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Background: This study was designed to explore the effect of transient inducible nitric oxide synthase (iNOS) overexpression via cationic liposome-mediated gene transfer on cardiac function, fibrosis, and microvascular perfusion in a porcine model of chronic ischemia., Methods and Results: Chronic myocardial ischemia was induced using a minimally invasive model in 23 landrace pigs. Upon demonstration of heart failure, 10 animals were treated with liposome-mediated iNOS-gene-transfer by local intramyocardial injection and 13 animals received a sham procedure to serve as control. The efficacy of this iNOS-gene-transfer was demonstrated for up to 7 days by reverse transcriptase-polymerase chain reaction in preliminary studies. Four weeks after iNOS transfer, magnetic resonance imaging showed no effect of iNOS overexpression on cardiac contractility at rest and during dobutamine stress (resting ejection fraction: control 27%, iNOS 26%; P = ns). Late enhancement, infarct size, and the amount of fibrosis were similar between groups. Although perfusion and perfusion reserve in response to adenosine and dobutamine were not significantly modified by iNOS-transfer, both vessel number and diameter were significantly increased in the ischemic area in the iNOS-treated group versus control (point score: control 15.3, iNOS 34.7; P < 0.05)., Conclusions: Our findings demonstrate that transient iNOS overexpression does not aggravate cardiac dysfunction or postischemic fibrosis, while potentially contributing to neovascularization in the chronically ischemic heart.

A trunk of human cytidylate-phosphate-deoxyguanylate-binding protein/CXXC finger protein 1 (CFP1), immobilized onto an aminohexyl-Sepharose column, can be used as a preanalytical tool for the selective enrichment of bacterial DNA from mixed solutions with high amounts of human background DNA for nucleic acid amplification technique-based detection of pathogens. The transcriptional activator protein exhibits a high affinity for nonmethylated CpG dinucleotide motifs, which are differentially distributed in prokaryotic and higher eukaryotic genomes. The feasibility of the affinity chromatography (AC) step was tested with DNA from severely septic patients. AC using 16S rRNA gene primers substantially increased PCR sensitivity. Approximately 90% of eukaryotic DNA was removed, which significantly increased the signal-to-noise ratio. Threshold cycle values revealed that sensitivity was elevated at least 10-fold. The change in the ratio of bacterial DNA to human DNA increased from 26% to 74% the likelihood of culture-independent PCR-based identification of bacterial presence. Compared to the results seen with blood culture (which is the clinical gold standard for systemic infections, exhibiting 28% positives), the combination of AC and PCR achieves a significant increase in sensitivity and contributes to shortening the time to results for the initiation of guided antibiotic therapy.

To further substantiate gestational age-related changes in oxalate excretion, we studied urinary oxalate excretion in 66 preterm infants born at 23.4-34.7 weeks of gestation. Spot urine of 66 preterm infants was analysed by ion chromatography as soon as they were completely orally fed with enriched breast milk and/or special preterm milk formula (days 7 to 57 of postnatal life). Infants with evidence of renal, gastrointestinal, muscular or metabolic disease were not included. Newborns on parenteral nutrition were excluded. Oxalate/creatinine ratios (Ox/Cr) decreased with gestational age (three age groups: group 1, 23 0/7-28 0/7; group 2, 28 1/7-32 0/7; and group 3, 32 1/7-35 0/7 weeks of gestation). The mean Ox/Cr was highest in group 1 (398.2 mmol/mol +/- 116.8; n = 21). Differences between groups 1 + 3 were statistically significant; p = 0.001; those between groups 1 + 2 and between groups 2 + 3 were not. Ox/Cr correlated inversely with gestational and maturational age (r = -0.41, p = 0.001; r = -0.33, p = 0.007) and positively with postnatal age (r = 0.32, p = 0.008). It correlated inversely with birth weight as well as actual weight at sample collection (r = -0.46 and -0.44, p < 0.001). Ox/Cr was significantly linked to energy and carbohydrate intake (r = 0.3 and 0.4, p = 0.03 and 0.001). These results were independent of sex. In the present study we show that urinary oxalate excretion in preterm infants depends on gestational age.

Chlamydia pneumoniae infection has been associated with chronic obstructive airway disease (COPD), asthma, and atherosclerosis. Inflammation and airway remodeling in asthma and COPD result in subepithelial fibrosis that is characterized by the deposition of interstitial collagens and fibronectin. The progression of atherosclerosis is also accompanied by an increased production of interstitial collagens in the intima. As shown by reverse transcription-PCR and immunoblotting, infection of human fibroblasts and smooth muscle cells by C. pneumoniae TW-183 downregulated the expression of type I and III collagen and fibronectin, whereas the level of type IV collagen remained unchanged. Conditioned medium from infected fibroblasts as well as epithelial WISH cells also reduced the expression of interstitial collagens and fibronectin in uninfected cells. In experiments using blocking antibodies, beta interferon was found to contribute to the inhibitory effects of conditioned medium collected from infected fibroblasts. In contrast, downregulation of matrix protein expression by conditioned medium from epithelial cells was caused by interleukin-1alpha, which was not secreted from fibroblasts following chlamydial infection. C. pneumoniae-mediated inhibition of collagen and fibronectin expression was diminished following transfection of fibroblasts with specific small interfering RNA targeting the transcription factor CCAAT/enhancer-binding protein beta. The downregulation of interstitial collagens and fibronectin by the Chlamydia-induced host cell cytokine response may modulate tissue remodeling processes in airway diseases. In atherosclerosis the inhibition of collagen synthesis by C. pneumoniae infection may promote plaque vulnerability, thereby increasing the risk of plaque rupture.

