Influence of thyroid hormones on the human ATP synthase β-subunit gene promoter

The action of thyroid hormones on the expression of the mitochondrial ATP synthase -subunit gene (ATPsyn) is controversial. We detected a binding site for the thyroid hormone receptor between-366 and-380 in the human ATPsyn gene by DNase I footprint analysis and band-shift assays. However, expression vectors in which the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the 5 upstream region of ATPsyn gene were unresponsive to T₃ when transiently transfected to HepG2 or GH4C1 cells. CAT constructs driven by the rat phosphoenolpyruvate carboxykinase (PEPCK) or the growth hormone (GH) promoters were stimulated several fold by T₃ in parallel experiments. It is proposed that the biological effects of thyroid hormones on the ATPsyn expression occur through indirect mechanisms.     

In vitro penetration assay of boar sperm fertility: effect of various factors on the penetrability of immature pig oocytes

The present study was designed 1) to examine the influence of cumulus cells, ovary storage time and oocyte size on the penetrability of immature pig oocytes, and 2) to investigate the effect of 2 methods of treating the semen from different boars on the inter-assay variability of homologous in vitro penetration tests of boar sperm fertility. In Experiment 1, cumulus oocyte complexes, oocytes with spontaneous loss of the cumulus cells during collection, and oocytes mechanically stripped of cumulus cells were used. No differences were observed in oocyte penetrability among the 3 types of oocyte, although mechanical removal of the cumulus caused an increase (P < 0.005) in the degeneration rate compared with the other oocyte types. In Experiment 2, the oocytes were recovered from ovaries kept in PBS (30 degrees C) for 2, 4 or 6 h after slaughter of prepuberal gilts. Ovary storage did not modify the penetrability of oocytes but increased (P < 0.02) their degeneration rates. In Experiment 3, the diameters of fresh oocytes were determined after co-incubation with spermatozoa. They were classified into 4 groups according to diameter: A) < 105 microm, B) 105-115 microm, C) 116-120 microm and D) > 120 microm. Oocytes from Groups C and D exhibited higher (P < 0.05) penetrability than oocytes from the other groups. In Experiment 4, stored, diluted spermatozoa from 4 boars were pretreated by centrifugation at 50 x g for 3 min and subsequent concentration of the supernatants at 1,200 x g for 3 min. The pellets were treated (washed twice and preincubated for 40 minutes) before co-incubation with immature oocytes or used directly as untreated samples (unwashed and non-preincubated). A boar effect (P < 0.001) was evident for the parameters of in vitro penetration, independently of sperm treatment. When the oocytes were inseminated with untreated spermatozoa, the effects of the replicate and the boar-by-replicate interaction on the variability in oocyte penetrability were not significant. The results of this study indicate that the use of standardized immature pig oocytes and stored untreated, diluted spermatozoa can provide a useful method for optimizing the homologous in vitro penetration (hIVP) assay of boar fertility.     

Effect of oocyte-sperm co-incubation on acrosome reaction in the goat

The induction of acrosome reaction of goat spermatozoa was investigated. The acrosomal status of spermatozoa was determined by a triple-staining technique. The effect of the presence of goat oocytes on the proportion of acrosome-reacted spermatozoa was also determined. Ovulated oocytes were obtained from superstimulated adult goats. Other sources of oocytes were adult and prepubertal goats; oocytes from both sources were maturated in vitro. There was an increase in the percentage of acrosome-reacted spermatozoa from 4% +/- 0.98 to 9% +/- 1.41 when oocytes from adult females were used. Similar induction rates were measured with prepubertal and adult oocytes maturated in vitro (10.4% +/- 2.06 and 8.75% +/- 1.06, respectively). The influence of several qualities of cumulus oophorus as well as the presence of zona pellucida was also investigated. No significant differences were obtained with any cumulus oophorus or zona pellucida oocyte complexes. Although oocyte quality is important for high fertilization rates, it does not seem to be crucial for the induction of acrosome reaction.     

Studies on Stenostomum grande Child, 1902 (Platyhelminthes,Catenulida): fine structure of the digestive tract and the endocytotic activity of the gastrodermis

Abstract The digestive tract and its endocytotic activity in the catenulid Stenostomum grande were studied by electron microscopy. The pharynx was typical of the simplex type. At the mouth, between the integumental epithelium and the pharyngeal epithelium proper, was a transition zone. Among the epithelial cells of this transition were monociliated sensory cells and the necks of bucco-pharyngeal secretory cells of two types. The pharyngeal epithelium proper was densely ciliated, with long ciliary rootlets and mitochondria. It was surrounded by two layers of muscles. The gastrodermis consisted of phagocytes and typical secretory Minotian cells. It was underlain by a delicate basal lamina and muscle fibers. Distinctive of the phagocytes was the presence of differentiated cilia, cup-shaped mitochondria, and vacuoles with dense inclusions. Morphological differences between pharyngeal and gastrodermal cilia suggest functional differences. Experiments using latex beads as tracers and the identification of acid phosphatase in cytoplasmic vacuoles pointed to a high level of endocytotic and digestive activity in the phagocytes. Our data demonstrate that the basic structure of the digestive tract in S. grande conforms well to that of other free-living platyhelminths, but it does have ultrastructural peculiarities.     

