Identification and transcriptional control of the genes encoding the Caulobacter crescentus ClpXP protease.

The region of the Caulobacter crescentus chromosome harboring the genes for the ClpXP protease was isolated and characterized. Comparison of the deduced amino acid sequences of the C. crescentus ClpP and ClpX proteins with those of their homologues from several gram-positive and gram-negative bacteria revealed stronger conservation for the ATPase regulatory subunit (ClpX) than for the peptidase subunit (ClpP). The C. crescentus clpX gene was shown by complementation analysis to be functional in Escherichia coli. However, clpX from E. coli was not able to substitute for the essential nature of the clpX gene in C. crescentus. The clpP and clpX genes are separated on the C. crescentus chromosome by an open reading frame pointing in the opposite direction from the clp genes, and transcription of clpP and clpX was found to be uncoupled. clpP is transcribed as a monocistronic unit with a promoter (PP1) located immediately upstream of the 5' end of the gene and a terminator structure following its 3' end. PP1 is under heat shock control and is induced upon entry of the cells into the stationary phase. At least three promoters for clpX (PX1, PX2, and PX3) were mapped in the clpP-clpX intergenic region. In contrast to PP1, the clpX promoters were found to be downregulated after heat shock but were also subject to growth phase control. In addition, the clpP and clpX promoters showed different activity patterns during the cell cycle. Together, these results demonstrate that the genes coding for the peptidase and the regulatory subunits of the ClpXP protease are under independent transcriptional control in C. crescentus. Determination of the numbers of ClpP and ClpX molecules per cell suggested that ClpX is the limiting component compared with ClpP.

[Postoperative cognition disorders in elderly patients. The results of the "International Study of Postoperative Cognitive Dysfunction" ISPOCD 1)]. / Postoperative Störungen der kognitiven Leistungsfähigkeit bei älteren Patienten. Die Ergebnisse der "International Study of Postoperative Cognitive Dysfunction" (ISPOCD 1).

OBJECTIVE: Cognitive dysfunction is a known problem after operations and may be especially relevant in the elderly. The aim of this international multicentre study was to investigate short- and long-term cognitive dysfunction in elderly patients and to elucidate the relevance of hypoxaemia and hypotension as causative factors. METHODS: 1218 patients aged 60 years and older and scheduled for major non-cardiac surgery under general anaesthesia were investigated. Oxygen saturation was measured by continuous pulse oximetry before surgery and throughout the day of and the first 3 nights after surgery. Blood pressure was recorded every 3 minutes during the operation and every 15-30 min for the rest of that day and night. Cognitive testing was performed before and 1 week and 3 months after the operation. Cognitive dysfunction was identified with neuropsychological tests compared with controls recruited from the UK (n = 176) and the same countries as study centres (n = 145). RESULTS: Postoperative cognitive dysfunction was present in 25.8% of patients 1 week after surgery and in 9.9% 3 months after surgery, compared with 3.4% and 2.8%, respectively, of the UK controls. Increasing age and duration of anaesthesia, little education, a second operation, postoperative infections, and respiratory complications were the risk factors for early postoperative cognitive dysfunction, but only age was a risk factor for long-term postoperative cognitive dysfunction. Hypoxaemia and hypotension were not significant risk factors at any time. CONCLUSION: With this investigation long-term cognitive dysfunction could be proven definitively for elderly patients after major operations under general anaesthesia. No factors with prophylactic or therapeutic influence were detectable so that aetiology and pathophysiology of POCD could not be further determined.

The relative increase in skin temperature after stellate ganglion block is predictive of a complete sympathectomy of the hand.