The influence of age and gender composition on group performance and self-reported health disorders was examined with data from 4,538 federal tax employees working in 222 natural work unit groups. As hypothesized, age diversity correlated positively with performance only in groups solving complex decision-making tasks, and this finding was replicated when analyzing performance data collected 1 year later. Age diversity was also positively correlated with health disorders--but only in groups working on routine decision-making tasks. Gender composition also had a significant effect on group performance, such that groups with a high proportion of female employees performed worse and reported more health disorders than did gender-diverse teams. As expected, effects of gender composition were most pronounced in large groups. Effects of age diversity were found when controlling for gender diversity and vice versa. Thus, age and gender diversity seem to play a unique role in performance and well-being. The moderating role of task complexity for both effects of age diversity and the moderating role of group size for both effects of gender diversity further suggest that the impact of these 2 variables depends on different group processes (e.g., knowledge exchange, variation in gender salience).

During the adaptive immune response, antigen challenge triggers a million-fold increase in mutation rates in the variable-region antibody genes. The frequency of mutation is causally and directly linked to transcription, which provides ssDNA and drives supercoiling that stabilizes secondary structures containing unpaired, intrinsically mutable bases. Simulation analysis of transcription in VH5 reveals a dominant 65nt secondary structure in the non-transcribed strand containing six sites of mutable ssDNA that have also been identified independently in human B cell lines and in primary mouse B cells. This dominant structure inter-converts briefly with less stable structures and is formed repeatedly during transcription, due to periodic pauses and backtracking. In effect, this creates a stable yet dynamic "mutability platform" consisting of ever-changing patterns of unpaired bases that are simultaneously exposed and therefore able to coordinate mutagenesis. Such a complex of secondary structures may be the source of ssDNA for enzyme-based diversification, which ultimately results in high affinity antibodies.

The role of secondary structures and base mutability at different levels of transcription and supercoiling is analyzed in variable region antibody genes VH5, VH94 and VH186.2. The data are consistent with a model of somatic hypermutation in which increasing levels of transcription and secondary structure stability correlate with the initial formation of successive mutable sites. Encoded differences exist in stem length and the number of GC pairs at low versus high levels of transcription in CDRs. These circumstances simplify the complexities of coordinating mutagenesis by confining this process to each mutable site successively, as they form in response to increasing levels of transcription during affinity maturation.

We report on a novel experimental approach for studying the dissipative spreading of collective motion in a metastable nuclear system, using, for the first time, highly fissile nuclei with spherical shape. This was achieved by fragmentation of 45 radioactive heavy-ion beams at GSI, Darmstadt. The use of inverse kinematics and a dedicated experimental setup allowed for the identification in atomic number of both fission fragments. From the width of their nuclear-charge distributions, a transient time of (3.3+/-0.7)x10(-21) s is deduced for initially spherical nuclei.

Four independent nonsense mutations were engineered into the Escherichia coli chromosomal lacZ gene, and reversion rates back to LacZ(+) phenotypes were determined. The mutation potential of bases within putative DNA secondary structures formed during transcription was predicted by a sliding-window analysis that simulates successive folding of the ssDNA creating these structures. The relative base mutabilities predicted by the mfg computer program correlated with experimentally determined reversion rates in three of the four mutants analysed. The nucleotide changes in revertants at one nonsense codon site consisted of a triple mutation, presumed to occur by a templated repair mechanism. Additionally, the effect of supercoiling on mutation was investigated and, in general, reversion rates increased with higher levels of negative supercoiling. Evidence indicates that predicted secondary structures are in fact formed in vivo and that directed mutation in response to starvation stress is dependent upon the exposure of particular bases, the stability of the structures in which these bases are unpaired and the level of DNA supercoiling within the cell.

The volcanic soils of southern Chile have demonstrated a high capacity to adsorb environmental pollutants, but for an industrial application, a stable solid material is necessary. The objective of this work was to produce a stable ceramic material through a process involving volcanic soil-polyurethane foam produced with recycled polyethylene terephthalate (PET)-polyols, and further thermal treatment. The selected foam formulation with 35.4% volcanic soil (< 63 microm) seems to be the most suitable for thermal treatment, with temperature steps at 700, 850, 1000 and 1200 degrees C. The porous ceramic material obtained has a stable solid form and an improved chlorophenols adsorption capacity (comparable to natural zeolites) that makes it suitable for advanced wastewater treatment and landfill leachate depuration.

