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Biomedical subjects

K H Schmidt

Publications and source records attributed to K H Schmidt.

At least 19 recordsLinked to original sources
PubMed2000

Genetic organisation of the M protein region in human isolates of group C and G streptococci: two types of multigene regulator-like (mgrC) regions.

In addition to beta-haemolytic streptococci belonging to Lancefield group A (Streptococcus pyogenes, GAS), human isolates of group C (GCS) and group G (GGS) streptococci (S. dysgalactiae subsp. equisimilis) have been implicated as causative agents in outbreaks of purulent pharyngitis, of wound infections and recently also of streptococcal toxic shock-like syndrome. Very little is known about the organisation of the genomic region in which the emm gene of GCS and GGS is located. We have investigated the genome sequences flanking the emm gene in GCS by sequencing neighbouring fragments obtained by inverse PCR. Our sequence data for GCS strains 25287 and H46A revealed two types of arrangement in the emm region, which differ significantly from the known types of mga regulon in GAS. We named this segment of the genome mgrC (for multigene regulon-like segment in group C streptococci). In strains belonging to the first mgrC type (prototype strain 25287) the emm gene is flanked up-stream by mgc, a gene that is 61% identical to the mga gene of GAS. A phylogenetic analysis of the deduced protein sequences showed that Mgc is related to Mga proteins of various types of GAS but forms a distinct cluster. Downstream of emm, the mgrC sequence region is bordered by rel. This gene encodes a protein that functions in the synthesis and degradation of guanosine 3',5' bipyrophosphate (ppGpp) during the stringent regulatory response to amino acid deprivation. In the second mgrC type (prototype strain H46A), the genes mgc and emm are arranged as in type 1. But an additional ORF (orf) is inserted in opposite orientation between emm and rel. This orf shows sequence homology to cpdB, which is present in various microorganisms and encodes 2',3' cyclo-nucleotide 2'-phosphodiesterase. PCR analysis showed that these two mgrC arrangements also exist in GGS. Our sequence and PCR data further showed that both types of mgrC region in GCS and GGS are linked via rel to the streptokinase region characterised recently in strain H46A. A gene encoding C5a peptidase, which is present at the 3' end of the mga regulon in GAS, was not found in the mgrC region identified in the GCS and GGS strains investigated here.

Amino Acid Sequence↗
PubMed2000

Two opposing effects of mismatch repair on CTG repeat instability in Escherichia coli.

The expansion of normally polymorphic CTG microsatellites in certain human genes has been identified as the causative mutation of a number of hereditary neurological disorders, including Huntington's disease and myotonic dystrophy. Here, we have investigated the effect of methyl-directed mismatch repair (MMR) on the stability of a (CTG)43 repeat in Escherichia coli over 140 generations and find two opposing effects. In contrast to orientation-dependent repeat instability in wild-type E. coli and yeast, we observed no orientation dependence in MMR- E. coli cells and suggest that, for the repeat that we have studied, orientation dependence in wild-type cells is mainly caused by functional mismatch repair genes. Our results imply that slipped structures are generated during replication, causing single triplet expansions and contractions in MMR- cells, because they are left unrepaired. On the other hand, we find that the repair of such slipped structures by the MMR system can go awry, resulting in large contractions. We show that these mutS-dependent contractions arise preferentially when the CTG sequence is encoded by the lagging strand. The nature of this orientation dependence argues that the small slipped structures that are recognized by the MMR system are formed primarily on the lagging strand of the replication fork. It also suggests that, in the presence of functional MMR, removal of 3 bp slipped structures causes the formation of larger contractions that are probably the result of secondary structure formation by the CTG sequence. We rationalize the opposing effects of MMR on repeat tract stability with a model that accounts for CTG repeat instability and loss of orientation dependence in MMR- cells. Our work resolves a contradiction between opposing claims in the literature of both stabilizing and destabilizing effects of MMR on CTG repeat instability in E. coli.

Adenosine Triphosphatases↗
PubMed2000

Palindromes as substrates for multiple pathways of recombination in Escherichia coli.

