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Decreased elasticity of the cardiovascular system is one of the hallmarks of the normal aging process of mammals. A potential explanation for this decreased elasticity is that glucose can react nonenzymatically with long-lived proteins, such as collagen and lens crystallin, and link them together, producing advanced glycation endproducts (AGEs). Previous studies have shown that aminoguanidine, an AGE inhibitor, can prevent glucose cross-linking of proteins and the loss of elasticity associated with aging and diabetes. Recently, an AGE cross-link breaker (ALT-711) has been described, which we have evaluated in aged dogs. After 1 month of administration of ALT-711, a significant reduction ( approximately 40%) in age-related left ventricular stiffness was observed [(57.1 +/- 6.8 mmHg x m(2)/ml pretreatment and 33.1 +/- 4.6 mmHg x m(2)/ml posttreatment (1 mmHg = 133 Pa)]. This decrease was accompanied by improvement in cardiac function.

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BACKGROUND: Collagen accumulation in the myocardial interstitium of diabetic animals is considered to promote diastolic stiffness through advanced glycosylation. Because in vitro data suggest that metformin can modify glycosylation, this study was undertaken in a canine diabetic model 4 months in duration. METHODS AND RESULTS: Untreated diabetics (group II) and diabetics treated with metformin alone (group III) or with insulin (group IV) were compared in the basal state and during volume infusion. Basal hemoglobin A(1c), heart rate, aortic pressure, and ejection fraction were comparable. Left ventricular end-diastolic pressure was significantly increased in the untreated diabetics of group II, associated with a reduced end-diastolic volume. By contrast these parameters in the metformin-treated diabetics of group III were comparable with those in the normals of group I. Similarly in group IV end-diastolic volume was higher than that in group II, but filling pressure, although lower, was not significantly so. Calculation of left ventricular chamber stiffness in the basal state indicated a higher level for group II compared with controls and the treatment groups. During the systemic infusion of dextran, the untreated diabetics of group II had the largest end-diastolic pressure increase and the smallest rise of end-diastolic volume of the treatment groups, consistent with a significantly greater chamber stiffness. Myocardial collagen concentration was increased in group II with an interstitial distribution on morphological exam. Levels of collagen-linked advanced glycosylation end products isolated from the left ventricular were significantly greater in group II than in group I. Treatment with metformin prevented the increment observed in the untreated diabetic but had no effect on the elevated collagen concentration. CONCLUSIONS: Untreated diabetics exhibited increased diastolic chamber stiffness associated with collagen-linked glycation in myocardium compared with control animals. Chronic metformin use prevented the abnormalities of function and composition.

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New therapies for vancomycin-resistant Enterococcus faecium (VREF) infections are urgently needed. We describe the treatment of 15 patients with VREF infection with quinupristin/dalfopristin (RP 59500), a new injectable streptogramin antibiotic. Primary infections treated were bacteremia (4), urinary tract (4), intraabdominal (5), otitis externa (1), and meningitis (1). Minimum inhibitory concentrations for quinupristin/dalfopristin ranged from 0.5 microgram/ml or less to 2 micrograms/ml, and minimum bactericidal concentrations were greater than 64 micrograms/ml for all VREF isolates tested. Peak serum inhibitory titers following infusion of quinupristin/dalfopristin ranged from 1:8 to 1:64; all bactericidal titers were less than 1:2. Development of resistance to quinupristin/dalfopristin during therapy was not observed. The only drug-related adverse effect noted was phlebitis in 4 patients; all had received quinupristin/dalfopristin by peripheral venous infusion. Three patients had clinical and bacteriologic cures. Relapses occurred in 5 patients with recovery of VREF from infected sites in post-treatment cultures. Ten patients died of severe underlying disease; VREF was believed to contribute directly to the death of only 1 patient. While evaluation of clinical efficacy was complicated by the severity of underlying disease in patients with VREF infection, our experience suggests that quinupristin/dalfopristin is a safe and potentially useful agent for the treatment of VREF infections.

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Measles virus (MV) mRNA transcription and replication are thought to be controlled by cis-acting sequence elements contained within the terminal MV genomic noncoding nucleotides. To validate these promoter and regulatory signal assignments, cDNAs were constructed allowing synthesis of RNAs corresponding to a MV genome in which all coding and intercistronic regions were replaced by the chloramphenicol acetyl transferase (CAT) coding sequence. Transcript production by T7 polymerase starting and ending precisely with the MV genome terminal residues was achieved by fusing the T7 polymerase promoter and the hepatitis delta virus genome ribozyme followed by tandem T7 polymerase termination sequences to the MV genomic 5' and 3' ends, respectively. Transfection of these negative polarity transcripts, mimicking natural defective interfering RNAs of the internal deletion type, into MV-infected 293 cells gave rise to CAT activity which could be serially transferred and massively amplified together with progeny helper virus in fresh cells. Transfer was blocked only by antibodies able to neutralize MV infectivity, indicating that the chimeric RNA not only was encapsidated, transcribed, and replicated, but also packaged into virions. Sequence analyses confirmed that both the expected chimeric antigenome and mRNA products were transcribed and replicated with fidelity during serial passage. Minor changes introduced in the transcription promoter markedly compromised function. This system now can be exploited to examine MV genomic cis-acting regulatory elements and extended to the development of full-length MV cDNAs.

