Endonuclease Activity of MutL Protein of the Rhodobacter sphaeroides Mismatch Repair System.

We have purified the MutL protein from Rhodobacter sphaeroides mismatch repair system (rsMutL) for the first time. rsMutL demonstrated endonuclease activity in vitro, as predicted by bioinformatics analysis. Based on the alignment of 1483 sequences of bacterial MutL homologs with presumed endonuclease activity, conserved functional motifs and amino acid residues in the rsMutL sequence were identified: five motifs comprising the catalytic site responsible for DNA cleavage were found in the C-terminal domain; seven conserved motifs involved in ATP binding and hydrolysis and specific to the GHKL family of ATPases were found in the N-terminal domain. rsMutL demonstrated the highest activity in the presence of Mn2+. The extent of plasmid DNA hydrolysis declined in the row Mn2+ > Co2+ > Mg2+ > Cd2+; Ni2+ and Ca2+ did not activate rsMutL. Divalent zinc ions inhibited rsMutL endonuclease activity in the presence of Mn2+ excess. ATP also suppressed plasmid DNA hydrolysis by rsMutL. Analysis of amino acid sequences and biochemical properties of five studied bacterial MutL homologs with endonuclease activity revealed that rsMutL resembles the MutL proteins from Neisseria gonorrhoeae and Pseudomonas aeruginosa.

An RpoHI-Dependent Response Promotes Outgrowth after Extended Stationary Phase in the Alphaproteobacterium Rhodobacter sphaeroides.

Under unfavorable growth conditions, bacteria enter stationary phase and can maintain cell viability over prolonged periods with no increase in cell number. To obtain insights into the regulatory mechanisms that allow bacteria to resume growth when conditions become favorable again (outgrowth), we performed global transcriptome analyses at different stages of growth for the alphaproteobacterium Rhodobacter sphaeroides The majority of genes were not differentially expressed across growth phases. After a short stationary phase (about 20 h after growth starts to slow down), only 7% of the genes showed altered expression (fold change of >1.6 or less than -1.6, corresponding to a log2 fold change of >0.65 or less than -0.65, respectively) compared to expression at exponential phase. Outgrowth induced a distinct response in gene expression which was strongly influenced by the length of the preceding stationary phase. After a long stationary phase (about 64 h after growth starts to slow down), a much larger number of genes (15.1%) was induced in outgrowth than after a short stationary phase (1.7%). Many of those genes are known members of the RpoHI/RpoHII regulons and have established functions in stress responses. A main effect of RpoHI on the transcriptome in outgrowth after a long stationary phase was confirmed. Growth experiments with mutant strains further support an important function in outgrowth after prolonged stationary phase for the RpoHI and RpoHII sigma factors.IMPORTANCE In natural environments, the growth of bacteria is limited mostly by lack of nutrients or other unfavorable conditions. It is important for bacterial populations to efficiently resume growth after being in stationary phase, which may last for long periods. Most previous studies on growth-phase-dependent gene expression did not address outgrowth after stationary phase. This study on growth-phase-dependent gene regulation in a model alphaproteobacterium reveals, for the first time, that the length of the stationary phase strongly impacts the transcriptome during outgrowth. The alternative sigma factors RpoHI and RpoHII, which are important regulators of stress responses in alphaproteobacteria, play a major role during outgrowth following prolonged stationary phase. These findings provide the first insight into the regulatory mechanisms enabling efficient outgrowth.

[Chronic complex tinnitus: does day care unit-based short-term psychotherapy help?]. / Chronisch komplexer Tinnitus : Hilft eine teilstationäre Kurzzeitpsychotherapie?