BACKGROUND AND OBJECTIVES: Although an increase in skin temperature of the hand implies sympathetic block after stellate ganglion block (SGB), it does not indicate complete sympathetic block unless accompanied by an absence of sweating because skin temperature may increase even with a partial sympathetic block. This study examined the efficacy of the SGB to block sweating in the hand and to determine if the magnitude of temperature change in the hand is predictive of a negative sweat test. METHODS: Fifty-nine SGBs were performed in 30 patients (15 women and 15 men) for diagnostic or therapeutic indications. Stellate ganglion block was performed via an anterior paratracheal approach at C6 using 15 mL 0.25% bupivacaine. Skin temperature was measured bilaterally on the index finger. A cobalt blue sweat test was performed bilaterally pre- and post-SGB on the middle finger. Successful sympathetic block after SGB was considered present when: (a) (change in ipsilateral temperature (postblock-preblock)] (Di)-[change in contralateral temperature] (Dc) > or = 1.5 degrees C; (b) Horner's syndrome present; and (c) sweat test changed from positive to negative. Logistical regression was applied to determine what value of Di - Dc could be used to predict a negative sweat test. RESULTS: Thirty-six percent (21/59) of blocks met all three criteria. Of the blocks where Di - Dc > or = 1.5 degrees C, 72% (21/29) had a negative sweat test post-SGB. Of the blocks where Di - Dc < 1.5 degrees C, 37% (11/30) had a negative sweat test postblock. If Di - Dc > or = 2.0 degrees C, a negative sweat test could be predicted with 69 +/- 12% sensitivity and 85 +/- 10% specificity. CONCLUSIONS: Stellate ganglion block often fails to increase skin temperature in the ipsilateral more than the contralateral hand. A value of Di - Dc > or = 2.0 degrees C was a good predictor of a sympathetic block, but was not sufficient to guarantee a complete sympathetic block of the hand after SGB in all cases. An apparently successful SGB as measured by "usual" clinical criteria may not result in a complete sympathectomy of the hand as is often assumed. Therefore, if obtaining a sympathectomy is important for diagnostic or therapeutic purposes, performing a sweat test provides important confirmatory evidence of the genuine success of the sympathetic block.

Regulation of the ADE2 gene from Saccharomyces cerevisiae.

Regulation of ADE2 gene expression was investigated in the yeast S. cerevisiae using translational fusions between this gene and the lacZ gene from E. coli. Expression was repressed in the presence of adenine and slightly increased under amino-acid starvation conditions. The promoters of the ADE2 gene, and of other genes involved in adenine biosynthesis, contain the hexanucleotide sequence TGACTC. A search for the hexanucleotide TGACTC in yeast promoter sequences revealed that many genes not related to amino-acid biosynthesis contain such sequences. We show here that these elements play a crucial role in ADE2 regulation since mutations in two such elements drastically reduced gene expression. Maximal expression required the transcriptional activators Bas1, Bas2 and Gcn4, whereas Yap1 had only minor effects.

A new yeast translation initiation factor suppresses a mutation in the eIF-4A RNA helicase.

We have isolated a gene, STM1, which encodes a new translation initiation factor from Saccharomyces cerevisiae. The gene acts, if present on a multicopy plasmid, as a suppressor of a temperature-sensitive mutation in eIF-4A. The single copy STM1 gene is not essential, but disruption causes a slow growth phenotype. Analysis of polysomes from a strain carrying a disrupted stm1 allele shows a clear defect in translation initiation as shown by a strong reduction in polysomes and an increase in the monosomes. Sequence analysis revealed interesting features of the putative Stm1 protein. Comparison of the entire protein sequence with databanks showed some similarity with the human eIF-4B protein. The Stm1 protein has potential RNP1 and RNP2 motifs characteristic for RNA-binding proteins. The protein also contains six highly conserved direct repeats of 21-26 amino acids and one partial repeat.

The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors.

We have determined the sequence of a DNA fragment encoding the ADE2 gene from Saccharomyces cerevisiae. A DNA fragment of 2241 bp capable of complementing ade2 mutations was modified so it is available as a single BglII fragment for use in yeast vectors or for gene disruptions. The minimal fragment codes for a putative protein which is highly similar to the protein encoded by the ADE6 gene from Schizosaccharomyces pombe and to the proteins encoded by the purEK operon of Escherichia coli.

Role of conserved sequence elements in yeast centromere DNA.

Conserved sequence features in Saccharomyces cerevisiae CEN DNA are confined to a region of approximately 120 bp. The highly conserved 8 bp at the left (PuTCACPuTG) constitute the left boundary of a functional CEN DNA as shown by the analysis of a series of Bal31 deletions. The right boundary of a functional CEN DNA lies within the conserved 25 bp at the right (TGT-T-TG--TTCCGAA-----AAA) or a few base pairs further outside of the 120-bp region. One mutant which just lacks the left conserved DNA element PuTCACPuTG can still assemble into a partially functional mitotic centromere and it assembles into a well functioning meiotic centromere. The sequences between the two conserved terminal DNA elements can be increased in length (+50%) or in GC content (from 6% to 12%) without measurable changes in mitotic and meiotic segregations of plasmids carrying such CEN mutations. The naturally occurring length and GC content of this centromere DNA sequence element is, therefore, not essential for centromere function. We discuss the possibility that it partly acts as a hinge region between two domains. Finally, we tested integrations of CEN DNA into the genome and found a toleration of wild-type CEN6 DNA when present 3' of the LYS2 gene.