There is compelling evidence that proliferating cell nuclear antigen (PCNA), a DNA sliding clamp, co-ordinates the processing and joining of Okazaki fragments during eukaryotic DNA replication. However, a detailed mechanistic understanding of functional PCNA:ligase I interactions has been incomplete. Here we present the co-crystal structure of yeast PCNA with a peptide encompassing the conserved PCNA interaction motif of Cdc9, yeast DNA ligase I. The Cdc9 peptide contacts both the inter-domain connector loop (IDCL) and residues near the C-terminus of PCNA. Complementary mutational and biochemical results demonstrate that these two interaction interfaces are required for complex formation both in the absence of DNA and when PCNA is topologically linked to DNA. Similar to the functionally homologous human proteins, yeast RFC interacts with and inhibits Cdc9 DNA ligase whereas the addition of PCNA alleviates inhibition by RFC. Here we show that the ability of PCNA to overcome RFC-mediated inhibition of Cdc9 is dependent upon both the IDCL and the C-terminal interaction interfaces of PCNA. Together these results demonstrate the functional significance of the beta-zipper structure formed between the C-terminal domain of PCNA and Cdc9 and reveal differences in the interactions of FEN-1 and Cdc9 with the two PCNA interfaces that may contribute to the co-ordinated, sequential action of these enzymes.

Molecular biological methods such as polymerase chain reaction (PCR) enable microbiologists to detect bacteria even if antibiotic treatment has already been started. Based on this case report of a 39-year-old man with multiple intracerebral lesions, we describe a PCR method called 16S rDNA-PCR which can be used to identify panbacterial DNA by focussing on the universal gene sequences for the bacterial 16S part of the ribosome.

Saccharomyces cerevisiae mutants lacking two of the three DNA helicases Sgs1, Srs2, and Rrm3 exhibit slow growth that is suppressed by disrupting homologous recombination. Cells lacking Sgs1 and Rrm3 accumulate gross-chromosomal rearrangements (GCRs) that are suppressed by the DNA damage checkpoint and by homologous recombination-defective mutations. In contrast, rrm3, srs2, and srs2 rrm3 mutants have wild-type GCR rates. GCR types in helicase double mutants include telomere additions, translocations, and broken DNAs healed by a complex process of hairpin-mediated inversion. Spontaneous activation of the Rad53 checkpoint kinase in the rrm3 mutant depends on the Mec3/Rad24 DNA damage sensors and results from activation of the Mec1/Rad9-dependent DNA damage response rather than the Mrc1-dependent replication stress response. Moreover, helicase double mutants accumulate Rad51-dependent Ddc2 foci, indicating the presence of recombination intermediates that are sensed by checkpoints. These findings demonstrate that different nonreplicative helicases function at the interface between replication and repair to maintain genome integrity.

Background/aims: The purpose of the study was to investigate the intracellular survival of Porphyromonas gingivalis as a possible mechanism for maintaining periodontitis., Methods: P. gingivalis strains, the strain ATCC 33277 and seven clinical isolates, were co-cultured with KB cells. The number of intracellular bacteria was determined up to 3 days after infection. In addition, the numbers of KB cells per well, the concentrations of the cytokines interleukin-1beta (IL-1beta), IL-6, IL-8 and tumour necrosis factor-alpha (TNF-alpha) and the arginine-specific amidolytic activity were measured. The 16S rRNA of P. gingivalis and the mRNA expression of IL-1beta, IL-6, IL-8, TNF-alpha and rgpA were also determined., Results: All the P. gingivalis strains studied were able to survive within KB cells. In contrast to the reduced values of colony-forming units at day 3, equal and higher levels of 16S rRNA were seen in comparison to day 0. Arginine-specific amidolytic activity declined in all samples during infection. Expression of mRNA for rgpA was not found after infection of KB cells by P. gingivalis strains. IL-8 was detectable in all samples 2 days after infection with P. gingivalis strains. Principal components analysis underlined a correlation between the arginine-specific amidolytic activity 1 h after infection and both the released IL-8 and the mRNA expression of IL-8. Associations were found between the cultivable numbers of intracellular P. gingivalis and the mRNAs of IL-1, IL-6 and TNF-alpha at the day of infection., Conclusion: The results indicate survival of P. gingivalis within epithelial cells, possibly in a non-cultivable stage. Invasion into cells modulates the virulence properties of P. gingivalis as well as the inflammatory response of the cells.

Sgs1 is a RecQ family DNA helicase required for genome stability in Saccharomyces cerevisiae whose human homologs BLM, WRN, and RECQL4 are mutated in Bloom's, Werner, and Rothmund Thomson syndromes, respectively. Sgs1 and mismatch repair (MMR) are inhibitors of recombination between similar but divergent (homeologous) DNA sequences. Here we show that SGS1, but not MMR, is critical for suppressing spontaneous, recurring translocations between diverged genes in cells with mutations in the genes encoding the checkpoint proteins Mec3, Rad24, Rad9, or Rfc5, the chromatin assembly factors Cac1 or Asf1, and the DNA helicase Rrm3. The S-phase checkpoint kinase and telomere maintenance factor Tel1, a homolog of the human ataxia telangiectasia (ATM) protein, prevents these translocations, whereas the checkpoint kinase Mec1, a homolog of the human ATM-related protein, and the Rad53 checkpoint kinase are not required. The translocation structures observed suggest involvement of a dicentric intermediate and break-induced replication with multiple cycles of DNA template switching.