BACKGROUND: Results of empirical research on the effectiveness of psychotherapeutic treatment of chronic complex tinnitus are still unclear and require further studies for clarification. OBJECTIVE: The objective of this study is to investigate the effectiveness of multimodal treatment of patients with chronic complex tinnitus in a day care unit setting. PATIENTS AND METHODS: In this prospective study, the effectiveness of an intense 5-day multimodal treatment delivered to 93 patients was assessed using a pre-post design. Tinnitus-specific effects were captured by the Mini-TQ-12 measurement instrument and psychiatric comorbidity assessed using the Brief Symptom Inventory (BSI). In statistical analyses, t-tests, χ(2) tests and Wilcoxon signed ranks tests were performed to determine statistical significance; effect sizes (ES) were calculated according to Cohen's d. RESULTS: The pre-post difference in Mini-TQ-12 was highly significant in t-test (p < 0.001); the ES was small (ES = 0.45). In BSI, the pre-post difference in the Global Severity Index (GSI) as a global measure was also highly significant in t-test (p < 0.001); ES was small (ES = 0.40). Nonparametric tests (Wilcoxon test) confirmed these results. CONCLUSION: This study demonstrates the statistically significant effectiveness of a multimodal therapeutic approach. Moreover, the results indicate that patients could benefit from more intensive therapeutic interventions.

[Multimorbidity and successful aging: the population-based KORA-Age study]. / Multimorbidität und erfolgreiches Altern: Ein Blick auf die Bevölkerung im Rahmen der KORA-Age-Studie.

BACKGROUND: The objective of the KORA-Age research consortium is to assess the determinants and consequences of multimorbidity in the elderly and to look into reasons for successful aging in the general public. PATIENTS AND METHODS: In the KORA-Age cohort study 9,197 persons were included who where born in the year 1943 or before and participants of previous KORA cohort studies conducted between 1984 and 2001 (KORA: Cooperative Health Research in the Region of Augsburg). The randomized intervention study KORINNA (Coronary infarct follow-up treatment in the elderly) tested a nurse-based case management program with 338 patients with myocardial infarct and included an evaluation in health economics. RESULTS: A total of 2,734 deaths were registered, 4,565 participants submitted a postal health status questionnaire and 4,127 participants were interviewed by telephone (response 76.2% and 68.9% respectively). A gender and age-stratified random sample of the cohort consisting of 1,079 persons took part in a physical examination (response 53.8%). CONCLUSION: The KORA-Age consortium was able to collect data in a large population-based sample and is contributing to the understanding of multimorbidity and successful aging.

11C-PiB PET studies in typical sporadic Creutzfeldt-Jakob disease.

OBJECTIVE: Brain amyloid imaging using positron emission tomography (PET) is of increasing importance in the premortem evaluation of dementias, particularly in relation to Alzheimer disease (AD). The purpose of this study was to explore the premortem diagnostic utility of (11)C-PiB PET in sporadic Creutzfeldt-Jakob disease (CJD). METHODS: Two patients, 72 and 59 years old, underwent evaluation for rapidly progressive cognitive decline, dying after illness durations of 5 and 7 months, respectively. As part of their comprehensive assessment, (18)F-FDG PET and (11)C-PiB PET studies were performed approximately 2-4 weeks prior to death, and the brain regional distributions compared with those from cohorts of healthy controls (HC) and AD patients. RESULTS: Routine investigations, including brain MRI scans, revealed changes typical of sporadic CJD, with the diagnosis confirmed at autopsy in both patients. The (18)F-FDG PET showed global hypometabolism in one patient and thalamic and frontal hypometabolism with unexpected hypermetabolism in the dentate nuclei of the cerebellum in the other. Neither patient displayed cerebral cortical (11)C-PiB PET retention above the levels observed in HC. CONCLUSIONS: No grey-matter (11)C-PiB retention was observed in two pathologically confirmed cases of typical sporadic CJD. We speculate that low PrP plaque density and small plaque size, as well as a relatively low affinity of the radioligand, explain the absence of (11)C-PiB retention. More studies to validate this hypothesis are warranted.

Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease.