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Acridine-induced frameshift mutations in bacteriophage T4 occur at the precise location in the DNA at which acridines stimulate DNA cleavage by the T4-encoded type II topoisomerase in vitro. The mutations are duplications or deletions that begin precisely at the broken phosphodiester bond. In vivo, acridine-induced frameshift mutagenesis is reduced nearly to background levels when the topoisomerase is genetically inactivated. These observations are consistent with a model in which cleaved DNA, induced by the topoisomerase and acridine, serves as the substrate for the production of frameshift mutations at the same site. Our model predicts that the specificity and frequency of cleavage direct the specificity and frequency of mutagenesis. This prediction was tested by examining the influence of DNA sequence changes on topoisomerase-mediated cleavage and on mutagenesis in the T4 rIIB gene. The model successfully predicted the results. When DNA sequence changes altered the position of acridine-induced, topoisomerase-mediated DNA cleavage in vitro, frameshift mutations were found at the new positions. DNA sequence changes that strongly decreased in vitro cleavage also reduced mutagenesis at that site. These results demonstrate that acridine-induced frameshift mutation specificity is directed by the characteristics of the acridine-topoisomerase reaction and do not suggest that slipped pairing in repeated sequences plays a major role in acridine-induced frameshifts in bacteriophage T4.

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Chromosome replication in the asymmetrically dividing bacteria Caulobacter crescentus is discontinuous with the new, motile swarmer cell undergoing an obligatory presynthetic gap period (G1 period) of 60 min before the initiation of DNA synthesis and stalk formation. To examine the regulation of the cell division cycle at the molecular level, we have cloned the DNA chain elongation gene dnaC from a genomic DNA library constructed in cosmid vector pLAFR1-7. To ensure that the cloned sequence corresponded to dnaC, we isolated the gene by genetic complementation of the temperature-sensitive allele dnaC303 on DNA fragment that contained a Tn5 insertion element tightly linked by transduction to dnaC. The size of the dnaC gene was estimated to be 1,500 bp or less based on the pattern of complementation by subcloned restriction and BAL 31 deletion fragments. Nuclease S1 assays were used to map the transcription start site and to determine the pattern of dnaC expression in the cell cycle. Large amounts of the dnaC transcript began to accumulate only in the late G1 period of the swarmer cell and then peaked early during chromosome replication. We confirmed that the gene is periodically transcribed by monitoring the rate of beta-galactosidase synthesis directed by a dnaC promoter-lacZ fusion in a synchronous cell culture. dnaC is the first C. crescentus cell cycle gene whose regulation has been reported, and the discontinuous pattern of its expression suggests that the DNA synthetic period in these dimorphic bacteria is regulated in part by the stage-specific expression of DNA replication genes.

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An enzyme that catalyzes the conversion of L-glutamic acid and 10-formyl-H4folic acid (also known as 10-formyl-H4pteroylglutamic acid) to 10-formyl-H4pteroyl-gamma-glutamylglutamic acid has been purified by 74-fold from extracts of Escherichia coli. ATP, Mg-2+, and a monovalent cation (K+ or NH-4, but not Na+) are required for the enzyme to function. Radioactive and bioautographic analyses revealed the formation of a single product. This product was identified as 10-formyl-H-4pteroyl-gamma-glutamylglutamic acid from its spectral characteristics, its ability to be used effectively as a growth faster for Lactobacillus casei 7469, and from radioactive analysis that indicated the incorporation into the product of 1 mol glutamate/mol of 10-formyl-H-4pteroylglutamic acid utilized. The enzyme functions optimally at pH 9.0-9.8 and at 50 degrees. Its molecular weight is estimated at 42,000-43,000. The Km values are 180 muM for L-glutamic acid and less than 2 muM for (-) 10-formyl-H-4pteroylglutamic acid. The only other naturally occurring folate compounds with significant activity as substrate are H-4pteroylglutamic acid and 5,10-methylene-H-4pteroylglutamic acid; however, these compounds are not used as effectively (K-m values are 10-12 mu-M) as 10-formyl-H-4pteroylglutamic acid.

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