Steroidogenic activity of chicken ovary during pause in egg laying

The present study was designed (i) to assess the changes in the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and cytochrome P450 aromatase (P450arom) in the ovaries of hens which are subjected to a pause in egg laying by fasting, and (ii) relate these changes with progesterone (P(4)) and estradiol (E(2)) production in the ovary. Hy-Line Brown laying hens (n=90) were fasted for 5 days with water deprivation only on day 3 and subsequently fed every second day up to day 13 and then ad libitum. Birds were euthanized (n=18) on day 0, 3, 6, 9 and 16 of the experiment. The activities of 3beta-HSD and P450arom were evaluated in stroma with cortical follicles (<1mm) and in the wall of white non-hierarchical (1-8 mm) and yellow hierarchical follicles (>8 mm) by histochemical and immunohistochemical method, respectively. Ovarian P(4) and E(2) were measured radioimmunologically. Hens stopped egg laying on day 4 of the experiment and pause in egg laying lasted up to day 12. The hens then began to gradually resume egg laying and on day 16 all hens laid eggs. It was found that during the pause in egg laying: (i) the activity of 3beta-HSD in stroma and normal white follicles was slightly decreased while P450arom activity was significantly increased; (ii) in yellow hierarchical follicles which became atretic and regressed, activity of both enzymes were markedly decreased; (iii) ovarian P(4) production dramatically decreased, whereas ovarian E(2) production after an initial decrease significantly increased. In white atretic follicles the activity of 3beta-HSD and P450arom was very weak during the whole experiment. In conclusion, the present results indicate that during a pause in egg laying white follicles become resistant to atresia.     

Analogues of cyclolinopeptide A containing alpha-hydroxymethyl amino acid residues

Linear and cyclic cyclolinopeptide A (CLA) analogues containing alpha-hydroxymethylleucine (HmL) in positions 1, 4, and 1&4, and alpha-hydroxymethylvaline (HmV) in position 5, were synthesized by the solid-phase peptide strategy and cyclized with the 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide/1-hydroxy-7-azabenzotriazole (EDC/HOAt) reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation assays (LPA). Only HmL-containing peptides demonstrated at about 25% lower immunosuppressive activity, but they are four times more soluble in water solutions than the native CLA. It seems from the LPA results that peptide [(HmL4)CLA] is the most promising for further studies. This peptide was characterized in solution, at room temperature in CDCl3, and the conformation compared with that observed for CLA in the solid state.     

Tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) as selective inhibitors of protein kinase CK2: evaluation of their effects on cells and different molecular forms of human CK2

The development of selective cell-permeable inhibitors of protein kinase CK2 has represented an important advance in the field. However, it is important to not overlook the existence of discrete molecular forms of CK2 that arise from the presence of distinct isozymic forms, and the existence of the catalytic CK2 subunits as free subunits and in complexes with the regulatory CK2beta subunits and, possibly, other proteins. This review examines two recently developed, and presently widely applied, CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBBt) and the related 4,5,6,7-tetrabromobenzimidazole (TBBz), the latter of which was previously shown to discriminate between different molecular forms of CK2 in yeast. We have shown, by spectrophotometric titration, that TBBt, with a pK(a) approximately 5, exists in solution at physiological pH almost exclusively (>99%) as the monoanion; whereas TBBz, with a pKa approximately 9, is predominantly (>95%) in the neutral form, both of obvious relevance to their modes of binding. In vitro, TBBt inhibits different forms of CK2 with Ki values ranging from 80 to 210 nM. TBBz better discriminates between CK2 forms, with Ki values ranging from 70 to 510 nM. Despite their general similar in vitro activities, TBBz is more effective than TBBt in inducing apoptosis and, to a lesser degree, necrosis, in transformed human cell lines. Finally, development of shRNA strategies for the selective knockdown of the CK2alpha and CK2alpha' isoforms reinforces the foregoing results, indicating that inhibition of CK2 leads to attenuation of proliferation.     

Tissue distribution of a menthyl-conjugated oligodeoxyribonucleotide antisense to PAI-1 mRNA

The inhibitory effect of numerous analogues of PO-16, an hexadecadeoxyribonucleotide antisense to sequences -22 to -17 of PAI-1 mRNA coding for a fragment of the signal peptide, on the expression of PAI-1 in endothelial cells, and physiological consequences of the subsequently reduced PAI-1 activity tested in vitro and in vivo, were described in our previous studies. Of particular interest was PO-16 5'-O-conjugated with menthyl phosphorothioate (MPO-16R). In this work, tissue localisation of MPO-16R labelled with [(35)S] phosphorothioate at the 3'-end, was determined. [(35)S]MPO-16R and control [(35)S]MPO-16R-SENSE oligonucleotides were administered intravenously into 22 rats and organ distribution of the labelled bioconjugates was assessed after 24 and 48 h. For this purpose, tissue sections were subjected to autoradiography, and quantitated by liquid scintillation after solubilisation. Overall clearance of radioactivity was already seen after 24 h, with the radioactivity recovered mainly in the kidney and liver. A smaller fraction of radioactivity was also retained in the spleen and heart. The kidney concentration of the labelled probe was higher than that of liver by 50%. The distribution of PAI-1 mRNA in untreated rat kidney, liver, spleen and heart established by two independent techniques: Ribonuclease Protection Assay and Real-Time PCR, shows the same pattern as that observed for [(35)S]MPO-16R antisense.     