Four mutations resulting in opal stop codons were individually engineered into a plasmid-borne chloramphenicol-resistance (cat) gene driven by the lac promoter. These four mutations were located at different sites in secondary structures. The mutations were analysed with the computer program mfg, which predicted their relative reversion frequencies. Reversion frequencies determined experimentally correlated with the mutability of the bases as predicted by mfg. To examine the effect of increased transcription on reversion frequencies, the lac promoter was replaced with the stronger tac promoter, which resulted in 12- to 30-fold increases in reversion rates. The effect of increased and decreased supercoiling was also investigated. The cat mutants had higher reversion rates in a topA mutant strain with increased negative supercoiling compared with wild-type levels, and the cat reversion rates were lower in a topA gyrB mutant strain with decreased negative supercoiling, as predicted.

Cells utilize numerous DNA metabolic pathways and cell-cycle checkpoints to maintain the integrity of their genome. Failure of these mechanisms can lead to genome instability, abnormal cell proliferation, and cell death. This chapter describes a method for the measurement of the rate of accumulating gross-chromosomal rearrangements (GCRs) in haploid cells of the yeast Saccharomyces cerevisiae. The isolation of cells with GCRs relies on the simultaneous loss of two counterselectable markers, CAN1 and URA3, within a nonessential region on the left arm of chromosome V. Healing of DNA breaks by de novo telomere addition, translocations, large interstitial deletions, and chromosome fusion has been detected using a PCR-based procedure for the mapping and amplification of breakpoint junctions, which is also described in detail here. This GCR analysis provides an effective tool for the assessment of the contribution by multiple cellular mechanisms to the maintenance of genome integrity.

We present an approach for quantitative analysis of changes in the composition and phosphorylation of protein complexes by MS. It is based on a new class of stable isotope-labeling reagent, the amine-reactive isotope tag (N-isotag), for specific and quantitative labeling of peptides following proteolytic digestion of proteins. Application of the N-isotag method to the analysis of Rad53, a DNA damage checkpoint kinase in Saccharomyces cerevisiae, led to the identification of dynamic associations between Rad53 and the nuclear transport machinery, histones, and chromatin assembly proteins in response to DNA damage. Over 30 phosphorylation sites of Rad53 and its associated proteins were identified and quantified, and they showed different changes in phosphorylation in response to DNA damage. Interestingly, Ser789 of Rad53 was found to be a major initial phosphorylation site, and its phosphorylation regulates the Rad53 abundance in response to DNA damage. Collectively, these results demonstrate that N-isotag-based quantitative MS is generally applicable to study dynamic changes in the composition of protein complexes and their phosphorylation patterns in a site-specific manner in response to different cell stimuli.

Aims: The dosage of fibrinolytic agents such as streptokinase must be controlled carefully to maximize therapeutic activity while avoiding adverse effects. Therefore, the integrity and activity of streptokinase products is likely to be clinically relevant. This study was conducted to compare the in vitro characteristics of different streptokinase preparations., Methods and Results: Sixteen streptokinase preparations (three of which were recombinant) were compared in a chromogenic substrate activity assay by native, and reducing, sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), and N-terminal sequencing. Deficiencies in streptokinase activity were observed in most of the products: only three fulfilled the minimum requirements of the European Pharmacopoeia. These were Icikinase (ICI Pharm Ltd, India, only one of two batches tested), Kabikinase (Pharmacia Upjohn, Sweden), and Streptase (Aventis Behring GmbH, Germany). The remaining products exhibited activities ranging from 20.8 to 86.6% of the label claim. Differences in composition and purity were demonstrated by both native and reducing SDS-PAGE. N-terminal sequencing of the recombinant preparations showed differences compared with the native protein--indeed, for one product, the 15 N-terminal amino acids bore no resemblance to streptokinase., Conclusion: There are wide variations in the activity, purity, and composition of the available streptokinase preparations.

From the albumin gland of the snail Cepaea hortensis we isolated and characterized a new N-acetyl-D-galactosamine/N-acetyl-D-glucosamine (GalNAc/GlcNAc) specific lectin (CHA-II) which was purified by a combination of affinity chromatography on GalNAc-agarose and gel filtration. The purified native lectin was found to be a multimeric protein, as revealed by SDS-PAGE and MALDI-TOF analysis. In SDS-PAGE the denatured and reduced lectin showed two bands of molecular masses with 17 and 15.5 kDa which reacted equally with anti-CHA-II rabbit antiserum. The lectin was O- and N-glycosylated with [(Gal)2-Man]2-Man-GlcNAc-GlcNAc-Asn as a probable structure for the oligosaccharide. Isoelectric focusing revealed a heterogeneous protein of at least four bands around pH 8.7. Tryptic peptides of CHA-II were N-terminally sequenced and highly degenerated gene specific oligonucleotide primers (GSPs) had been constructed. Using total RNA isolated from albumin glands, cDNAs were produced by the running race technique. Specific PCR fragments were obtained by PCR using GSPs, the universal primer and 5'- or 3'-RACE-cDNAs. The amplified fragments were cloned into the vector pDrive and were sequenced. The resulting total cDNA sequence consisted of 496 base pairs including an open reading frame of 360 base pairs which encoded a protein of 120 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to comprise 99 amino acid residues with a calculated molecular weight of 11,239 Da. The PCR fragment encoding the mature protein was cloned into the vector pQE30 and expressed in E. coli. Recombinant CHA-II lectin was produced as inclusion bodies and extracted by 6 M guanidine hydrochloride. After refolding, the recombinant CHA-II agglutinated specifically human red blood cells of groups A and AB. In immunodiffusion experiments using rabbit antiserum raised against the native lectin, the protein showed a precipitation line of identity with the native lectin.

