proteins in several oral spirochetes. A prominent heat-modifiable Msp-like protein having an apparent molecular mass of between 43 and 64 kDa was present in all oral spirochete strains tested. Antibodies raised against the ATCC 35405 Msp reacted strongly with the Msp proteins of T. denticola ATCC 35404 and T. vincentii, reacted very weakly with the Msp protein of T. denticola ATCC 33520, and did not react with T. denticola OTK, T. socranskii, and T. pectinovorum. The msp loci of the T. denticola strains and T. vincentii were identified in analyses using PCR with oligonucleotide primers derived from the DNA sequence flanking msp in ATCC 35405. Southern blot analysis showed at least three groups of related msp DNA sequences. Comparison of DNA sequences of the 5' and 3' ends of the msp genes showed high sequence homology in the flanking regions and signal peptide coding regions, while the homologies between regions encoding the mature peptide were as low as 50%. The entire msp DNA sequences of T. denticola ATCC 33520 and OTK were determined, and the deduced Msp amino acid sequences were compared to the sequence of the previously reported Msp of ATCC 35405. The results show that the msp locus is conserved in oral treponemes but that there are significant differences between the mature Msp peptides of different strains. Further studies of the antigenic domains, functional domains, and physical structures of Msp proteins, based on these results, will enhance understanding of the role of Msp in the cytopathology associated with oral spirochetes.
Amino Acid Sequence↗
Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids.
Angiogenesis is important in the pathophysiology of endometriosis, a condition characterized by implantation of ectopic endometrium in the peritoneal cavity. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor involved in physiological and pathological angiogenesis, and elevated levels of VEGF are found in peritoneal fluid of patients with endometriosis. Our aim was to investigate the site of expression and regulation of VEGF in endometriosis. VEGF immunoreactivity was found in tissue macrophages present in ectopic endometrium and in activated peritoneal fluid macrophages. Macrophage activation was highest in women with endometriosis, and media conditioned by peritoneal fluid macrophages from these women caused a VEGF-dependent increase in endothelial cell proliferation above that seen from normal women. Peritoneal fluid macrophages secreted VEGF in response to ovarian steroids, and this secretion was enhanced after activation with lipopolysaccharide. Peritoneal fluid macrophages expressed receptors for steroid hormones. VEGF receptors flt and KDR (kinase domain receptor) were also detected, suggesting autocrine regulation. During the menstrual cycle, expression of flt was constant but that of KDR was increased in the luteal phase, at which time the cells migrated in response to VEGF. KDR expression and the migratory response were significantly higher in patients with endometriosis. This study demonstrates that activated macrophages are a major source of VEGF in endometriosis and that this expression is regulated directly by ovarian steroids.
Adult↗
Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola.
The gene encoding the major outer sheath protein (Msp) of the oral spirochete Treponema denticola ATCC 35405 was cloned, sequenced, and expressed in Escherichia coli. Preliminary sequence analysis showed that the 5' end of the msp gene was not present on the 5.5-kb cloned fragment described in a recent study (M. Haapasalo, K. H. Müller, V. J. Uitto, W. K. Leung, and B. C. McBride, Infect. Immun. 60:2058-2065,1992). The 5' end of msp was obtained by PCR amplification from a T. denticola genomic library, and an open reading frame of 1,629 bp was identified as the coding region for Msp by combining overlapping sequences. The deduced peptide consisted of 543 amino acids and had a molecular mass of 58,233 Da. The peptide had a typical prokaryotic signal sequence with a potential cleavage site for signal peptidase 1. Northern (RNA) blot analysis showing the msp transcript to be approximately 1.7 kb was consistent with the identification of a promoter consensus sequence located optimally upstream of msp and a transcription termination signal found downstream of the stop codon. The entire msp sequence was amplified from T. denticola genomic DNA and cloned in E. coli by using a tightly regulated T7 RNA polymerase vector system. Expression of Msp was toxic to E. coli when the entire msp gene was present. High levels of Msp were produced as inclusion bodies when the putative signal peptide sequence was deleted and replaced by a vector-encoded T7 peptide sequence. Recombinant Msp purified to homogeneity from a clone containing the full-length msp gene adhered to immobilized laminin and fibronectin but not to bovine serum albumin. Attachment of recombinant Msp was decreased in the presence of soluble substrate. Attachment of T. denticola to immobilized laminin and fibronectin was increased by pretreatment of the substrate with recombinant Msp. These studies lend further support to the hypothesis that Msp mediates the extracellular matrix binding activity of T. denticola.