New analogue of cyclolinopeptide B modified by amphiphilic residue of alpha-hydroxymethylmethionine

In order to evaluate the role and influence of the methionine residue on the biological activity of cyclolinopeptide B, an analogue with methionine residue in position 7 replaced by the amphiphilic (S)-alpha-hydroxymethylmethionine residue was synthesized. This peptide exhibits high immunosuppressive activity in the cellular, and to a lesser degree in the humoral immune response, comparable to that of CsA. In addition, the peptide was devoid of toxicity, even at high doses.     

Disappearance of differences in antibiotic resistance of Staphylococcus aureus strains isolated in hospitals and in general practice

There is general opinion that Staphylococcus aureus strains isolated in hospitals are more frequently resistant to antibiotics than community strains, however, the increasing resemblance between hospital and community strains has been recently reported. The aim of the study was to compare the antibiotic resistance and phage-type pattern of S. aureus strains isolated from patients treated either in hospitals or in general practice in northern part of Poland. The study was conducted on 771 S. aureus strains isolated from different specimens. Phage typing was performed according to the method of Blair and Williams. The drug susceptibility was determined by the disc-diffusion method. There were no significant differences in antibiotic resistance or phage-type pattern when hospital and community methicillin-sensitive S. aureus (MSSA) strains were compared. The most MSSA were resistant to penicillin (84.6% and 82.1% respectively) and doxycycline (49.3% and 50.4% respectively) whereas they were rarely resistant to other antibiotics. The predominance of phage group II was found in both hospitals (28.0%) and general practice (29.9%). Phage group III, usually associated with hospitals, occurred in small percentage (12.9% and 9.4% respectively) while to this group predominantly (76.6%) multiresistant methicillin resistant S. aureus (MRSA) isolated in hospitals belonged. These results suggest, that there is only slight difference in antibiotic resistance between hospital and community S. aureus strains. Antibiotic resistance pattern mainly results from frequency of appearance of MRSA, mostly occurring in hospitals.     

Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16INK4a-mediated premature senescence.

Much has been learned about the mechanisms underlying cellular senescence. The pathways leading to senescence appear to vary, depending on the cell type and cell culture conditions. In this respect, little is known about senescence of human peritoneal mesothelial cells (HPMC). Previous studies have significantly differed in the reported proliferative lifespan of HPMC. Therefore, in the present study, we have examined how HPMC enter state of senescence under conditions typically used for HPMC culture. HPMC were isolated from omentum and grown into senescence. The cultures were assessed for the growth rate, the presence of senescence markers, activation of cell-cycle inhibitors, and the oxidative stress. HPMC were found to reach, on average, six population doublings before senescence. The terminal growth arrest was associated with decreased expression of Ki67 antigen, increased percentage of cells in the G1 phase, reduced early population doubling level cDNA-1 mRNA expression, and the presence of senescence-associated beta-galactosidase. Compared with early-passage cells, the late-passage HPMC exhibited increased expression of p16INK4a but not of p21Cip1. In addition, these cells generated more reactive oxygen species and displayed increased presence of oxidatively modified DNA (8-hydroxy-2'-deoxyguanosine). These results demonstrate that early onset of senescence in omentum-derived HPMC may be associated with oxidative stress-induced upregulation of p16INK4a.     

Generation of cloned and chimeric embryos/offspring using the new methods of animal biotechnology

The article summarizes results of studies concerning: 1/ qualitative evaluation of pig nuclear donor cells to somatic cell cloning, 2/ developmental potency of sheep somatic cells to create chimera, 3/ efficient production of chicken chimera. The quality of nuclear donor cells is one of the most important factors to determine the efficiency of somatic cell cloning. Morphological criteria commonly used for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Therefore, different types of somatic cells being the source of genomic DNA in the cloning procedure were analyzed on apoptosis with the use of live-DNA or plasma membrane fluorescent markers. It has been found that morphological criteria are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. Developmental potencies of sheep somatic cells in embryos and chimeric animals were studied using blastocyst complementation test. Fetal fibroblasts stained with vital fluorescent dye and microsurgically placed in morulae or blastocysts were later identified in embryos cultured in vitro. Transfer of Polish merino blastocysts harbouring Heatherhead fibroblasts to recipient ewes brought about normal births at term. Newly-born animals were of merino appearance with dark patches on their noses, near the mouth and on their clovens. This overt chimerism shows that fetal fibroblasts introduced to sheep morulae/blastocysts revealed full developmental plasticity. To achieve the efficient production of chicken chimeras, the blastodermal cells from embryos of the donor breeds, (Green-legged Partridgelike breed or GPxAraucana) were transferred into the embryos of the recipient breed (White Leghorn), and the effect of chimerism on the selected reproductive and physiological traits of recipients was examined. Using the model which allowed identification of the chimerism at many loci, it has been found that 93.9% of the examined birds were chimeras. The effect of donor cells on the reproduction and physiology of the recipients was evident.     

Reactivity difference between protolytic forms of some macrocyclic chromium(III) complexes in ligand substitution and electron transfer processes

The review provides insight into the mechanism of ligand substitution and electron transfer (from chromium(III) to iron(III)) by comparison of the reactivity of some tetraazamacrocyclic chromium(III) complexes in the conjugate acid-base forms. Use of two geometrical isomers made possible to estimate the influence of geometry and protolytic reactions in trans and cis position towards the leaving group on the rate enhancement. Studies on the reaction rates in different media demonstrated the role played by outer sphere interactions in a monodentate ligand substitution.