To validate the provisional findings of a number of smaller studies and explore additional determinants of characteristic diagnostic investigation results across the entire clinical spectrum of sporadic Creutzfeldt-Jakob disease (CJD), an international collaborative study was undertaken comprising 2451 pathologically confirmed (definite) patients. We assessed the influence of age at disease onset, illness duration, prion protein gene (PRNP) codon 129 polymorphism (either methionine or valine) and molecular sub-type on the diagnostic sensitivity of EEG, cerebral MRI and the CSF 14-3-3 immunoassay. For EEG and CSF 14-3-3 protein detection, we also assessed the influence of the time point in a patient's illness at which the investigation was performed on the likelihood of a typical or positive result. Analysis included a large subset of patients (n = 743) in whom molecular sub-typing had been performed using a combination of the PRNP codon 129 polymorphism and the form of protease resistant prion protein [type 1 or 2 according to Parchi et al. (Parchi P, Giese A, Capellari S, Brown P, Schulz-Schaeffer W, Windl O, Zerr I, Budka H, Kopp N, Piccardo P, Poser S, Rojiani A, Streichemberger N, Julien J, Vital C, Ghetti B, Gambetti P, Kretzschmar H. Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol 1999; 46: 224-233.)] present in the brain. Findings for the whole group paralleled the subset with molecular sub-typing data available, showing that age at disease onset and disease duration were independent determinants of typical changes on EEG, while illness duration significantly influenced positive CSF 14-3-3 protein detection; changes on brain MRI were not influenced by either of these clinical parameters, but overall, imaging data were less complete and consequently conclusions are more tentative. In addition to age at disease onset and illness duration, molecular sub-type was re-affirmed as an important independent determinant of investigation results. In multivariate analyses that included molecular sub-type, time point of the investigation during a patient's illness was found not to influence the occurrence of a typical or positive EEG or CSF 14-3-3 protein result. A typical EEG was most often seen in MM1 patients and was significantly less likely in the MV1, MV2 and VV2 sub-types, whereas VV2 patients had an increased likelihood of a typical brain MRI. Overall, the CSF 14-3-3 immunoassay was the most frequently positive investigation (88.1%) but performed significantly less well in the very uncommon MV2 and MM2 sub-types. Our findings confirm a number of determinants of principal investigation results in sporadic CJD and underscore the importance of recognizing these pre-test limitations before accepting the diagnosis excluded or confirmed. Combinations of investigations offer the best chance of detection, especially for the less common molecular sub-types such as MV2 and MM2.

Molecular Subtyping of PrPres in Human Sporadic CJD Brain Tissue.

Across the spectrum of sporadic human prion diseases (also known as transmissible spongiform encephalopathies: TSE), there is considerable phenotypic diversity. Cumulative scientific evidence supports that prions, the infectious agents of prion diseases, are constituted predominantly, if not exclusively, by misfolded, typically protease-resistant, disease-associated isoforms of the prion protein (PrPres). Consequently, tissue deposition of PrPres is considered a hallmark of prion disease pathology, and this can be visualized by Western blotting after tissue homogenization and treatment with proteinases, particularly proteinase K (PK). Indeed, Western blot profiles of PrPres are utilized as one marker of different prion strains, with such strains thought to contribute to at least part of the phenotypic variation observed in sporadic human prion disease. Typically, Western blotting of PrPres demonstrates three bands of different electrophoretic mobility, depicting the di-glycosylated, mono-glycosylated and unglycosylated species although further subclassification and the delineation of novel sporadic disease subtypes, such as variably protease-sensitive prionopathy, has contributed greater complexity. Nevertheless, it is the mobility of the unglycosylated PrPres band, the relative abundance of the two glycosylated bands or overall profile of the banding post-PK, in combination with the prion protein gene (PRNP) codon 129 genotype that allows the categorisation of molecular subtypes of sporadic human prion disease. These subtypes appear to correlate with distinct clinico-pathological profiles of sporadic Creutzfeldt-Jakob disease.

Myocardial edema in acute myocarditis: relationship of T2 relaxometry and late enhancement burden by using dual-contrast turbo spin-echo MRI.