Amino Acid Sequence↗
[Modified treatment concept in fractures of the humeral head in elderly patients].
Between 1990 and 1994, 94 patients over 65 years with subcapital humerus fractures were treated by plate osteosynthesis. All patients were operated on within 48 h after trauma. Intensive physical exercising was begun in the early postoperative period. Three months after the operation all osteosynthesis materials were removed with differentiated treatment of the subacromial area. If deemed necessary on the basis of the intraoperative findings (i.e. symptoms of impingement) treatment included a Neer acromioplasty. Sixty-nine of the 94 patients were followed up, including clinical, radiological and ultrasonographic examinations. Twenty-two patients showed an excellent, 26 a good, 9 a satisfactory and 12 an unsatisfactory result, i.e. in more than 82% of patients functional result was excellent to satisfactory. Therefore, we recommended plate osteosynthesis of subcapital humerus fractures in the elderly, in combination with our standard postoperative regimen.
Aged↗
[Results of a differentiated therapy concept for dislocated subcapital humeral fractures in elderly patients].
Between 1990 and 1994 94 patients over 65 years of age with subcapital humeral fractures were treated by plate osteosynthesis combined with a modified postoperative treatment including intensive physical exercise in the early postoperative period. Three months after surgery all osteosynthesis materials were removed using a differentiated treatment of the subacromial area. In the follow-up study 69 of the 94 patients were examined; 22 patients showed an excellent, 26 a good, 9 a satisfactory, and 12 an unsatisfactory result. Thus, we recommend plate osteosynthesis of subcapital humeral fractures in elderly people in combination with our standardized postoperative regimes.
Aged↗
Analysis of the heavy metal-responsive transcription factor MTF-1 from human and mouse.
Heavy metal-induced transcription in mammalian cells is conferred by the metal-responsive 70 kDa transcription factor MTF-1 which contains six zinc fingers and at least three activation domains. In previous cell transfection experiments we have shown that the zinc finger region confers an about 3 fold metal inducibility of transcription, due to its differential zinc binding. However, we also noted that human MTF-1 was more metal-responsive than the mouse factor (about 10 fold versus 3 fold, respectively). Here we analyze this difference in more detail by using chimeric human-mouse factors and narrow the critical region to a 64 amino acid stretch immediately downstream of the zinc fingers, overlapping with the acidic activation domain. A short human segment of this region (aa 313-377) confers efficient metal induction to the mouse MTF-1 when replacing the corresponding mouse region. However, high metal inducibility requires an unaltered MTF-1 and is lost when human MTF-1 is fused to the general activation domain of herpesvirus VP16. Wild type and truncation mutants of MTF-1 fused to VP16 yield chimeras of high transcriptional activity, some exceeding the wildtype regulator, but only limited (about 3 fold) heavy metal inducibility.
3T3 Cells↗
Applications of SNMS in archaeometry.
The recently developed High Frequency Mode HFM of electron gas SNMS allows investigations on insulating samples with the well known advantages of the SNMS Direct Bombardment Mode DBM for the analysis of conducting samples. HFM has been applied to analyses of different historic ceramic and glass samples in order to demonstrate the possibilities of SNMS in this field. It is shown that manufacturing places of ceramic samples could be distinguished by SNMS mass spectra ("fingerprints"). Furthermore questions of the constituents of colour remains on a painted ceramic ("Kaisermedaillon") could be answered by our SNMS analyses. SNMS investigations have been also applied to corrosion phenomena on different glass samples.
Journal Article↗
[Rupture of the Achilles tendon--case report of a rare form of Achilles tendon damage].
