Colorimetric assay of blood coagulation factor XIII in plasma

In this new colorimetric assay for Factor XIII in plasma, 5-(biotinamido)pentylamine is used as the amine substrate. Factor XIII, a zymogen, is transformed by thrombin and Ca2+ to active Factor XIIIa, and the incorporation of 5-(biotinamido)pentylamine into N,N-dimethylcasein is used to measure catalytically active Factor XIIIa. The biotinylated enzymatic product is immobilized onto 96-well microtiter plates, complexed with streptavidin-beta-galactosidase, and the absorbance at 405 nm is monitored for production of p-nitrophenol from p-nitrophenyl-beta-D-galactopyranoside. Concentrations of N,N-dimethylcasein, 5-(biotinamido)pentylamine, Ca2+, and thrombin were chosen to allow near-maximum velocity of amine incorporation. A linear relationship was obtained between assay product and plasma volume, from 0.5 to 50 microL of plasma. Results correlated well (r greater than 0.924) with those from the most frequently utilized radiometric filter-paper assay for Factor XIII. The method appears to be ideal for routine diagnostic estimation of Factor XIII in plasma because of its simplicity, its lack of use of radioisotopes, and its potential for assay of large numbers of samples by use of microtiter plates and automated plate readers.     

Transforming Growth Factor-β1 Induces Apoptosis through Down-Regulation of c-mycGene and Overexpression of p27Protein in Cervical Carcinoma

Transforming growth factor-β1 (TGF-β1) is known to be a potent growth inhibitor for many cell types, including most epithelial cells. In skin keratinocytes, TGF-β1 has been shown to inhibit growth and to rapidly reduce c-mycexpression. However, the molecular mechanism of TGF-β1 action on cell growth of cervical carcinoma has not yet been elucidated. We thus assessed the effect of TGF-β1 on the growth of cervical carcinoma cell lines. Two cervical squamous carcinoma cell lines, CUMC-3 and CUMC-6, were incubated with varying concentrations of TGF-β1, and growth inhibition was evaluated with tetrazolium-based colorimetric assay. After culture in TGF-β1 for 24 h, inhibition of growth was detected in a dose-dependent manner at concentrations of 0.1–10 ng/ml in both cell lines. This effect of TGF-β1 on cultured carcinoma cells was associated with apoptotic process including oligonucleosomal ladder DNA and apoptotic body formations. Northern blot analysis revealed c-mycmRNA expression was suppressed by 10 ng/ml of TGF-β1 following 3 h of treatment in both cell lines. Western blot analysis showed that the level of p27^Kip1protein was increased after TGF-β1 treatment in both cell lines. These results suggest that the mechanisms by which TGF-β1 inhibits the growth of cervical carcinoma are complex and may include effects on down-regulation of c-mycgene, and overexpression of p27^Kip1protein.     

Use of intraoperative ultrasonography during hepatic transplantation.

The aim of this study was to evaluate if intraoperative vascular ultrasonography is of clinical value in the perioperative management of hepatic transplant patients. Fifteen intraoperative ultrasonographic examinations were performed on 13 patients (five female, eight male) during transplantation. These patients had clinically suspected vascular compromise. Among the 13 patients studied intraoperatively, five were correctly diagnosed as having hemodynamically significant vascular compromise. Of the intraoperative vascular sonographic examinations, the results of 13 were in concordance with the surgical impression as to whether further intervention was necessary or if the procedure could be terminated. Intraoperative sonography demonstrates potential to be of aid to the surgeon in recognition of vascular compromise.     

C2H2-171 : A novel human cDNA representing a developmentally regulated poz domain/zinc finger protein preferentially expressed in brain

We describe a novel human zinc finger cNDA, C2H2-171. This cDNA represents an mRNA which encodes a protein of 484 amino acids and a calculated molecular weight of 54 kD. Four zinc finger-like domains are found in the C-terminal end of the protein. At the N-terminus, C2H2-171 contains a POZ/tramtrack-like domain similar to that found in the tumor associated zinc finger proteins LAZ-3/BCL-6 and PLZ-F, as well as in non-zinc finger proteins. C2H2-171 RNA is preferentially expressed in the brain, and increases during the course of murine development, with maximal expression in the adult. C2H2-171 RNA is differentially expressed in brain regions, with the highest level of expression in the cerebellum. C2H2-171 RNA was expressed at high levels in primary cerebellar granule cell neurons compared to astrocytes. The gene encoding C2H2-171 is highly conserved in vertebrates, and maps to the terminus of human chromosome 1 (1q44-ter). This chromosomal location is associated with a number of cytogenetic aberrations including those involving brain developmental anomalies and tumorigenesis. These data suggest that C2H2-171 may play an important role in vertebrate brain development and function.     