To quantify myocardial edema by using a T2 relaxometry approach with a dual-contrast turbo spin-echo (dcTSE) sequence in patients with acute myocarditis regarding focal late gadolinium enhancement (LGE) burden. CMR T2 relaxometry was performed in 39 patients (age 41 ± 19 years; 36% women) with LGE in a typical myocarditis pattern and in ten healthy volunteers (age 46 ± 12; 60% woman). dcTSE sequence (echo time 29 and 75 ms, respectively) was used for T2 mapping, analysis were performed on the basis of region of interest (ROI). Myocardial T2 relaxation times (T2 RT) in patients-ROI with focal LGE were significantly (p < 0.001) higher than T2 RT in patients-ROI without apparent LGE pattern (65 ms (IQR 36-95) vs. 60 ms (IQR 26-88), respectively). T2 RT in healthy volunteers [55 ms (IQR 35-71)] were significantly lower than in patients ROI with or without focal LGE-pattern (p < 0.001, respectively). T2 RT assessed by dcTSE are significantly higher in patients segments with and without focal LGE compared to normal controls, supporting a global myocardial inflammatory process in acute myocarditis. Furthermore, this quantitative T2-mapping approach highlights the potential to identify patients with diffuse myocarditis.

An mRNA degrading complex in Rhodobacter capsulatus.

An RNA degrading, high molecular weight complex was purified from Rhodobacter capsulatus. N-terminal sequencing, glycerol-gradient centrifugation, and immunoaffinity purification as well as functional assays were used to determine the physical and biochemical characteristics of the complex. The complex comprises RNase E and two DEAD-box RNA helicases of 74 and 65 kDa, respectively. Most surprisingly, the transcription termination factor Rho is a major, firmly associated component of the degradosome.

mRNA degradation in bacteria.

Messenger RNAs in prokaryotes exhibit short half-lives when compared with eukaryotic mRNAs. Considerable progress has been made during recent years in our understanding of mRNA degradation in bacteria. Two major aspects determine the life span of a messenger in the bacterial cell. On the side of the substrate, the structural features of mRNA have a profound influence on the stability of the molecule. On the other hand, there is the degradative machinery. Progress in the biochemical characterization of proteins involved in mRNA degradation has made clear that RNA degradation is a highly organized cellular process in which several protein components, and not only nucleases, are involved. In Escherichia coli, these proteins are organized in a high molecular mass complex, the degradosome. The key enzyme for initial events in mRNA degradation and for the assembly of the degradosome is endoribonuclease E. We discuss the identified components of the degradosome and its mode of action. Since research in mRNA degradation suffers from dominance of E. coli-related observations we also look to other organisms to ask whether they could possibly follow the E. coli standard model.

The relationship between plasma free fatty acids and liver mitochondrial function in vivo.

P/O ratio, state 3 and 4 respiration rates, and acceptor control index (ACI) were assessed in rat liver mitochondria following an overnight fast and single bout of treadmill exercise of 30-180 min. P/O was unaffected by fasting and 30 min of exercise; however, ACI was reduced because of an increase in state 4 respiration. Fasting, followed by running for 1 h or more decreased P/O approx. 40% and ACI by 50%, an effect that could be attributed to a reduction in state 3 respiration. The decrease in P/O was reversed 15 min after the cessation of exercise, whereas ACI remained depressed. All these functional alterations were mimicked by incubation of isolated mitochondria with palmitate and reversed by washing them with albumin. No direct correlation between plasma free fatty acids and the alterations in mitochondrial respiration was apparent. These data demonstrate that the decrease in the normal coupling of oxidation and phosphorylation in liver mitochondria produced by fasting/exercise is reversed rapidly in vivo. Furthermore, it is apparent that, if fatty acids act as a regulatory agent under these conditions, they do not do so solely on the basis of their plasma concentration.

Alterations in liver mitochondrial function as a result of fasting and exhaustive exercise.