The great increase in the number of persons engaged in sports in the 30-50 years age group has led to a marked increase in injuries of the tendon of Achilles. In this case there is tearing loose of the Achilles tendon; the bone and the tendon come apart, as in a rupture of Sharpey's fibres. The tack was made in webbing the tendon of Achilles with polydioxanone ligaments to the tuberosity of calcaneus. The question about prophylactic webbing of the degenerated tendon of Achilles on the other side has to be discussed. Assessment of this injury from the viewpoint of the accident insurance company is explained.
Achilles Tendon↗
Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola.
A 53-kDa protein from the outer sheath of the oral spirochete Treponema denticola was purified to homogeneity and shown to reconstitute channels in black lipid bilayer model membranes. The channel had a single-channel conductance of 1.8 nS in 0.1 M KCl, making this the largest porin channel observed to date (estimated diameter, 3.4 nm). Electron micrographs of 53-kDa-protein-containing outer sheaths of T. denticola showed a regular hexagonal array of darker staining pits.
Antigens, Bacterial↗
Characterization of three fimbrial genes, sefABC, of Salmonella enteritidis.
Salmonella enteritidis produces thin, filamentous fimbriae designated SEF14. A 3.9-kb region of a 5.3-kb fragment encoding genes responsible for SEF14 biosynthesis was sequenced and found to contain three genes, sefABC. sefA encoded a novel fimbrin, the structural subunit of SEF14 fimbriae. sefB and sefC encoded proteins homologous to Escherichia coli and Klebsiella pneumoniae fimbrial periplasmic chaperone proteins and fimbrial outer membrane proteins, respectively, and are the first such genes to be characterized from Salmonella spp. in vitro expression directed by the 5.3-kb DNA fragment identified SefA, SefB, and SefC as approximately 14,000-, 28,000-, and 90,000-M(r) proteins, respectively, which correlated with their predicted amino acid sequences. sefB and sefC were not expressed in the absence of sefA. Primer extension analysis of sefABC revealed two major transcription start sites located upstream of sefA. Transcription of sefBC also initiated from the sefA promoter region. Secondary-structure analysis of the mRNA transcript for sefABC predicted the formation of two stable stem-loop structures in the intercistronic region between sefA and sefB indicative of differential regulation of SefA, SefB, and SefC translation. E. coli cells carrying the 5.3-kb DNA fragment of S. enteritidis DNA were unable to assemble distinguishable SEF14 fimbriae; however, immunogold-labelled SEF14 fimbriae were displayed on E. coli clones containing a 44-kb DNA fragment which encompassed the 5.3-kb region. Therefore, sefABC genes make up part of a complex sef operon responsible for the expression and assembly of SEF14 fimbriae.
Amino Acid Sequence↗
Characterization, cloning, and binding properties of the major 53-kilodalton Treponema denticola surface antigen.
Treponema denticola surface proteins were studied for their biochemical and biological characteristics. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of detergent extracts of whole cells revealed a major protein of 53 kDa and a number of minor proteins. Antiserum raised against whole cells of T. denticola ATCC 35405 reacted with the 53-kDa protein and a 72-kDa protein but not with the other proteins. Immunoelectron microscopy with anti-53-kDa-protein antibodies showed that the 53-kDa protein is located on the surface of the cell. SDS-PAGE analysis of unheated samples indicated that the 53-kDa protein is the major component of oligomers with molecular masses ranging from 130 to 300 kDa. Western blot (immunoblot) analysis showed that the high-molecular-mass oligomers reacted with whole-cell antiserum and anti-53-kDa-protein antibody. The aggregates dissociated into their subunits after heating to 70 degrees C. Isoelectric focusing followed by SDS-PAGE indicated that the 53-kDa protein was separated into several forms with apparent pI values ranging from 8.0 to 5.5. The oligomeric forms were highly resistant to proteolysis by trypsin and proteinase K, whereas the monomeric proteins were readily digested. A clone expressing a 53-kDa antigen in Escherichia coli was isolated from a lambda ZAP II DNA library of T. denticola ATCC 35405. The recombinant protein had exactly the same molecular mass as the major 53-kDa T. denticola surface protein and reacted with antisera raised against this protein. The role of T. denticola ATCC 35405 surface proteins in attachment to laminin, fibronectin, gelatin, fibrinogen, and bovine serum albumin (BSA) was studied by a modified Western blot binding assay. Fibronectin, laminin, and fibrinogen attached to the 53-kDa surface protein of T. denticola as well as to a 72-kDa protein, whereas no attachment to gelatin or BSA was observed. Attachment could be inhibited by pretreating the blots with fibrinogen but not with gelatin or BSA. Our results suggest that the 53-kDa major surface protein of T. denticola may play a role in the attachment to host proteins and may thus be an important virulence determinant of this species.