A 246-bp imperfect palindrome has the potential to form hairpin structures in single-stranded DNA during replication. Genetic evidence suggests that these structures are converted to double-strand breaks by the SbcCD nuclease and that the double-strand breaks are repaired by recombination. We investigated the role of a range of recombination mutations on the viability of cells containing this palindrome. The palindrome was introduced into the Escherichia coli chromosome by phage lambda lysogenization. This was done in both wt and sbcC backgrounds. Repair of the SbcCD-induced double-strand breaks requires a large number of proteins, including the components of both the RecB and RecF pathways. Repair does not involve PriA-dependent replication fork restart, which suggests that the double-strand break occurs after the replication fork has passed the palindrome. In the absence of SbcCD, recombination still occurs, probably using a gap substrate. This process is also PriA independent, suggesting that there is no collapse of the replication fork. In the absence of RecA, the RecQ helicase is required for palindrome viability in a sbcC mutant, suggesting that a helicase-dependent pathway exists to allow replicative bypass of secondary structures.

Bacterial Proteins↗
PubMed2000

Production of basic fibroblast growth factor and interleukin 6 by human smooth muscle cells following infection with Chlamydia pneumoniae.

Chlamydia pneumoniae infection has been associated with asthma and atherosclerosis. Smooth muscle cells represent host cells for chlamydiae during chronic infection. In this study we demonstrated that C. pneumoniae infection of human smooth muscle cells in vitro increased production of interleukin 6 (IL-6) and basic fibroblast growth factor (bFGF) as shown by reverse transcription-PCR, immunoblotting, and enzyme-linked immunosorbent assay. In contrast, levels of platelet-derived growth factor A-chain mRNA were not affected after infection. The stimulation of bFGF and IL-6 production was most effective when viable chlamydiae were used as inoculum. Furthermore, inhibition of bacterial protein synthesis with chloramphenicol prevented up-regulation of IL-6 and bFGF in infected cells. Addition of IL-6 antibody to infected cultures diminished bFGF expression, indicating involvement of produced IL-6. These findings suggest that chlamydial infection of smooth muscle cells elicits a cytokine response that may contribute to structural remodeling of the airway wall in chronic asthma and to fibrous plaque formation in atherosclerosis.

Arteriosclerosis↗
PubMed1999

Expression of interferon regulatory factors and indoleamine 2,3-dioxygenase in Chlamydia trachomatis-infected synovial fibroblasts.

Synovial fibroblasts probably represent host cells for Chlamydia trachomatis during initial intra-articular infection in reactive arthritis. In vitro synovial cells produce interferon-beta (IFN-beta) in response to chlamydial infection. IFN-beta expression can be activated by interferon regulatory factor-1 (IRF-1) and interferon-stimulated gene factor 3gamma (ISGF3gamma). In this study, we demonstrate that infection of synovial fibroblasts with C. trachomatis serotype D induced the expression of IRF-1 mRNA as shown by reverse transcription-PCR. Tumor necrosis factor-alpha (TNF-alpha) stimulation enhanced IRF-1 mRNA levels in infected cells and was required to detect IRF-1 protein by immunoblotting. The level of constitutively expressed IRF-2 was not significantly affected after infection. C. trachomatis was found to cause an up-regulation of ISGF3gamma protein in synovial cells. Induction of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is an important mechanism of the host cell response to control intracellular infection by chlamydiae. It has been described that IRF-1 can induce IDO gene expression. Infection of synovial fibroblasts alone in the absence of exogenous cytokine induced the expression of IDO mRNA which was enhanced by TNF-alpha treatment. The stimulation of IRF-1, ISGF3gamma, and IDO expression was most effective when viable chlamydiae were used as inoculum. Neutralization of IFN-beta in the culture medium of infected cells diminished but did not abrogate expression of IRF-1, ISGF3gamma, and IDO. The increased production of IRF-1 and ISGF3gamma in C. trachomatis-infected synovial fibroblasts may contribute to induction of IFN-beta and IDO.

Antibodies↗
PubMed1999

Febrile lady with acute renal failure and desquamating erythema.

A 63-year-old woman developed acute renal failure and streptococcal toxic shock syndrome caused by streptococcus group G. Initially, an erythema resembling vasculitis was misleading. The subsequent clinical course, however, was typical for streptococcal toxic shock syndrome and met the criteria put forward by The Working Group on Severe Streptococcal Infections. In patients infected with streptococcus group G, toxic shock syndrome is rare. The streptococcus group G strains isolated from this patient did not produce pyrogenic exotoxins. Instead they produced an M-like protein related to group C and G streptococci that do not act as superantigens.