Spallation residues and fission fragments from 1A GeV 238U projectiles irradiating a liquid hydrogen target were investigated by using the fragment separator at GSI for magnetic selection of reaction products including ray-tracing, energy-loss and time-of-flight techniques. The longitudinal-momentum spectra of identified fragments were analyzed, and evaporation residues and fission fragments could be separated. For 1385 nuclides, production cross sections down to values of 10 microb with a mean accuracy of 15%, velocities in the uranium rest frame and kinetic energies were determined. In the reaction all elements from uranium to nitrogen were found, each with a large number of isotopes.

A new experimental approach is introduced to investigate the relaxation of the nuclear deformation degrees of freedom. Highly excited fissioning systems with compact shapes and low angular momenta are produced in peripheral relativistic heavy-ion collisions. Both fission fragments are identified in atomic number. Fission cross sections and fission-fragment element distributions are determined as a function of the fissioning element. From the comparison of these new observables with a nuclear-reaction code a value for the transient time is deduced.

Objectives: The objective of the study was to evaluate the in vitro activity of ciprofloxacin, gatifloxacin and moxifloxacin against 16 Porphyromonas gingivalis strains., Methods: MICs of the quinolones were established by Etest and the agar dilution technique. Experiments focused on determination of the spontaneous mutation rate and the induction of resistant strains, using 0.25-fold MIC of antibiotic. Fragments of gyrA and gyrB as well as of parC were sequenced., Results: Moxifloxacin and gatifloxacin had very low MIC values. Subinhibitory concentrations of the fluoroquinolones rapidly induced mutations. The spontaneous mutation rate was strain- and quinolone-dependent; the lowest rate was encountered after moxifloxacin. The predicted serum concentrations of the quinolones were bactericidal to wild-type strains, but 100 mg/L of each tested quinolone was insufficient to kill a mutant exhibiting moderate resistance. Often the mutants exhibited high resistance to >/=32 mg/L. All these mutants bore a Ser-83-->Phe substitution in GyrA., Conclusions: DNA gyrase is the primary target of fluoroquinolones in P. gingivalis. In terms of the achievable level in the gingival fluid and the MICs, moxifloxacin might prevent the development of resistance and may be an alternative in the antibiotic treatment of P. gingivalis-associated periodontitis.

Chlamydia pneumoniae may modulate the proliferation of smooth muscle cells (SMC) in atherosclerotic plaques. Conditioned medium from C. pneumoniae-infected SMC decreased the proliferation of uninfected SMC. Treatment of infected cells with the cyclooxygenase-2 inhibitor NS-398 [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide] suppressed the up-regulation of prostaglandin E(2) (PGE(2)) and abolished the antimitogenic effect of conditioned medium, suggesting that C. pneumoniae can decrease SMC proliferation via stimulation of PGE(2) synthesis.

Escherichia coli auxotrophs of leuB and argH were examined to determine if higher rates of transcription in derepressed genes were correlated with increased reversion rates. Rates of leuB and argH mRNA synthesis were determined using half-lives and concentrations, during exponential growth and at several time points during 30 min of amino acid starvation. Changes in mRNA concentration were primarily due to increased mRNA synthesis and not to increased stability. Four strains of E. coli amino acid auxotrophs, isogenic except for relA and argR, were examined. In both the leuB and argH genes, rates of transcription and mutation were compared. In general, strains able to activate transcription with guanosine tetraphosphate (ppGpp) had higher rates of mRNA synthesis and mutation than those lacking ppGpp (relA2 mutants). argR knockout strains were constructed in relA(+) and relA mutant strains, and rates of both argH reversion and mRNA synthesis were significantly higher in the argR knockouts than in the regulated strains. A statistically significant linear correlation between increased rates of transcription and mutation was found for data from both genes. In general, changes in mRNA half-lives were less than threefold, whereas changes in rates of mRNA synthesis were often two orders of magnitude. The results suggest that specific starvation conditions target the biosynthetic genes for derepression and increased rates of transcription and mutation.

Highly degenerated gene-specific oligonucleotide primers (GSPs) were constructed from the amino acid sequence of tryptic fragments produced from the purified sialic acid-specific lectin of the garden snail Cepaea hortensis. From the albumin glands, the total RNA or the mRNA was prepared. Combination of a universal primer with the GSPs delivered gene-specific fragments of about 650, 620 and 280 bp by polymerase chain reaction (PCR). These fragments were cloned into the vector pDrive (Qiagen) and sequenced. The resulting cDNA sequence consisted of 744 bp, including an open reading frame of 480 bp. The encoded protein consists of 159 amino acids, including the putative signal sequence peptide. The mature protein should comprise 141 amino acid residues with a calculated molecular mass of 15,529 Da. The expression of the recombinant lectin in Escherichia coli resulted in a soluble protein reacting specifically with rabbit antiserum raised against the native lectin.

