Intracellular events determine the fate of antithrombin Utah

We sought to determine whether intracellular or extracellular events contribute to the decrease in circulating antithrombin (AT) levels that is seen in subjects with the Utah mutation (Pro 407 to Leu). Site- directed mutagenesis was used to recreate this mutation within a previously characterized rabbit AT cDNA. Cell-free expression of the mutated cDNA yielded an AT protein that failed to react with thrombin. Expression of the rabbit AT-Utah protein in transiently transfected Cos cells resulted in a 10-fold decrease in the amount of AT antigen detected in the conditioned media, as compared with that seen with the wild-type recombinant AT. This effect was not caused by variations in transfection efficiency, because AT levels were normalized to the product of a cotransfected plasmid, chloramphenicol acetyl transferase. Moreover, on Northern blot analysis, AT mRNA levels were comparable in cells expressing either the rabbit AT-Utah or wild-type recombinant rabbit AT. Immunoblots of conditioned media from the two populations of transfected cells showed that the recombinant AT-Utah protein was intact. The results obtained with Cos cells were reproduced using permanently transfected Chinese hamster ovary (CHO) cells. Pulse-chase experiments with the CHO lines showed that both initial levels of rabbit AT-Utah after the pulse labeling and the rate of subsequent secretion during the chase period were reduced compared with that seen with cells expressing the wild-type AT. The observed reduction in AT secretion was also observed for the AT-Oslo mutation (Ala 404 to Thr) when recreated in the rabbit AT background, and expressed in Cos cells. In these experiments, the media levels of mutant AT were reduced by 50%, compared with wild-type. These results show that intracellular events, as opposed to accelerated clearance or other extracellular causes, contribute to the paucity of AT secretion seen in these strand 1C AT mutants.     

新吩嗪化合物K＿3－Ye的分离和结构鉴定

自一株基因工程链霉菌Ｋ３的发酵液中分离得到一吩嗪类新化合物，经光谱数据（ＵＬ，ＩＲ，１ＨＮＭＲ，１３ＣＮＭＲ，ＤＥＰＴ）分析，确定其结构为１－吩嗪氧基乙酸甲酯，代号Ｋ３－Ｙｅ。经初步测定它有较强的抗核苷转运活性。     

Development and Evaluation of a Chromatographic Procedure for Partial Purification of Substance P with Quantitation by an Enzyme Immunoassay

We have developed a simple chromatographic procedure for the partial purification of substance P (SP) from acidified plasma and serum samples. We have evaluated a sensitive antigen competition enzyme immunoassay (EIA) for the quantitation of SP. The chromatographic procedure has recovery efficiencies ranging from 94.8 to 125%. The immunoreactivity of unknown amounts of purified SP subjected to the preparative procedure yielded a coefficient of variance of 9.4%. The EIA yielded reproducible standard curves having an interassay (n = 8) correlation coefficient of 0.984. The evaluation of normal adult control serum yielded a mean value of 51 pg/ml (range, 35 to 61 pg/ml). The evaluation of 3.33× concentrates of serum-derived partially purified SP provided uncorrected SP values of 117 to 201 pg/ml, which fell within the midpoint of the three-decalog standard curve. These studies indicate that both the preparative and quantitative procedures are required for the detection of SP in plasma or serum samples collected from patients with several clinical disorders.     

Evaluation of GeneOhm VanR and Xpert vanA/vanB molecular assays for the rapid detection of vancomycin-resistant enterococci

Rapid diagnosis is critical for treating and preventing infections due to vancomycin-resistant enterococci (VRE). We assessed the performance of GeneOhm VanR and Xpert vanA/vanB assays that detect vanA and vanB, the two most important genes encoding vancomycin resistance, utilizing 50 stool samples from renal dialysis patients, as well as well-characterized VRE strains. Stool samples were screened for the presence of VRE by culture followed by polymerase chain reaction (PCR) for the detection of van genes in isolates. Furthermore, the direct detection of vanA/vanB from aerobically and anaerobically pre-enriched stools was performed by in-house PCR sequencing. GeneOhm was less sensitive (43.5% vs. 73.9%) and more specific (100% vs. 92.6%) than Xpert in detecting vanA from stool samples. vanB detection by GeneOhm was more sensitive than Xpert (100% vs. 87.5%), but equally non-specific (20.6% vs. 14.7%). A further estimation on log-serial dilutions of VRE strains showed that Xpert was more sensitive at detecting VRE at low concentrations (10–100 colony forming units [cfu]/ml).     

Current trends in culture-based and molecular detection of extended-spectrum-β-lactamase-harboring and carbapenem-resistant Enterobacteriaceae

The ever-expanding role of extended-spectrum-β-lactamase (ESBL)-harboring and carbapenem-resistant Enterobacteriaceae in causing serious infections poses a grave public health threat because these organisms also tend to be multidrug resistant, for which very few (if any) antibiotic options remain available. Rapid detection of such panresistant organisms offers one of the best solutions to improve patient screening and hospital infection control practices as well as curb inappropriate antibiotic use. This review discusses and compares primarily the current state-of-the-art culture-based and molecular methods that are commercially available for detection or screening of ESBL-harboring and carbapenem-resistant Enterobacteriaceae.     

Intraoperative US as an aid to intraoperative electron beam radiation therapy

Intraoperative electron-beam radiation therapy (IRT) is being used to treat certain abdominal malignancies. Accurate determination of tumor size, depth, and extent is necessary to select a treatment cone of appropriate size and appropriate electron beam energy for the treatment portal. Fourteen patients undergoing evaluation for this treatment were examined with intraoperative ultrasonography (IUS) to aid therapy planning. IUS study required little additional time and readily delineated tumor size, depth, and relationship to adjacent structures. In two patients, the US and operative findings provided information that precluded use of IRT. The traditional method of tumor evaluation by intraoperative palpation and inspection was consistently enhanced by the use of IUS findings. IUS is a valuable adjunct that improves the accuracy of therapy planning for IRT.