Androgenesis and homozygous gynogenesis in muskellunge (Esox masquinongy): Evaluation using flow cytometry

The purpose of this work was to study the effects of ultraviolet (UV) irradiation on denucleation of eggs and investigate the heat-shock conditions for diploidization for induction of androgenesis in muskellunge, Esox masquinongy. Several egg incubation media, including saline, Ringer's solution, and Ringer's solution supplemented with bovine serum albumin (BSA), were found suitable to maintain the egg fertility as high as in muskellunge ovarian fluid. The optimal doses of UV radiation were 660–1320 J/m², at which 100% haploid larvae were produced at a hatching rate of 22.5 ± 2.8%. UV irradiation at low doses (165–330 J/m²) generated abnormal larvae, which were morphologically identical to haploids. Using a flow cytometry method, it was found that cellular DNA content of these larvae was close to that of diploids but significantly lower in value and had a wider distribution (expressed as coefficient of variation) than that of control fish. This suggested that a low dose of UV irradiation might cause gene mutations, alteration of chromosomal conformation and fragmentation, but did not prevent maternal DNA from participating in mitotic division. Interference of maternal DNA residues could be another reason for the poor viability of androgenetic fish. A high dose of UV radiation (1980 J/m²) caused development of severely deformed embryos, indicating that UV radiation also damaged molecules in the eggs other than the denucleation. Our results suggest that classic color and allozyme markers might not be sufficient to prove a complete androgenesis. In order to optimize time and duration of shock for induced diploidization, we investigated the heat-shock conditions for inhibiting the first mitotic cleavage through induction of homozygous gynogenesis. We found that heat-shock treatment at 31°C for 9 min starting at 1.4τ₀ (a dimensionless factor describing progress in embryo development) after fertilization produced the highest percentage of diploids at hatching. Mol. Reprod. Dev. 49:10–18, 1998. © 1998 Wiley-Liss, Inc.     

Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain

Most glucoamylases (alpha-1,4-D-glucan glucohydrolase, EC 3.2.1.3) have structures consisting of both a catalytic and a starch binding domain. The structure of a glucoamylase from Saccharomycopsis fibuligera HUT 7212 (Glu), determined a few years ago, consists of a single catalytic domain. The structure of this enzyme with the resolution extended to 1.1 A and that of the enzyme-acarbose complex at 1.6 A resolution are presented here. The structure at atomic resolution, besides its high accuracy, shows clearly the influence of cryo-cooling, which is manifested in shrinkage of the molecule and lowering the volume of the unit cell. In the structure of the complex, two acarbose molecules are bound, one at the active site and the second at a site remote from the active site, curved around Tyr464 which resembles the inhibitor molecule in the 'sugar tongs' surface binding site in the structure of barley alpha-amylase isozyme 1 complexed with a thiomalto-oligosaccharide. Based on the close similarity in sequence of glucoamylase Glu, which does not degrade raw starch, to that of glucoamylase (Glm) from S. fibuligera IFO 0111, a raw starch-degrading enzyme, it is reasonable to expect the presence of the remote starch binding site at structurally equivalent positions in both enzymes. We propose the role of this site is to fix the enzyme onto the surface of a starch granule while the active site degrades the polysaccharide. This hypothesis is verified here by the preparation of mutants of glucoamylases Glu and Glm.     

Atresia of large ovarian follicles of the rat

In the rat, at the beginning of pregnancy a cohort of antral follicles develops until the preovulatory stage. However, these follicles, differentiating in the hyperprolactinemic milieu, produce only small amount of estradiol, do not ovulate and undergo rapid degeneration. They constitute an interesting physiological model of atresia. In the present study, we analysed the development and subsequent degeneration of such follicles. The study was performed on Wistar female rats killed in succession between days 1-9 of pregnancy. Excised ovaries were submitted to a routine histological procedure. Paraffin sections were subjected to hematoxylin and eosin staining or in situ DNA labelling. Histological and TUNEL staining revealed that the investigated group of follicles grew slower than that on the corresponding days of the estrous cycle and reached a preovulatory size and morphological appearance on day 5 of pregnancy. They did not ovulate and between days 6 and 9 of pregnancy an increasing number of apoptotic cells appeared within these follicles. They were localized predominantly in the antral granulosa layer, especially near the cumulus oophorus complex (COC) and in the region linking the COC with the follicular wall. The COC and the theca layer were much less affected. In late stages of atresia, also cumulus cells became apoptotic but degenerating oocytes did not exhibit positive TUNEL staining. Only limited number of the theca cells have undergone apoptosis and generally they were not hypertrophied. Our findings indicate that much smaller than normal amount of intrafollicular estradiol was sufficient to support a normal, according to the morphological criteria, although slower development of antral follicles to the late preovulatory stage.     