The Rrm3 DNA helicase of Saccharomyces cerevisiae interacts with proliferating cell nuclear antigen and is required for replication fork progression through ribosomal DNA repeats and subtelomeric and telomeric DNA. Here, we show that rrm3 srs2 and rrm3 sgs1 mutants, in which two different DNA helicases have been inactivated, exhibit a severe growth defect and undergo frequent cell death. Cells lacking Rrm3 and Srs2 arrest in the G(2)/M phase of the cell cycle with 2N DNA content and frequently contain only a single nucleus. The phenotypes of rrm3 srs2 and rrm3 sgs1 mutants were suppressed by disrupting early steps of homologous recombination. These observations identify Rrm3 as a new member of a network of pathways, involving Sgs1 and Srs2 helicases and Mus81 endonuclease, suggested to act during repair of stalled replication forks.

The neuropeptide galanin is known to have an antinociceptive effect under neuropathic conditions. After axotomy, galanin is upregulated in sensory neurons, presumably in the capsaicin-sensitive ones. Here, the sensitivity to capsaicin and the expression of galanin were simultaneously examined by double-staining in individual, dissociated rat dorsal root ganglion neurons (1) after axotomy of the sciatic nerve for up to 14 days and (2) in culture for up to 4 days without prior nerve injury. Ten days after axotomy, the proportion of capsaicin-sensitive neurons had decreased by 36 percentage points (from 63% to 27%), whereas the proportion of galaninergic neurons had increased by 33 percentage points (from 3% to 36%). These changes were also observed in neurons kept in culture, where the regulation was attenuated by the addition of nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) to the medium. After axotomy, galaninergic neurons had a soma size-distribution profile similar to the capsaicin-sensitive neurons, but there was no colocalization of capsaicin sensitivity and galanin expression in individual neurons. In culture, some neurons showed colocalization after 30 h and 48 h, but not after 6 h or 96 h. We conclude that the upregulation of galanin in an individual neuron is preceded by downregulation of its capsaicin sensitivity both in NGF-dependent peptidergic and in GDNF-dependent non-peptidergic neurons, indicating a change in phenotype.

A total of 305 Ixodes ricinus ticks collected from three areas of Thuringia in central Germany were investigated for infection with Borrelia burgdorferi sensu lato species and Anaplasma phagocytophila. Overall, 11.1% were infected with Borrelia burgdorferi and 2.3% with Anaplasma phagocytophila. Adult ticks showed a significantly higher rate of infection with both borreliae and Anaplasma phagocytophila. Borrelia garinii (55.9%) was detected most frequently, followed by Borrelia burgdorferi sensu stricto (32.4%), Borrelia afzelii (17.6%), and Borrelia valaisiana (5.9%). Four ticks had dual infection with Borrelia garinii and Borrelia burgdorferi sensu stricto. Two of the Borrelia-positive ticks were coinfected with Anaplasma phagocytophila.

Precise momentum distributions of identified projectile fragments, formed in the reactions 238U+Pb and 238U+Ti at 1A GeV, are measured with a high-resolution magnetic spectrometer. With increasing mass loss, the velocities first decrease as expected from previously established systematics, then level off, and finally increase again. Light fragments are on the average even faster than the projectiles. This finding is interpreted as the response of the spectators to the participant blast. The reacceleration of projectile spectators is sensitive to the nuclear mean field and provides a new tool for investigating the equation of state of nuclear matter.

This work provides evidence that, during transcription, the mutability (propensity to mutate) of a base in a DNA secondary structure depends both on the stability of the structure and on the extent to which the base is unpaired. Zuker's DNA folding computer program reveals the most probable stem-loop structures (SLSs) and negative energies of folding (-DeltaG) for any given nucleotide sequence. We developed an interfacing program that calculates (i) the percentage of folds in which each base is unpaired during transcription; and (ii) the mutability index (MI) for each base, expressed as an absolute value and defined as -follows: MI = (% total folds in which the base is unpaired) x (highest -DeltaG of all folds in which it is unpaired). Thus, MIs predict the relative mutation or reversion frequencies of unpaired bases in SLSs. MIs for 16 mutable bases in auxotrophs, selected during starvation in derepressed genes, are compared with 70 background mutations in lacI and ebgR that were not derepressed during mutant selection. All the results are consistent with the location of known mutable bases in SLSs. Specific conclusions are: (i) Of 16 mutable bases in transcribing genes, 87% have higher MIs than the average base of the sequence analysed, compared with 50% for the 70 background mutations. (ii) In 15 of the mutable bases of transcribing genes, the correlation between MIs and relative mutation frequencies determined experimentally is good. There is no correlation for 35 mutable bases in the lacI gene. (iii) In derepressed auxotrophs, 100% of the codons containing the mutable bases are within one codon's length of a stem, compared with 53% for the background mutable bases in lacI. (iv) The data suggest that environmental stressors may cause as well as select mutations in derepressed genes. The implications of these results for evolution are discussed.