The effect of exercise upon liver mitochondria structure and function was examined in fasted and fed rats, following a single run to exhaustion on a motor-driven treadmill. Exercise alone and exercise coupled with fasting both produced a significant decrease in the amount of hexokinase bound to the mitochondria, as well as reduction in the ADP/O ratio and acceptor control index measured in the presence of succinate. The mitochondria of the exercised animals, when exposed to freeze-fracture analysis while in state 3, displayed fewer deflections in the fracture plane between the inner and outer membrane than those isolated from control animals. This suggests that fewer contacts existed between the two membranes. Measurements based upon the binding of 8-anilinonaphthalene 1-sulphonate indicated that there was an increase in the net negative charge on the surface of the mitochondrial membranes of the exercised animals. All of these effects could be mimicked by incubation of mitochondria from control animals with free fatty acids. This fact, coupled with the observation that washing of the mitochondria with a solution comprising 5% (w/v) albumin could reverse all of the consequences of exercise, suggests that these alterations in mitochondrial structure and function may be the result of the increase in plasma free fatty acids that accompanies long-term exercise. Furthermore, the observation that the exercise-induced changes are dynamic and readily reversible indicates that the mitochondria were not necessarily damaged, but rather that the coupling of oxidative phosphorylation may be subject to physiological regulation.

The rate of decay of Rhodobacter capsulatus-specific puf mRNA segments is differentially affected by RNase E activity in Escherichia coli.

In Rhodobacter capsulatus the puf operon encodes proteins of the photosynthetic apparatus. The polycistronic puf mRNA is comprised of segments that show differential stability. Here, we show that the rate of decay of the 2.7-kb pufBALMX mRNA species in Escherichia coli depends on the activity of ribonuclease E (RNase E), whereas the degradation of the 0.5-kb pufBA mRNA segment is not affected by a mutation in the rne gene. The RNase E-promoted decay of the pufLMX mRNA depends on the presence of a 1.4-kb pufLM mRNA segment, in which rate-limiting endonucleolytic cleavage was postulated to occur in R. capsulatus. The insertion of 185 bp of this 1.4-kb segment into pufB results in an RNase E-dependent decay of the modified pufBA mRNA segment in E. coli. Our findings suggest that in R. capsulatus an RNase E-like activity is responsible for the rate-limiting endonucleolytic cleavage occurring within the pufLM mRNA segment, whereas the 0.5-kb pufBA mRNA segment is degraded by a different RNase E-independent decay mechanism.

Cloning, nucleotide sequence and characterization of the rpoD gene encoding the primary sigma factor of Rhodobacter capsulatus.

A synthetic oligodeoxynucleotide probe based on a highly conserved region of the sigma factors was used to identify and clone the rpoD gene encoding the principal sigma factor of R. capsulatus. The deduced polypeptide contains 674 amino acids and has a predicted molecular mass of 75,942 Da. The deduced amino acid sequence of R. capsulatus RpoD protein exhibits 46.2% and 45.7% identity with housekeeping sigma factors of Pseudomonas and E. coli, respectively. Unsuccessful attempts to inactivate the single chromosomal rpoD gene of R. capsulatus by gene replacement technique indicate that this gene is essential for cell survival, as expected for the primary sigma factor. The rpoD transcript 5'-end was mapped by primer extension analysis, 74 bp upstream of the initiation codon and DNA sequence analysis has identified a motif resembling the delta 70 E. coli consensus promoter sequences at the expected distance from this proposed transcription start site. rpoD gene expression, as measured by the activity of the phi (rpoD'-lacZ+) translational fusion, was found to be constant throughout exponential and early plateau phases, but significantly increased at later times of the stationary phase.

Rapid reduction in parvalbumin concentration during chronic stimulation of rabbit fast twitch muscle.

Chronic indirect stimulation of fast twitch rabbit muscle induced a rapid reduction in parvalbumin concentration. When compared to the unstimulated contralateral muscle, parvalbumins were reduced to 55% following 6 days of stimulation. Prolonged stimulation further reduced parvalbumins so that they were undetectable after 28 days. The time course of these changes appears to be related with the previously observed changes in the sarcoplasmic reticulum.

Both N-terminal catalytic and C-terminal RNA binding domain contribute to substrate specificity and cleavage site selection of RNase III.