The biosynthesis of the lantibiotics epidermin,gallidermin, Pep5 and epilancin K7

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.Abbreviations Dha 2,3-didehydroalanine - Dhb 2,3-didehydrobutyrine - Lan lanthionine - Melan methyllanthionine     

Anaerobic digestion of palm oil mill effluent and condensation water waste: an overall kinetic model for methane production and substrate utilization

The process of anaerobic digestion is viewed as a series of reactions which can be described kinetically both in terms of substrate utilization and methane production. It is considered that the rate limiting factor in the digestion of complex wastewaters is hydrolysis and this cannot be adequately described using a Monod equation. In contrast readily assimilable wastewaters conform well to this approach. A generalized equation has thus been derived, based on both the Monod and Contois equations, which serves extreme cases. The model was verified experimentally using continuous feed anaerobic digesters treating palm oil mill effluent (POME) and condensation water from a thermal concentration process. POME represents a complex substrate comprising of unhydrolyzed materials whereas the condensation water is predominantly short chain volatile fatty acids. Substrate removal and methane production in both cases could be predicted accurately using the generalized equation presented.List of Symbols A (=K_skY/K_h) Kinetic parameter - B Specific methane yield, 1 of CH₄/g of substrate added B₀ Maximum specific methane yield, 1 of CH₄/g of substrate added at infinity - C Empirical constant in Contois equation - F Volumetric substrate removal rate, g/l day - k Hydrolysed substrate transport rate coefficient, 1/days - K (=YC) Kinetic parameter in Chen-Hashimoto equation - K _h Substrate hydrolysis rate coefficient, 1/days - K _s Half-saturation constant for hydrolysed substrate, g/l - M _v Volumetric methane production rate, 1 of CH₄/l day - MS Mineral solids, g/l - MSS Mineral suspended soilds, g/l - POME Palm oil mill effluent - R (=S_r/S_T0) Refractory coefficient - S _h Concentration of hydrolysed substrate, g/l - S _u Intracellular concentration of hydrolysed substrate, g/l - S ₀ Input biodegradable substrate concentration, g/l - S Biodegradable substrate concentration in the effluent or in the digester, g/l - S _r Refractory feed substrate concentration, g/l - S _T0 (=S₀+S_r) Total feed substrate concentration, g/l - S _T (S+S_r) Total substrate concentration in the effluent, g/l - TS Total solids, g/l - TSS Total suspended solids, g/l - VFA Total volatile fatty acids, g/l - VS Volatile solids, g/l - VSS Volatile suspended solids, g/l - X Biomass concentration, g/l - Y Biomass yield coefficient, biomass/substrate mass - Hydraulic retention time, days. - Specific growth rate of microorganisms, l/days - _m Maximum specific growth rate of microorganisms, l/days The authors wish to express their gratitude to the Departamento de Postgrado y Especialización del CSIC and to the Consejería de Educación y Ciencia de la Junta de Andalucia for their financial support of this work.     

Studies on homoveratric acid transformation by the ligninolytic fungus Pleurotus eryngii

Homoveratric acid (HVA) degradation was observed in cultures of Pleurotus eryngii lacking lignin peroxidase (LiP) activity. Extracellular enzymes seemed responsible for this transformation, and the lack of activity after ultrafiltration of the culture liquid suggests that the presence of some low-molecular-size compounds is required. This hypothesis is supported by rapid HVA transformation after addition of the synthetic laccase substrate 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to the ultrafiltered liquid. HVA transformation by the extracellular enzymes from P. eryngii takes place via C-C breakdown and formation of veratryl alcohol, which is further transformed into veratraldehyde. The same major compounds were found during HVA transformation by LiP from Phanerochaete chrysosporium, but this reaction was not stimulated by ABTS. Although the involvement of other enzymes cannot be ruled out, purified laccase from Pleurotus eryngii caused the same HVA transformation pattern in presence of ABTS. Moreover, veratryl alcohol oxidation by P. eryngii laccase was demonstrated in the presence of ABTS. These results suggest that enzymatic systems lacking LiP could be responsible for natural degradation of lignin.