Proliferating cell nuclear antigen (PCNA) plays an essential role in eukaryotic DNA replication, and numerous DNA replication proteins have been found to interact with PCNA through a conserved eight-amino acid motif called the PIP-box. We have searched the genome of the yeast Saccharomyces cerevisiae for open reading frames that encode proteins with putative PIP-boxes and initiated testing of 135 novel candidates for their ability to interact with PCNA-conjugated agarose beads. The first new PCNA-binding protein identified in this manner is the 5' to 3' DNA helicase RRM3. Yeast two-hybrid tests show that N-terminal deletions of RRM3, which remove the PIP-box but leave the helicase motifs intact, abolish the interaction with PCNA. In addition, mutating the two phenylalanine residues in the PIP-box to alanine or aspartic acid reduces binding to PCNA, confirming that the PIP-box in RRM3 is responsible for interaction with PCNA. The results presented here suggest that the RRM3 helicase functions at the replication fork.

Chlamydia trachomatis infection can cause reactive arthritis that is associated with the persistence of chlamydial organisms in the joint. Fibroblasts of the synovial membrane represent host cells for Chlamydia during articular infection. In this study we investigated the expression of HLA class I molecules in synovial fibroblasts following infection with C. trachomatis D. The expression of HLA class I heavy chain (HLA-I) was up-regulated in infected cultures as shown by reverse transcription-PCR and immunoblotting. The increase in cell surface expression of HLA-I and beta(2) microglobulin on infected fibroblasts was demonstrated by flow cytometric analysis. Suppression of enhanced production of interferon-stimulated gene factor 3gamma (ISGF3gamma) in infected cell cultures by antisense oligonucleotide treatment reduced the level of HLA-I. Blocking antibodies to beta interferon (IFN-beta) inhibited the Chlamydia-induced enhancement of both ISGF3gamma and HLA-I. These findings show that the up-regulation of HLA-I in synovial fibroblasts infected with C. trachomatis is caused by the induction of IFN-beta, which in turn stimulates the synthesis of ISGF3gamma, a transcription factor participating in the regulation of the HLA-I gene. The IFN-beta-mediated expression of HLA-I on Chlamydia-infected cells may be a regulatory factor in the immune response in chlamydial infections.

A DNA folding analysis indicates that the most hypermutable bases in exons 5, 7, and 8 of the p53 tumor suppressor gene are located immediately next to stems in stable DNA stem-loop structures. On the basis of the highest negative energy (-DeltaG) value of the structures containing each mutable bases and on the extent to which each base is unpaired during transcription, their relative mutabilities are calculated using a new computer algorithm. These predicted mutation frequencies correlate well with those observed in 14,000 human cancers (R(2) = 0.76), whereas there is no such correlation (R(2) = 0.0005) for nearby control bases. The correlation of hypermutable base frequencies with -DeltaG values is poor (R(2) = 0.19), indicating that the extent to which a base is unpaired during transcription is a significant contribution to predicting mutation frequencies.

Streptococcus pyogenes (GAS) causes about 90% of streptococcal human infections while group C (GCS) and G (GGS) streptococci can be pathogenic for different mammalians. Especially the human pathogenic GCS and GGS, Streptococcus dysgalactiae, subsp. equisimilis, account for 5-8% of the human streptococcal diseases like wound infections, otitis media, purulent pharyngitis and also streptococcal toxic shock syndrome. A defined superantigen so far was not identified in GCS and GGS strains. In the present investigation we screened DNA of GCS and GGS human isolates for the presence of genes for streptococcal pyrogenic exotoxins (spe) by hybridisation with probes that stand for the GAS genes speA, speC, speZ (smeZ), speH, speG, speI, speJ and ssa. In many GCS and GGS strains we found positive reactions with the probes speG, speJ and ssa, but not with the probes for the remaining genes under investigation. PCR amplification with subsequent sequence analysis of the PCR fragments revealed only the presence of the gene speG in GCS and GGS strains, while no DNA fragments specific for speJ and ssa could be amplified. Additionally, the upstream and downstream regions flanking speG in GGS strain 39072 were sequenced. Remarkable differences were found in the neighbourhood of speG between GAS and GGS sequences. Downstream of speG we identified in strain GGS 39072 two new open reading frames encoding proteins with no similarity to protein sequences accessible in the databases so far. In the compared GAS strains SF370 and MGAS8232, this segment, apart from some small fragments, had been deleted. Our analysis suggests that a gene transfer from GGS to GAS has preceded following deletion of the two genes orf1 and orf2 in GAS.

A sialic acid-specific lectin was isolated from the albumin glands of the garden snail Cepaea hortensis by affinity chromatography on fetuin-Sepharose following gel filtration on Superdex 200. The purified native lectin showed a molecular mass of about 95 kDa by gel filtration and 100 kDa by SDS electrophoresis. It was cleaved by boiling in buffer containing SDS in three serological identical bands corresponding to molecular masses of about 24, 20 and 16 kDa, respectively. From these three fragments, only the 24- and the 20-kDa bands were found to be glycosylated. Only the three sugars mannose, galactose and N-acetylglucosamine could be detected in a molar ratio of 3:8.6:2. The oligosaccharide moieties seem to be N- and partially O-glycosidic bound. Isoelectric focusing (IEF) of the purified lectin revealed a heterogeneous pattern with bands in the pH range of 4.3-5.0. Isolated bands of different isoelectric points showed in SDS electrophoresis the same three fragments with molecular masses of 24, 20 or 16 kDa. The heterogeneity of the lectin was revealed either by IEF or amino acid sequencing of internal tryptic peptides.

