The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/- 8.1 cells, which increased to 84.4 +/- 5.7 and 125.5 +/- 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/- 6.0 and 40.3 +/- 5.0, respectively) and then doubled on day 7 (80.6 +/- 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/- 4.0 and 41.9 +/- 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/- 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.     

Increase in plasma 5 alpha-androstane-3 alpha,17 beta-diol glucuronide as a marker of peripheral androgen action in hirsutism: a side-effect induced by cyclosporine A

Dose-dependent hypertrichosis is a common dermatological side-effect affecting the majority of patients treated with cyclosporine A (CSA). Previous studies have not demonstrated the influence of CSA on specific sex hormone levels. The aim of this study is to investigate whether CSA increases the activity of 5 alpha-reductase, an enzyme which transforms androgens into dihydrotestosterone in peripheral tissues. The metabolite which best reflects this activity is 5 alpha-androstane-3 alpha,17 beta-diol glucuronide (Adiol G). The study was carried out on 49 insulin-dependent diabetes patients participating in the double-blind "Cyclosporine-Diabète-France" clinical trial, of which 28 were treated with CSA (16 males and 12 females), and 21 received only placebo (10 males and 11 females). All patients underwent extensive clinical and laboratory evaluations prior to and during the present study. In addition to Adiol G, testosterone (T), dehydroepiandrosterone sulfate (DHEA S) and sex hormone-binding globulin (SHBG) were assayed. Levels of Adiol G increased significantly in CSA-treated groups: males, 11.86 +/- 2.58 vs 7.83 +/- 2.30 nmol/l; females, 4.48 +/- 2.70 vs 2.10 +/- 1.22 nmol/l; P less than 0.02 (comparison of means). There were no significant differences in this parameter before and during treatment in either the male or female placebo groups (paired t-test). During the treatment period, T, DHEA S, SHBG and the T/SHBG ratio did not significantly change with respect to their baseline values in any of the groups studied (comparison of means). Comparison (using paired t-test) showed a significant increase of DHEA S in CSA-treated groups: males, delta = 3.08 +/- 3.33 nmol/l, P less than 0.01; females, delta = 0.98 +/- 1.13 nmol/l, P less than 0.05. In conclusion, it is possible that CSA induces hypertrichosis or hirsutism by increasing 5 alpha-reductase activity in peripheral tissues. Nevertheless the role of increased DHEA S as a possible Adiol G precursor cannot be excluded.     

The multidrug resistance P-glycoprotein modulates cell regulatory volume decrease.

Cell volume is frequently down-regulated by the activation of anion channels. The role of cell swelling-activated chloride channels in cell volume regulation has been studied using the patch-clamp technique and a non-invasive microspectrofluorimetric assay for changes in cell volume. The rate of activation of these chloride channels was shown to limit the rate of regulatory volume decrease (RVD) in response to hyposmotic solutions. Expression of the human MDR1 or mouse mdr1a genes, but not the mouse mdr1b gene, encoding the multidrug resistance P-glycoprotein (P-gp), increased the rate of channel activation and the rate of RVD. In addition, P-gp decreased the magnitude of hyposmotic shock required to activate the channels and to elicit RVD. Tamoxifen selectively inhibited both chloride channel activity and RVD. No effect on potassium channel activity was elicited by expression of P-gp. The data show that, in these cell types, swelling-activated chloride channels have a central role in RVD. Moreover, they clarify the role of P-gp in channel activation and provide direct evidence that P-gp, through its effect on chloride channel activation, enhances the ability of cells to down-regulate their volume.     

Cloning and Expression of a 5-Hydroxytryptamine7 Receptor Positively Coupled to Adenylyl Cyclase

Abstract: In a number of different cell types, phosphorylation of a 63-kDa protein has been shown to increase rapidly in response to stimuli that lead to an increase in intracellular calcium. Here, a stimulus-sensitive protein at this molecular weight is identified in PC12 cells and rat cortical synaptosomes as phosphoglucomutase. In addition, the added phosphate is shown to be in an oligosaccharide terminating in phosphodiester-linked glucose. In synaptosomes, incorporated radioactivity, following incubation with [¹⁴C]glucose or the [β-³⁵S]phosphorothioate analogue of UDP-glucose, was found to increase within 5 s of stimulation and return to baseline within 25 s. Despite the many pathways utilizing glucose, this was the only detectable protein glycosylation observed in synaptosomes. These results indicate that cytoplasmic glycosylation is reversible and rapidly regulated, and suggest that phosphoglucomutase undergoes an alteration in function and/or topography in response to increases in intracellular calcium.     

Thermophilic glucoamylase from Talaromyces flavus

The heat-resistant mold, Talaromyces flavus , was found to produce a thermophilic glucoamylase that exhibited the highest activity at 50°C and in the pH range of 4.0–4.8. The K _m and V _max values of the crude enzyme for amylopectin were 0.21% and 16.7 mg glucose 1^-1, min^-1, respectively. The molecular weight of the enzyme as estimated by the gel filtration method was 42 kDa.     

Novel (E)-3-(3-Oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides as Histone Deacetylase Inhibitors: Design,Synthesis and Bioevaluation

Herein, we report the design, synthesis and evaluation of novel (E)-3-(3-oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides ( 4 a – i , 7 a – g ) targeting histone deacetylases. Three human cancer cell lines were used to test the cytotoxicity of the synthesized compounds (SW620, colon; PC-3, prostate; NCI−H23, lung cancer); inhibitory activity towards HDAC; anticancer activity; as well as their impact on the cell cycle and apoptosis. As a result, compounds 4 a – i bearing the alkyl substituents seemed to be less potent than the benzyl-containing compounds 7 a – g in all biological assays. Compounds 7 e – f were found to be the most active HDAC inhibitors with IC₅₀ of 1.498±0.020 μM and 1.794±0.159 μM, respectively. In terms of cytotoxicity and anticancer assay, 7 e and 7 f also showed good activity with IC₅₀ values in the micromolar range. In addition, the cell cycle and apoptosis of SW620 were affected by compound 7 f in almost a similar manner to that of reference compound SAHA. Docking assays were carried out for analysis the binding mode and selectivity of this compound toward 8 HDAC isoforms. Overall, our data confirmed that the inhibition of HDAC plays a pivotal role in their anticancer activity.