Acute Kidney Injury↗
PubMed1999

Characterization of nra, a global negative regulator gene in group A streptococci.

During sequencing of an 11.5 kb genomic region of a serotype M49 group A streptococcal (GAS) strain, a series of genes were identified including nra(negative regulator of GAS). Transcriptional analysis of the region revealed that nra was primarily monocistronically transcribed. Polycistronic expression was found for the three open reading frames (ORFs) downstream and for the four ORFs upstream of nra. The deduced Nra protein sequence exhibited 62% homology to the GAS RofA positive regulator. In contrast to RofA, Nra was found to be a negative regulator of its own expression and that of the two adjacent operons by analysis of insertional inactivation mutants. By polymerase chain reaction and hybridization assays of 10 different GAS serotypes, the genomic presence of nra, rofA or both was demonstrated. Nra-regulated genes include the fibronectin-binding protein F2 gene (prtF2) and a novel collagen-binding protein (cpa). The Cpa polypeptide was purified as a recombinant maltose-binding protein fusion and shown to bind type I collagen but not fibronectin. In accordance with nra acting as a negative regulator of prtF2 and cpa, levels of attachment of the nra mutant strain to immobilized collagen and fibronectin was increased above wild-type levels. In addition, nra was also found to regulate negatively (four- to 16-fold) the global positive regulator gene, mga. Using a strain carrying a chromosomally integrated duplication of the nra 3' end and an nra-luciferase reporter gene transcriptional fusion, nra expression was observed to reach its maximum during late logarithmic growth phase, while no significant influence of atmospheric conditions could be distinguished clearly.

Adhesins, Bacterial↗
PubMed1999

Validation of a questionnaire for assessing physical work load.

OBJECTIVES: Reliable, valid, and compatible methods are required for exploring the complex interactive effects of psychosocial and physical stressors on complaints and disorders. An instrument for assessing physical work load that integrates information from a biomechanical model of lumbar load is presented and validated. METHODS: Four hundred and fifty-five people working in nursing homes for elderly people in Germany filled out the developed questionnaire 3 times within 1 year. Test-retest reliability was calculated, and validity was checked several times. Relationships with other, theoretically related and unrelated variables were examined. RESULTS: The test-retest reliability of the questionnaire measures was about 0.65. The convergent and discriminant validity was satisfactory, and the questionnaire was able to separate professional subgroups with different physical work loads. The Spearman rank-order correlations between physical load and musculoskeletal complaints were about 0.30. CONCLUSIONS: The method developed in this study is a reliable and valid instrument for assessing physical work load. The integration of statistical methods from psychological testing and theory in the development of methods exploring the effects of physical work load is advocated.

Adult↗
PubMed1998

Keratinocyte growth inhibition by streptococcal proteins.

M proteins are receptor proteins and one of the virulence factors of streptococci. M proteins seem to play a role in inflammatory skin disorders such as psoriasis. It is however unknown whether M proteins have a direct influence on proliferative activity of human keratinocytes. In the present study human HaCaT keratinocytes were exposed to M proteins (M1, M3, M5, M12) and the proliferative and proinflammatory response was analyzed. We found a dose-dependent inhibition of keratinocyte proliferation with crude extract of strain M3 4/55. Following affinity chromatography we found inhibitory activity for keratinocyte proliferation with a maximum of 80% at 10-8 M in the M protein. Additionally tested M1 protein preparation showed an inhibitory activity of 55% whereas other M preparations (5 and 12) did not show any effect. In supernatants from HaCaT cultures IL-1alpha, IL-1beta, IL-6, IL-8, TNFalpha and ICAM-1 were measured by ELISA. The levels of IL-8 were high and TNFalpha was upregulated, whereas ICAM-1 was decreased from around 20 ng/ml to almost zero. In contrast to the streptococcal-derived M3 protein preparation the recombinant M3 did not interfere with the proliferation of HaCaT cells. Because neither recombinant M3 protein nor M3 protein purified by ion exchange chromatography on a Q-resource column had any antiproliferative activity on keratinocytes we suggest, that a component different from M3 protein was responsible.

Antigens, Bacterial↗
PubMed1997

C repeats of the streptococcal M1 protein achieve the human serum albumin binding ability by flanking regions which stabilize the coiled-coil conformation.

