Effect of transport and storage temperature of ovaries on in vitro maturation of bitch oocytes

In this study, the effects of ovary transport and storage temperature on in vitro maturation of bitch oocytes were investigated. Ovaries were collected from 23 mature bitches and one randomly selected ovary of each pair (n=23 pairs) was transported in physiologic saline at 4 degrees C, while the other one at 35-38 degrees C for 2-4h. A total of 316 cumulus oocyte complexes (COCs) were obtained from the 4 degrees C group and 301 COCs from the 35-38 degrees C group. All COCs were matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), essential and non-essential amino acids at 38 degrees C in a humidified 5% CO2, 5% O2, and 90% N2 atmosphere for 72 h. At the end of the in vitro maturation period, nuclear maturation of oocytes was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), undetermined nuclear maturation (UDNM), and MI+MII. The nuclear maturation rates to MI, MII, and MI+MII stages were 60.44%, 10.75%, and 71.20% in the 4 degrees C group and 37.20%, 7.64%, and 45.85% in the 35-38 degrees C group, respectively. The data demonstrated that oocytes obtained from ovaries transported at 4 degrees C had higher maturation rates than from the ones transported at 35-38 degrees C (p<0.001).     

Differential in vitro inhibition of polyphenoloxidase from a wild edible mushroom Lactarius salmonicolor

The polyphenol oxidase (LsPPO) from a wild edible mushroom Lactarius salmonicolor was purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. At the optimum pH and temperature, the K_M and V_Max values of LsPPO towards catechol, 4-methylcatechol and pyrogallol were determined as 0.025 M & 0.748 EU/mL, 1.809 × 10^{? 3} M & 0.723 EU/mL and 9.465 × 10^{? 3} M & 0.722 EU/mL, respectively.

Optimum pH and temperature values of LsPPO for the three substrates above ranged between the pH 4.5–11.0 and 5–50°C. Enzyme activity decreased due to heat denaturation with increasing temperature. Effects of a variety of classical PPO inhibitors were investigated opon the activity of LsPPO using catechol as the substrate. IC₅₀ values for glutathione, p-aminobenzenesulfonamide, L-cysteine, L-tyrosine, oxalic acid, β-mercaptoethanol and syringic acid were determined as 9.1 × 10^{? 4}, 2.3 × 10^{? 4} M, 1.5 × 10^{? 4} M, 3.8 × 10^{? 7} M, 1.2 × 10^{? 4} M, 4.9 × 10^{? 4} M, and 4 × 10^{? 4} M respectively. Thus L-tyrosine was by far the most effective inhibitor. Interestingly, sulfosalicylic acid behaved as an activator of LsPPO in this study.     

Genome sequences of two stress-tolerant Campylobacter jejuni poultry strains, 305 and DFVF1099

Campylobacter jejuni is a food-borne pathogen with a high prevalence in poultry meat, which in fresh unfrozen condition is the major source of campylobacteriosis. C. jejuni strains DFVF1099 and 305 are considered tolerant to several environmental stresses (T. Birk et al., J. Food Prot. 73:258-265, 2010; S. L. On et al., Int. J. Med. Microbiol. 296:353-363, 2006). Here, we report the genome sequences of C. jejuni 305 and DFVF1099, a turkey and a chicken isolate, respectively.     

Carbonic anhydrase inhibitors: Inhibition of the β-class enzyme from the yeast Saccharomyces cerevisiae with sulfonamides and sulfamates

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, has been cloned, purified, characterized kinetically and investigated for its inhibition with a series of sulfonamides and one sulfamate. The enzyme showed high CO₂ hydrase activity, with a k_cat of 9.4 × 10⁵ s^?1, and k_cat/K_M of 9.8 × 10⁷ M^?1 s^?1. Simple benzenesulfonamides substituted in 2-, 4- and 3,4-positions of the benzene ring with amino, alkyl, halogeno and hydroxyalkyl moieties were weak scCA inhibitors with K_Is in the range of 0.976–18.45 μM. Better inhibition (K_Is in the range of 154–654 nM) was observed for benzenesulfonamides incorporating aminoalkyl/carboxyalkyl moieties or halogenosulfanilamides; benzene-1,3-disulfonamides; simple heterocyclic sulfonamides and sulfanilyl-sulfonamides. The clinically used sulfonamides/sulfamate (acetazolamide, ethoxzolamide, methazolamide, dorzolamide, topiramate, celecoxib, etc.) generally showed effective scCA inhibitory activity, with K_Is in the range of 82.6–133 nM. The best inhibitor (K_I of 15.1 nM) was 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. These inhibitors may be useful to better understand the physiological role of β-CAs in yeast and some pathogenic fungi which encode orthologues of the yeast enzyme and eventually for designing novel antifungal therapies.     

Carbonic anhydrase inhibitors: purification and inhibition studies of pigeon (Columba livia var. domestica) red blood cell carbonic anhydrase with sulfonamides

A carbonic anhydrase (CA, EC 4.2.1.1) from red blood cells of pigeons (Columba livia var. domestica), clCA, was purified to homogeneity. Its kinetic parameters for the CO(2) hydration reaction were measured. With a k(cat)/K(m) of 1.1?×?10(8) M(-1) s(-1), and a k(cat) of 1.3?×?10(6) s(-1), clCA has a high activity, similar to that of the human isoform hCA II. A group of 25 aromatic/heterocyclic sulfonamides incorporating the sulfanilamide, homosulfanilamide, benzene-1,3-disulfonamide, and acetazolamide scaffolds showed variable inhibitory activity against the pigeon enzyme, with K(I)s in the range of 1.9-3460?nM. Red blood cells of pigeons, like those of ostriches, contain thus just one CA isoform, unlike the blood of mammals, which normally contain two isoforms, one of low (CA I-like) and one of very high activity (CA II-like). However, from the sulfonamide inhibition viewpoint, the pigeon enzyme was more similar to hCA II than to the ostrich enzyme.     

Extreme Entropy-Enthalpy Compensation in a Drug-Resistant Variant of HIV-1 Protease

The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug-resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5-15 kcal/mol, while losing only 1-3 kcal/mol in total binding free energy for any of six FDA-approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wild-type protease and another drug-resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug-resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design.     

Enhanced differentiation of splenic plasma cells but diminished long-lived high-affinity bone marrow plasma cells in aged mice

In the present work, we have dissected the mechanisms responsible for the impaired humoral responses in aging. We found that there was a substantially higher level of Ab-forming cells in the spleens of aged mice than that of young controls. However, the number of high-affinity, class-switched Ab-forming cells was severely decreased in the spleen of aged mice. The accumulation of low-affinity IgM Ab-forming cells in the spleens of aged animals was not due to a deficiency in isotype switching because the number of total IgG1 splenic plasma cells was not significantly reduced. Remarkably, plasma cells of both low and high affinity were significantly diminished in the bone marrow of aged mice compared with that of young mice. The results from reconstitution experiments showed that aged bone marrow was less supportive for plasma cells derived from young splenic B cells. These findings suggest that humoral immune deficiency in aging results from at least two mechanisms: the inability to generate sufficient numbers of high-affinity Ab-forming cells, which is a result of diminished germinal center reaction, and the defective bone marrow environment that has diminished ability to support the selection and survival of long-term Ab-forming cells.