Effective reconstitution of sarcoplasmic reticulum Ca2+-ATPase using lubrol PX]

Ca2(+)-ATPase of sarcoplasmic reticulum was reconstituted in the proteoliposomes by the salting out procedure. Triton X-100, C12E8 and Lubrol PX were used for the solubilization of the Ca2(+)-ATPase. Using fluorescent probes (diS-C3-(5), chlortetracycline) as well pH-measuring method, the functional of the reconstituted Ca2(+)-ATPase was comparatively studied in three types of proteoliposomes. The efficiency of Ca2(+)-ATPase grew in the following detergent order: Triton X-100, C12E8, Lubrol PX.     

Flow cytometry analysis of DNA degradation in thymocytes of gamma-irradiated or hydrocortisone treated rats

The pattern of DNA degradation in thymocytes of irradiated or hydrocortisone-treated rats has been studied by means of flow cytometry of the cells, treated with probes specifically bound to the AT or GC-pairs of DNA. It has been shown that the death of thymocytes is accompanied by a decrease in their DNA content. The main features of the occurrence and accumulation of cells with a DNA content less than the normal diploid level correspond with those of internucleosomal DNA fragmentation: such cells appear after a 1 hour lag-period, their accumulation is prevented by cycloheximide injection and is lower at 300 Gy than at doses of 10 to 30 Gy. At the same time, no increase in permeability of the cell membrane to ethidium bromide was observed up to the sixth hour after irradiation. Most of the thymocytes dying under the action of irradiation or hydrocortisone are in the G0 or G1 phases of the cell cycle. The method used allows detection of the cells with cleaved but not removed DNA.     

The Latex Serologic Test for Syphilis—A New Screening Procedure

A new macroscopic screening test for syphilis, the Latex-sts test, is extraordinarily simple. After inactivation of the patient''s serum for 30 minutes at 56°C the test is performed by mixing the patient''s serum with latex particles coated with cardiolipin and a protein fraction obtained from the non-pathogenic Reiter strain of Treponema pallidum. Two to three minutes after mixing, the result of the test is observed on a ringed serologic plate. The sensitivity, specificity and reproducibility of the new test are equivalent to those of the qualitative Venereal Disease Research Laboratory tube test. The advantages of the Latex-sts are that it can be done in a short time, it is simple and it requires a minimum of laboratory equipment. The coated latex particles are stable for 12 months.     

Proteasome activity imaging and profiling characterizes bacterial effector syringolin A

Syringolin A (SylA) is a nonribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv syringae that can inhibit the eukaryotic proteasome. The proteasome is a multisubunit proteolytic complex that resides in the nucleus and cytoplasm and contains three subunits with different catalytic activities: β1, β2, and β5. Here, we studied how SylA targets the plant proteasome in living cells using activity-based profiling and imaging. We further developed this technology by introducing new, more selective probes and establishing procedures of noninvasive imaging in living Arabidopsis (Arabidopsis thaliana) cells. These studies showed that SylA preferentially targets β2 and β5 of the plant proteasome in vitro and in vivo. Structure-activity analysis revealed that the dipeptide tail of SylA contributes to β2 specificity and identified a nonreactive SylA derivative that proved essential for imaging experiments. Interestingly, subcellular imaging with probes based on epoxomicin and SylA showed that SylA accumulates in the nucleus of the plant cell and suggests that SylA targets the nuclear proteasome. Furthermore, subcellular fractionation studies showed that SylA labels nuclear and cytoplasmic proteasomes. The selectivity of SylA for the catalytic subunits and subcellular compartments is discussed, and the subunit selectivity is explained by crystallographic data.     

Changes in Ovaries and Uterus after Aglepristone Administration in the Third Week of Luteal Phase of Non-Pregnant Bitches

Objective

The mechanism of aglepristone action in the placentation time in the bitch remains unclear. The aim of this study was to describe the mechanism by which aglepristone influences ovaries and uterus and to measure the levels of steroid sex hormones in non-pregnant bitches.

Materials and Methods

Fourteen bitches assigned to a study (n=9) and control (n=5) group were given aglepristone and saline solution, respectively, on the 19th and 20th day after LH peak. On the 26th day after LH peak an ovariohysterectomy was performed. Blood samples were screened for estradiol and progesterone concentrations. Ovaries and uterine horns and bodies were isolated for histological and morphometrical diagnosis and immunohistochemistry analysis of α-estrogen and progesterone receptor expression.

Results

A decrease of progesterone (p<0.01) and no differences in total estrogen level in the study group were observed. There were no significant differences either in the histomorphometry or α-estrogen and progesterone receptors expression in ovaries. Increase in expression of progesterone receptors in endometrium without surface epithelium of horns (p<0.05), endometrial surface epithelium (p<0.05), myometrium of uterine body (p<0.01) and estrogen receptors in endometrium without surface epithelium of horns (p<0.05) was observed. Elevated estrogen receptors probably increased sensitivity of tissues to estrogens in the bloodstream and led to notable inflammation, haemorrhages, and hyperplasia in endometrium with mononuclear immune cell infiltration. The myometrium of horns and endometrium of uterine body of study bitches were significantly thicker than in the control group (p<0.05 and p<0.01). Furthermore myometrium of uterine body was thicker than myometrium of horns (p<0.001) and expression of progesterone receptors was higher in uterine body (p<0.01). No differences were observed between endometrium of horns and body within groups.

Conclusion

To the knowledge of the authors this is the first study, which describes the inflammatory effect developing in uterus in response to aglepristone administration, and attempts to elucidate its mechanisms.     

Gestational diabetes mellitus (GDM) decreases butyrylcholinesterase (BChE) activity and changes its relationship with lipids

Many conditions interfere with butyrylcholinesterase (BChE) activity, e.g., pregnancy or presence of the BCHE gene variant −116A can decrease activity whereas obesity and types I and II diabetes mellitus can increase activity. In this study, we examined BChE activity, −116A and 1615A BCHE gene variants, and anthropometric and biochemical variables associated with diabetes in patients with gestational diabetes mellitus (GDM) and in healthy pregnant women. BChE activity was measured spectrophotometrically using propionylthiocholine as substrate and genotyping of the −116 and 1615 sites of the BCHE gene was done with a TaqMan SNP genotyping assay. Three groups were studied: 150 patients with GDM, 295 healthy pregnant women and 156 non-pregnant healthy women. Mean BChE activity was significantly lower in healthy pregnant women than in women from the general population and was further reduced in GDM patients. BChE activity was significantly reduced in carriers of −116A in GDM patients and healthy pregnant women. Although GDM patients had a significantly higher mean body mass index (BMI) and triglycerides than healthy pregnant women, they had lower mean BChE activity, suggesting that the lowering effect of GDM on BChE activity was stronger than the characteristic enhancing effect of increased BMI and triglycerides.     

Determination of the radiosensitivity of interphase-dying cells in the thymus, spleen and bone marrow of rats by flow cytometry

The flow cytofluorometry of cells stained with a DNA-specific probe was used to determine the share of dying cells (containing less than 2C DNA) in thymus, spleen and bone marrow cells of irradiated rats. The cell death curves for spleen and bone marrow had a plateau by the 6th h, and for thymus, by the 10th h following irradiation with different doses. On the basis of the dose-response relationship the share of cells dying in the interphase was determined in each organ under study, and dose-response curves shaped. All the curves had no shoulder. Do was 3.0, 3.0 and 3.7 Gy for thymus, spleen, and bone marrow cells, respectively.