The double-stranded RNA-specific endoribonuclease III (RNase III) of bacteria consists of an N-terminal nuclease domain and a double-stranded RNA binding domain (dsRBD) at the C-terminus. Analysis of two hybrid proteins consisting of the N-terminal half of Escherichia coli RNase III fused to the dsRBD of the Rhodobacter capsulatus enzyme and vice versa reveals that both domains in combination with the particular substrate determine substrate specificity and cleavage site selection. Extension of the spacer between the two domains of the E. coli enzyme from nine to 20 amino acids did not affect cleavage site selection.

Decrease in myosin light chain kinase activity of rabbit fast muscle by chronic stimulation.

Analysis of myosin light chain kinase (MLCK) activity in tibialis anterior muscles of the rabbit revealed that chronic stimulation at a frequency of 10 Hz for 24 h per day reduced the enzyme activity in a time-dependent manner. Since fast twitch muscle contains significantly more myosin light chain kinase than slow twitch muscle, the observed reductions are consistent with the type of fast-to-slow transformation observed for other type-specific muscle characteristics. The present data also indicate that the stimulation-induced decrease in MLCK activity precedes the fast-to-slow conversion of the myosin molecule as judged by pyrophosphate-polyacrylamide gel electrophoresis.

Autosomal recessive hydrocephalus with third ventricle obstruction.

Here we report a brother and sister who presented in the neonatal period with hydrocephalus. Ultrasonography showed marked dilatation of the lateral ventricles but not the third ventricle. One child with postnatal onset was shunted and had normal development at 3 years. The other child had severe hydrocephalus at birth and was not treated. Neuropathologic studies demonstrated dilatation of the lateral ventricles and marked narrowing of the posterior part of the third ventricle but no other malformations other than those that result directly from hydrocephalus. The potential for a good prognosis is emphasized.

Cerebrospinal fluid shunt infections in children.

We reviewed cerebrospinal fluid (CSF) shunt infections treated in the Royal Children's Hospital, Melbourne from 1981 to 1991. Forty-one episodes of CSF shunt infection were found after 900 shunt operations, an infection rate of 4.5%. Clinical symptoms were nonspecific in 31.7% of episodes, and in 17.1% of episodes the initial CSF sample was normal on microscopy and biochemistry, although a pathogen was isolated on culture. Most episodes occurred within 4 months of the last operation on the shunt, the exception being infections caused by Haemophilus influenzae. Four patients died during treatment, but none could be attributed to infection alone. Treatment of suspected CSF shunt infection should not be withheld because of lack of firm clinical diagnosis or normal CSF microscopy, and episodes occurring more than 4 months after the last operation on the CSF shunt should receive antibiotic cover for H. influenzae.

Effects of exercise of varying duration on sarcoplasmic reticulum function.

Sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+-Mg2+-ATPase activity were examined in muscle homogenates and the purified SR fraction of the superficial and deep fibers of the gastrocnemius and vastus muscles of the rat after treadmill runs of 20 or 45 min or to exhaustion (avg time to exhaustion 140 min). Vesicle intactness and cross-contamination of isolated SR were estimated using a calcium ionophore and mitochondrial and sarcolemmal marker enzymes, respectively. Present findings confirm previously reported fiber-type specific depression in the initial rate and maximum capacity of Ca2+ uptake and altered ATPase activity after exercise. Depression of the Ca2+-stimulated ATPase activity of the enzyme was evident after greater than or equal to 20 min of exercise in SR isolated from the deep fibers of these muscles. The lowered ATPase activity was followed by a depression in the initial rate of Ca2+ uptake in both muscle homogenates and isolated SR fractions after greater than or equal to 45 min of exercise. Maximum Ca2+ uptake capacity was lower in isolated SR only after exhaustive exercise. Ca2+ uptake and Ca2+-sensitive ATPase activity were not affected at any duration of exercise in SR isolated from superficial fibers of these muscles; however, the Mg2+-dependent ATPase activity was increased after 45 min and exhaustive exercise bouts. The alterations in SR function could not be attributed to disrupted vesicles or differential contamination in the SR from exercise groups and were reinforced by similar changes in Ca2+ uptake in crude muscle homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)