The M and M-like proteins of Streptococcus pyogenes are fibrous cell surface proteins. They have multiple binding sites for several human proteins and are composed of the C-terminal anchor domain, the alpha-helical coiled-coil domain, and the N-terminal non-coiled-coil domain. The coiled-coil domain of the M1 protein consists of repeat units called B, C, and D and a spacer unit S between B and C. Recombinant fragments A-B-S-C-D, A-B-S, B-S-C, S-C, S-C-D, C-D, and C of the coiled-coil domain were studied by analyzing their secondary structures and binding affinities to human serum albumin (HSA). As shown by circular dichroism, all fragments are in an alpha-helical conformation. C-D and S-C-D form coiled coils at room temperature and bind below 37 degrees C with high affinity to HSA. C-D and S-C-D unfold in two steps with Tm values of approximately 31 and approximately 65 degrees C; complex formation with HSA increases the unfolding temperatures. B-S-C has a lower alpha-helical content, a less pronounced coiled-coil conformation, and a reduced thermal stability, binds HSA weaker, and is only slightly stabilized by HSA binding in comparison to C-D and S-C-D. C and S-C are less stable than the other fragments and are not organized as coiled coils showing some features of alpha-helical single strands only below 20 degrees C, and binding of HSA was not observed. The results indicate that the formation of coiled-coil structures, supported by flanking D regions and, to a lesser extent also B regions, is essential for the binding of C repeat units to HSA.

Antigens, Bacterial↗
PubMed1997

Inactivation of single genes within the virulence regulon of an M2 group A streptococcal isolate result in differences in virulence for chicken embryos and for mice.

An M2 streptococcal isolate and isogenic mutants in which either the emm or mrp gene was insertionally inactivated were tested for virulence using either a mouse model or a chicken embryo model. The results of the studies using the mouse model demonstrated that neither the emm nor mrp gene products had a significant effect on virulence when mice were challenged via the i.p. route. However, when the bacteria were injected into the skin the emm gene product was identified as a virulence factor. In parallel studies in the chicken embryo model the mrp gene product was found to be a major virulence factor, while a minor contribution to virulence could also be attributed to the emm gene product. The importance of these gene products to virulence was noted when the chicken embryo were injected either i.v or when the bacteria were placed on top of the chorioallantoic membrane. The direct comparison of a single wild type group A organism and its paired isogenic mutants in two animal models suggests that different combinations of bacterial factors are required to overcome host defense strategies associated with different animal species.

Animals↗
PubMed1996

Expression of both M protein and hyaluronic acid capsule by group A streptococcal strains results in a high virulence for chicken embryos.

The human pathogenic microorganism Streptococcus pyogenes can resist against phagocytic attack of human granulocytes. Streptococcal M protein and hyaluronic acid were identified as virulence factors involved in this protection. So far, no experiments have been reported which describe the contribution of both components together in one system. We used the chicken embryo as an in vivo phagocytosis model to investigate the role of both components on the virulence of streptococci. For this, isogeneic mutants of group A streptococcal strains (GAS) which lack hyaluronic acid capsule (cap-) or M protein (M-) expression were used for infection and their virulence was compared with laboratory strains which had lost their ability to produce one or both virulence factors after long-time laboratory passages on blood agar. The experiments revealed that strains producing both M protein and hyaluronic capsule were highly virulent. Only 1-10 colony-forming units were enough to cause a 50% lethality of 12-day-old chicken embryos. Those strains lacking one of these components showed a significant decrease in virulence. Finally, strains which failed to express either hyaluronic acid or M protein showed an additional tenfold decrease in virulence. This indicates a partial contribution of both M protein and hyaluronic acid to the virulence of GAS in the chicken embryo.

Animals↗
PubMed1996

What is the size of the group A streptococcal vir regulon? The Mga regulator affects expression of secreted and surface virulence factors.

