Interactions of glutaredoxins, ribonucleotide reductase, and components of the DNA replication system of Escherichia coli

A strain of Escherichia coli missing three members of the thioredoxin superfamily, thioredoxins 1 and 2 and glutaredoxin 1, is unable to grow, a phenotype presumed to be due to the inability of cells to reduce the essential enzyme ribonucleotide reductase. Two classes of mutations can restore growth to such a strain. First, we have isolated a collection of mutations in the gene for the protein glutaredoxin 3 that suppress the growth defect. Remarkably, all eight independent mutations alter the same amino acid, methionine-43, changing it to valine, isoleucine, or leucine. From the position of the amino acid changes and their effects, we propose that these alterations change the protein so that its properties are closer to those of glutaredoxin 1. The second means of suppressing the growth defects of the multiply mutant strain was by mutations in the DNA replication genes, dnaA and dnaN. These mutations substantially increase the expression of ribonucleotide reductase, most likely by altering the interaction of the regulatory protein DnaA with the ribonucleotide reductase promoter. Our results suggest that this increase in the concentration of ribonucleotide reductase in the cell allows more effective interaction with glutaredoxin 3, thus restoring an effective pool of deoxyribonucleotides. Our studies present direct evidence that ribonucleotide reductase is the only essential enzyme that requires the three reductive proteins missing in our strains. Our results also suggest an unexpected regulatory interaction between the DnaA and DnaN proteins.     

Recent increase in antibiotic-resistant microorganisms in patients with spontaneous bacterial peritonitis adversely affects the clinical outcome in Korea

BACKGROUND AND AIM: Recently, antibiotic-resistant microorganisms have been increasingly noted in Korean patients with spontaneous bacterial peritonitis (SBP). The present study investigated the changing pattern of antibiotic resistance and its effects on the clinical outcome in treating SBP. METHODS: The present study retrospectively analyzed 87 episodes of SBP in 1995, 222 in 1998, and 271 in 1999. The isolated microorganisms and their antibiotic susceptibility were compared, and prognostic factors for survival were analyzed. RESULTS: Microorganisms were isolated in 41% of total episodes. The three most frequently isolated organisms were Escherichia coli (48%), Klebsiella pneumoniae (15%), and Aeromonas (8%). Strains that were resistant to cefotaxime in Gram-negative bacilli significantly increased from 7% in 1995 to 28% in 1999, and those to ciprofloxacin increased from 10% to 32%. Treatment failure also increased from 6% to 23%. Combined hepatocellular carcinoma and SBP caused by extended-spectrum beta-lactamase-producing strains were two independent prognostic factors for survival. CONCLUSION: Considering the increase in antibiotic-resistant microorganisms related to SBP, measures to prevent the in-hospital spread of resistant strains and the indiscriminate use of antibiotics should be instituted more stringently.     

Ischemia-reperfusion model in rat spinal cord: cell viability and apoptosis signaling study

This work aimed at determining the ideal ischemia time in an in vitro ischemia-reperfusion model of spinal cord injury. Rat spinal cord slices were prepared and then exposed or not to oxygen deprivation and low glucose (ODLG) for 30, 45, 60, 75 and 90 minutes. Cell viability was assessed by triphenyltetrazolium (TTC), lactate dehydrogenase (LDH) release, and fluorochrome dyes specific for cell dead (ethidium homodimer) using the apotome system. Glutamate release was enzymatically measured by a fluorescent method. Gene expression of apoptotic factors was assessed by real time RT-PCR. Whereas spinal cord slices exposed to ODLG exhibited mild increase in fluorescence for 30 minutes after the insult, the 45, 60, 75 and 90 minutes caused a 2-fold increase. ODLG exposure for 45, 60, 75 or 90 minutes, glutamate and LDH release were significantly elevated. nNOS mRNA expression was overexpressed for 45 minutes and moderately increased for 60 minutes in ODLG groups. Bax/bcl-xl ratio, caspase 9 and caspase 3 mRNA expressions were significantly increased for 45 minutes of ODLG, but not for 30, 60, 75 and 90 minutes. Results showed that cell viability reduction in the spinal cord was dependent on ischemic time, resulting in glutamate and LDH release. ODLG for 45 minutes was adequate for gene expression evaluation of proteins and proteases involved in apoptosis pathways.     

The crowding effect in Ancylostoma ceylanicum: density-dependent effects on an experimental model of infection

This study compared the course of Ancylostoma ceylanicum infection in hamsters infected with different inocula and the consequences for the host and helminth populations. The average of adult worms recovered, according to the number of third stage larva used, were 28.0, 24.8, 24.6, and 24.8 % to inocula size of 25 L3, 75 L3, 125 L3, and 250 L3, respectively. The size of the inoculum did not affect the establishment, survival, or fecundity of adult helminths. Reductions in the red blood cell and hemoglobin levels in the infected group were inversely proportional to the number of white blood cells. Moreover, differential cell counting revealed a positive correlation between the worm load and leucocyte numbers. The humoral response against excretion-secretion antigens was more robust and sensitive compared with the response against crude extract, with no direct linear correlation with the number of worms. The effect of the population density was more evident in females.