Inter- and intra-individual variability of non-invasive tear break-up time in Hong Kong Chinese

Non-invasive tear break-up time (NITBUT) has been proposed as a measure of tear film integrity which is superior to the more commonly used tear break-up time (TBUT), since it does not alter the volume or the physicochemical properties of the tear layer by the addition of fluorescein. We measured NITBUT by measuring the time taken for distortions or discontinuities to appear in the reflected image of a grid pattern which covered about 80 per cent of the corneal surface. NITBUT measures were made 100 times on seven Hong Kong Chinese subjects with up to 20 consecutive measures being made on a single day. We also measured NITBUT on one occasion on an unselected population of 52 Hong Kong Chinese subjects. NITBUT shows a skewed distribution in all subjects, with many shorter values and some extremely long values. There are statistically significant variations in NITBUT from day to day, and from subject to subject. The group of 52 subjects also had a skewed NITBUT distribution with many short values and some very long values. The arithmetic mean does not adequately represent NITBUT data, either for individual subjects or for this group of subjects. As many as five to eight measures may be necessary to gain a stable estimate of the NITBUT and stability of the measure is improved if extreme values are omitted. We recommend the use of nonparametric statistics to compare NITBUT values from day to day in or between subjects.     

The role of office hysteroscopy in in vitro fertilization.

Twenty-eight patients participated prospectively in a study to evaluate the impact of hysteroscopically detected uterine and cervical anomalies on the success rate of ET in an IVF-ET program. All participants had a normal intrauterine cavity by standard HSG. All the patients had a diagnostic office hysteroscopy under paracervical block before commencing COH. Because our IVF program does not include hysteroscopy as a requirement before undergoing IVF and because the significance of mild intrauterine abnormalities is not yet known, the hysteroscopic findings were not relayed to the personnel involved in the IVF-ET procedure. Sixteen patients (group I) had a normal hysteroscopic evaluation. Twelve patients (group II) had abnormal hysteroscopic findings including small uterine septa, small submucous fibroids, uterine hypoplasia and cervical ridges. Although no difference in patients or cycle characteristics was present, there was a significant difference in the clinical PR between patients in groups I and II. In conclusion, in an IVF-ET program patients with normal hysterography but abnormal hysteroscopic findings had a significantly lower clinical PR, demonstrating the importance of performing hysteroscopy before IVF-ET.     

Isolation and analysis of nephritic-producing immune complexes in Plasmodium berghei-infected mice.

A nephritic condition was developed by infecting Swiss Webster albino mice with the malarial parasite Plasmodium berghei NK 65. These animals were tested for urinary protein and the presence of circulating immune complexes using reagent strips and a polyethylene glycol (PEG) precipitation assay. The circulating immune complexes were isolated from the sera using both affinity chromatography and PEG precipitation and from the kidney by acid elution. The isolated complexes were dissociated into their individual components and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The components of the complexes were transferred to nitrocellulose sheets and probed for the presence of malarial antigens using a rabbit anti-P berghei antisera. The overall humoral response to the malarial parasite was evaluated using a radial immunodiffusion assay. The present study confirmed that the malarial-infected animals not only developed the nephritic condition (as evident by the high levels of proteinuria) but also, as indicated by the PEG assay, have the presence of high levels of circulating immune complexes in their serum. The apparent absence in the SDS gels of any abnormal protein bands followed by the inability of the Western blot to reveal any malarial antigens provides some of the strongest evidence to date that these malarial proteins are not directly involved in the circulating immune complexes believed to be responsible for producing this nephritic condition.