Polish variant of glucose-6-phosphate dehydrogenase (G-6-PD lublin)

Summary A new, favism-inducing variant of glucose-6-phosphate dehydrogenase in erythrocytes is described in a Polish family. The enzyme activity has been 0–4% of normal. The enzyme displayed normal heat stability, electrophoretic mobility 105–110% of normal, K_m for NADP: 16–22 M, K_m for G-6-P: 26 M, and the utilization of 2-deoxy-G-6-P: 2–3%.     

Effects of Chromium Brewer’s Yeast Supplementation on Body Mass,Blood Carbohydrates,and Lipids and Minerals in Type 2 Diabetic Patients

Chromium(III) is considered as an essential element for carbohydrate and lipid metabolism. The aim of this clinical study was to evaluate the efficacy of Cr brewer’s yeast supplementation on body mass, carbohydrate, lipids and mineral indices in type 2 diabetic patients. Twenty adult type 2 diabetic subjects (11 males and 9 females aged 37–63) were supplemented with Cr brewer’s yeast in dosages of 500 μg Cr/person/day or placebo for 8 weeks in a double-blind, placebo-controlled crossover design. It was found that supplemental Cr did not affect body mass, blood lipid profile, resistin levels, and the serum and hair Zn, Fe, and Cu levels, but increased serum Cr (by 116%) and hair Cr (by 20.6%) concentrations and improved some blood carbohydrate indices (significant increase in the β cell function index by 18.8%) in type 2 diabetic patients. In conclusion, Cr brewer’s yeast has a weak hypoglycemic potential, but does not affect body mass, blood biochemical profile, and microelement levels in type 2 diabetic subjects.     

FMRFamide and membrane stretch as activators of the Aplysia S-channel.

The long-standing distinction between channels and transporters is becoming blurred, with one pump protein even able to convert reversibly to a channel in response to osmotic shock. In this light, it is plausible that stretch channels, membrane proteins whose physiological roles have been elusive, may be transporters exhibiting channel-like properties in response to mechanical stress. We recently described a case, however, where this seems an unlikely explanation. An Aplysia K channel whose physiological pedigree is well established (it is an excitability-modulating conductance mechanism) was found able to be activated by stretch. Here we establish more firmly the identity of this Aplysia conductance, the S-channel, as a stretch channel. We show that the permeation and fast kinetic properties of the stretch-activated channel and of the FMRFamide-activated S-channel are indistinguishable. We have also made progress in extending the kinetic analysis of the stretch channel to situations of multiple channel activity. This analysis implements a novel renewal theory approach and is therefore explained in some detail.     

Effects of inhibition of RNA or protein synthesis on CHO cell cycle progression

Chinese hamster ovary (CHO) cells, synchronized by selective detachment at mitosis, were treated with various concentrations of actinomycin D (AMD) or cycloheximide (CHX) either immediately, or 1, 2, or 3 hr after mitosis. Since the minimum duration of G₁ phase in these cultures was 3.4 hr, the addition of RNA or protein synthesis inhibitors took place at the beginning, first third, second third, or end (G₁–S boundary) of G₁ phase. The kinetics of exit from G₁ phase, the rate and extent of traverse of S phase, and the reaccumulation of RNA were estimated under each set of growth conditions by flow cytometry of acridine orange-stained cells. A mathematical model was constructed to describe the trajectories of the cell populations with respect to their increase in RNA and DNA content in the absence or presence of the inhibitor. The chronologic synchrony imposed on the CHO cell population began to decay within 3 hr, resulting in stochastic entrance of cells into S phase in the absence of inhibitor. Addition of AMD or CHX at 0, 1, 2, or 3 hr after mitosis, regardless of the inhibitor concentration, did not provide evidence of a critical restriction point in G₁ beyond which cells were committed to enter S phase and were no longer sensitive to moderate suppression of RNA or protein synthesis. The observed kinetics of cell entrance into and traverse of S phase were consistent with an inherently heterogenous response to serum stimulation occurring at or just after cell division.