The vir regulon of group A streptococci (GAS) organizes the expression of several bacterial virulence factors under the control of the Mga regulator. Previously, the genes encoding the Mga regulator (mga), M and M-related proteins (emm, mrp, enn) and C5a peptidase (scpA) were reported to be clustered on the streptococcal genome in a core vir regulon. In the present study, the genomic regions of a serotype M49 strain upstream of mga and downstream of scpA were sequenced to assess the boundaries of the vir regulon. In the upstream region, an operon was identified that may be potentially involved in substrate transport and is independent from Mga regulation. In the downstream region, another Mga-controlled, scpA-cotranscribed gene was detected. This gene termed orfX encoded a 385-amino acid (aa) potential surface protein of unknown function. No binding of serum proteins to a recombinant ORFX was detectable and phagocytosis resistance of an orfX mutant remained unchanged. Downstream of orfX, another Mga-independent gene determined the 3' end of the core vir regulon. Utilizing the M49 wild type, a mga- mutant and comparative Northern blot hybridization, genes encoding the capsule synthesis machinery, streptokinase and streptolysin O, as well as erythrogenic toxin A and DNase C were found to be Mga independent. In contrast, expression of the genes encoding the cysteine protease SpeB, streptococcin A and the oligopeptide permease was reduced in the mga- mutant. This indicated that in addition to the core vir regulon, Mga directly or indirectly controls a number of genes dispersed throughout the GAS genome.

Amino Acid Sequence↗
PubMed1996

Molecular characterization of group A streptococcal (GAS) oligopeptide permease (opp) and its effect on cysteine protease production.

Bacterial oligopeptide permeases are membrane-associated complexes of five proteins belonging to the ABC-transporter family, which have been found to be involved in obtaining nutrients, cell-wall metabolism, competence, and adherence to host cells. A lambda library of the strain CS101 group A streptococcal (GAS) genome was used to sequence 10,192 bp containing the five genes oppA to oppF of the GAS opp operon. The deduced amino acid sequences exhibited 50-84% homology to pneumococcal AmiA to AmiF sequences. The operon organization of the five genes was confirmed by transcriptional analysis and an additional shorter oppA transcript was detected. Insertional inactivation was used to create serotype M49 strains which did not express either the oppA gene or the ATPase genes, oppD and oppF. The mutation in oppA confirmed that the additional shorter oppA transcript originated from the opp operon and was probably due to an intra-operon transcription terminator site located downstream of oppA. While growth kinetics, binding of serum proteins, and attachment to eukaryotic cells were unaffected, the oppD/F mutants showed reduced production of the cysteine protease, SpeB, and a change in the pattern of secreted proteins. Thus, the GAS opp operon appears to contribute to both protease production and export/processing of secreted proteins.

Amino Acid Sequence↗
PubMed1996

Impregnation of collagen corneal shields with liposomes: uptake and release of hydrophilic and lipophilic marker substances.

PURPOSE: Liposomes and collagen corneal shields (CCS) have been used as ophthalmic drug delivery devices. With regard to a possibly combined application, we studied the effects of surface charge and bilayer fluidity of liposomes on their uptake and release by CCS. METHODS: 12-hours-CCS were soaked in large unilamellar liposomes, which had been labelled with 4,5-carboxyfluorescein (CF) and N-(lissamine rhodamine B sulfonyl)-diacyl-phosphatidylethanolamine (PE-RhB) in the aqueous space and in the liposome bilayer, respectively. Released fluorophores were determined fluorometrically in the elution buffer at intervals from 1 to 240 min after immersion. RESULTS: The CF concentration in the CCS soaked in a CF solution was two to seven times higher than immersion in the liposome suspensions. Among those, the negatively charged, cholesterol-containing preparation led to the highest CF concentration in the CCS. The PE-RhB concentration was highest after soaking the CCS in neutral, cholesterol-free liposomes. All types of liposomes were found inside the CCS by freeze fracture electron microscopy. The release kinetics data indicate a first order release. More than 90% of CF was released by the CCS within the first 30 min. This was equal after soaking the CCS in the CF solution or in liposomes. With DOPC-liposomes, the maximal release was already attained after 10 min. In general, the differences in the release kinetics of both hydrophilic and lipophilic markers, obtained by the various liposome types were small. CONCLUSIONS: Our results indicate that surface charge and bilayer fluidity are of minor importance for the interaction with collagen corneal shields. However, since the release kinetics of a liposome-encapsulated hydrophilic or lipophilic substance are similar to the release of a non-encapsulated drug, the combination of liposomes with collagen shields may be useful mainly with respect to the encapsulation of drugs which do not penetrate the ocular surface as well as to prolong corneal contact time of the liposomes.

Biological Dressings↗