Animals↗
Type 1 fimbriae of Salmonella enteritidis.
Salmonella enteritidis was previously shown to produce fimbriae composed of 14,000-molecular-weight (Mr) fimbrin monomers (J. Feutrier, W. W. Kay, and T. J. Trust, J. Bacteriol. 168:221-227, 1986). Another distinct fimbrial structure, comprising 21,000-Mr fimbrin monomers, has now been identified. These fimbriae are simply designated as SEF 14 and SEF 21, respectively (for S. enteritidis fimbriae and the Mr [in thousands] of the fimbrin monomer). A simple method for the purification of both structures was developed by using the different biochemical properties of these fimbriae. SEF 21 remained intact after being boiled in sodium dodecyl sulfate but readily dissociated into subunits of 21,000 Mr at pH 2.2. The overall amino acid composition and the N-terminal amino acid sequence of the SEF 21 fimbrin were distinct from those of SEF 14 but were virtually identical to the predicted sequence for type 1 fimbrin of Salmonella typhimurium. Immunoelectron microscopy of S. enteritidis clearly revealed fimbrial structures that reacted with immune serum specific to the 21,000-Mr fimbrin. Immune sera raised against this subunit were cross-reactive with type 1 fimbrins found in whole-cell lysates of S. typhimurium, Salmonella illinois, and Salmonella cubana. However, there was no cross-reaction with Escherichia coli type 1 fimbriae or with other fimbrins produced by S. enteritidis. Under certain growth conditions, S. enteritidis produced both SEF 14 and SEF 21. However, when S. enteritidis was grown at 30 degrees C or lower, only the 21,000-Mr SEF 21 fimbrin could be detected. There was a direct correlation between mannose-sensitive hemagglutination and the presence of SEF 21.
Amino Acid Sequence↗
Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis.
Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.
Amino Acid Sequence↗
Fimbriation genes of Salmonella enteritidis.
From a cosmid clone, a 5.3-kilobase (kb) HindIII fragment of Salmonella enteritidis DNA containing the fimA gene was subcloned into bacteriophage T7 promoter vectors in both orientations. Predominantly mature fimbrin (14,000 Mr) was produced by clones containing the 5.3-kb insert, whereas the original cosmid clone predominantly accumulated a prefimbrin precursor (16,500 Mr). T7 RNA polymerase-dependent expression of the 5.3-kb insert and of a series of nested deletions revealed several membrane-localized polypeptides (80,000, 40,000, 29,000, 25,000, and 16,500 Mrs) transcribed in the same direction as fimA as well as a single polypeptide (9,000 Mr) transcribed in the opposite direction. Mini-Mu and TnphoA (Tn5 IS50L::phoA) transposon mutagenesis was used to identify a 2- to 3.5-kb region downstream of fimA that affected fimbrin production and processing. A more distant region (greater than 7 kb), revealed by Tn10 and Mu dI mutagenesis, was also required for fimbriation but did not hybridize with the 5.3-kb fragment. Yet another distant region did hybridize to the 5.3-kb fragment, suggesting the existence of other fimbriation-related genes.
Bacterial Proteins↗
[Urea in dermatology I].
In the main this first part of a review of the literature was already published 10 years ago. Here the importance of urea in the normal and diseased skin and its various dermopharmacologic properties is dealth with. For the time being the publications of the seventies are given special attention. In them particularly the utilization of the keratolytic and hydrating properties, i.e. water-binding capacity, of urea in the horny layer and also its significance in the treatment of hyperkeratosis and xerotic dermatosis without serious side effects is emphasized.
Animals↗
[Urea in dermatology II].
Publications from the last few years on urea in dermatology are discussed comprehensively as supplement to a review article which was published 10 years ago.
Clinical Trials as Topic↗