A total of 305 Ixodes ricinus ticks (243 nymphs and 62 adults) were collected from three different regions of Thuringia in Middle Germany which are known to be endemic for Borrelia burgdorferi. Our aim was to investigate the carrier rate of ticks for granulocytic Ehrlichia species. The presence of ehrlichial 16S ribosomal DNA was investigated by polymerase chain reaction. Using primers specific for the Ehrlichia phagocytophila group PCR fragments of 151 bp and 943 bp, respectively, were produced in positive samples. Adult ticks showed a significantly higher infection rate (4/62; 6.5%) compared to nymphs (3/243; 1.2%). Prevalence rates varied between 0 and 3.8% regarding the different areas under investigation. The nucleotide sequences showed high similarity (between 97.5% and 99% identity) to the known sequences of the three E. phagocytophila group members HGE agent, E. phagocytophila and Ehrlichia equi. The sequence data did not allow a final classification to a particular member of this group.

The mitogenic factor (MF) of group A streptococci has been reported to be a superantigen stimulating human T cells carrying Vbeta2, 4 and 8 and has been designated streptococcal pyrogenic exotoxin F (SPEF). MF was also shown to possess DNase activity. Here we have purified MF from culture supernatants of different Streptococcus pyogenes strains. Surprisingly, the MF preparations from different strains showed different Vbeta specificities depending on the expression of SPEC or SMEZ3 by the producing strain. Their mitogenic activity decreased upon further purification. In addition, the mitogenic activity could be only neutralized by antibodies against the basic streptococcal superantigens SPEC or SPEX (SMEZ3) but not by antibodies against MF itself although the latter were able to neutralize completely the DNase activity of MF. We found that streptodornase type B (SDB) was expressed in two molecular forms (SDBI and SDBII), differing only by one additional N-terminal arginine at SDBI. MF was found identical to the enzyme SDBII but is devoid of superantigenic properties and should no longer be called a superantigen or a pyrogenic exotoxin.

In this study we have investigated whether streptolysin O contributes to the virulence of group A streptococci. For this purpose we generated M-negative and SLO-negative mutants by insertion mutagenesis into the chromosome of an M type 1 strain. The inactivation of M1 protein expression was achieved by the construction of the integrative plasmid pSFABS, which contains the internal fragment abs of the emm1 gene. Integration of pSFABS by homologous recombination into the chromosome of strain 38 541 resulted in the generation of mutant EMM1. Inactivation of slo with plasmid pFWSLOD resulted in two different mutant forms. The homologous recombination with plasmid pFWSLOD carrying the two slo fragments slo1 (899 base pairs in the 5' region) and slo2 (709 base pairs in the downstream part) resulted in mutants SLO3, SLO4 and SLO17. In SLO17 a double crossover event took place with insertion of the spectinomycin resistance gene aad9 between the slo fragments 1 and 2. In mutants SLO3 and SLO4 the homologous recombination with the same plasmid led to the integration of the whole plasmid construct into the chromosome of strain 38 541. Both forms of mutation failed to express SLO. In mutant SLO4 additionally M1 protein expression was significantly decreased. The mutants EMM1 (M-, SLO+) and SLO4 (M decreased, SLO-) showed a reduced binding to collagen-coated surfaces. In contrast the mutants SLO3 and SLO17 (both M+, SLO-) and the wild-type strain 38 541 (M+, SLO+) showed an affinity to collagen similar to purified M1 protein. All mutants were less virulent for chicken embryos compared to the wild-type strain after infection by intravenous injection as well as by application onto the chorioallantoic membrane. The results show that besides M protein SLO can also influence virulence of group A streptococci. Moreover, it became obvious that streptococci need more than one tool to fully develop their infectious potential.

The object of our study is the evaluation of care needs of senior citizens and the adjustment of certain stage of care according to the German Health Care Act ("Pflegeversicherungsgesetz"). For assessment of care needs we developed a new rating scale (BPS). Factor analysis of the rating scale showed 5 dimensions: (1) "Psychoorganic Syndrome" (Dementia), (2) "Physical Care Needs", (3) "Lack of Social Skills", (4) "Aggressiveness" and (5) "Depression". The sample size is N = 1739, mean age 82.5 years. A statistically significant partial correlation of the extent of care needs as indicated by the stage of care described by BPS dimensions was found only for dimension (2) "Physical Care Needs" (r = 0.50). Cluster analysis resulted in distribution of the whole sample into 6 discriminable groups, according to their syndrome specificity (BPS profiles). Additionally clusters are different regarding the severity of need for care. It is obvious, analyzing the adjustment of stages of care within clusters, that psychiatric relevant disturbances contribute substantially to this adjustment. Nevertheless this finding is valid only for aged people, who receive regular care by ambulant or home services, and can not be transferred to senior citizens cared by their families.

Spallation residues produced in 1 GeV per nucleon 208Pb on proton reactions have been studied using the Fragment Separator facility at GSI. Isotopic production cross sections of elements from 61Pm to 82Pb have been measured down to 0.1 mb with a high accuracy. The recoil kinetic energies of the produced fragments were also determined. The obtained cross sections agree with most of the few existing gamma-spectroscopic data. The data are compared with different intranuclear-cascade and evaporation-fission models. Drastic deviations were found for a standard code used in technical applications.

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.

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.

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.

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.

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.

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.

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.

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.

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.