Circulating cornea specific antibodies and keratoplasty

The presence of corneal antibodies in blood sera was investigated in 130 patients by immunoblotting analysis. Sera were obtained from patients before the first keratoplasty, repeated corneal transplantation or keratoplasty of the other eye. Baseline levels of antibodies in 15 healthy blood donors served to establish a positivity threshold. Patients were divided according to diagnosis and number of keratoplasties. Corneal antigens were prepared from donor corneas not suitable for surgery. Hyperimmune sera obtained by immunization of rabbits were used as positive controls. Significant increase in corneal antibodies specific to epithelial antigens was found in patients (62.5% positive) with keratitis of microbial origin. The presence of circulating antibodies to endothelial or stromal proteins was without significant changes. Depending on the number of keratoplasties no statistical difference in corneal antibodies production was observed. The group of patients with increased anti-corneal antibodies already before transplantation had to take immunosuppressive and anti-inflammatory therapy after keratoplasty.     

Biochemical properties of Hsp70 chaperone system from Meiothermus ruber

The guanidine-hydrochloride (Gdn-HCl) and thermally induced unfolding of Hsp70 from Meiothermus ruber (Mru.Hsp70) were analysed using tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. The ANS binding to Mru.Hsp70 showed both the increase in fluorescence intensity and a shift in emission maximum. Analysis of the unfolding profile of Mru.Hsp70 indicated that Gdn-HCl induced unfolding of Mru.Hsp70 occurred through intermediate species. The tryptophan and ANS fluorescence emission spectra revealed that ATP induced conformational change increased the thermal stability of Mru.Hsp70. The data obtained are similar to those of Escherichia coli DnaK. The ATP-ase activity of chaperones is fundamental for their biological activity. It this paper we demonstrate that, in contrast to Thermus thermophilus, both Mru.Hsp40 and Mru.Hsp22 co-chaperones affect the ATP-ase activity of Mru.Hsp70. The use of truncated Mru.Hsp40 proteins showed that full-length Mru.Hsp40 is required for stimulation of ATP-ase activity of Mru.Hsp70. E. coli GrpE could act as nucleotide exchange factor the in thermophilic Hsp70 ATP hydrolysis reaction. However, the role of E. coli DnaJ in the M. ruber ATP cycle needs further analysis. We selected the new substrate laccA suitable for determination of refolding activity of thermophilic chaperones.     

Sperm binding properties and secretory activity of the bovine oviduct immediately before and after ovulation

The possibility that differences in hormonal regimes between the two oviducts in the cow around ovulation affects secretory activity of the oviduct epithelial cells and/or sperm-oviduct binding was studied. Oviducts were collected immediately after slaughter at 6 hr before to 5 hr after timed ovulation of 14 normally cyclic cows that had been inseminated (n = 6) or not (n = 8) and material obtained from the same cows was processed in three ways. First, in vivo, after artificial insemination of the cows, low numbers of sperm cells (approx. 15 per oviduct) were found within the entire oviducts as observed by scanning electron microscopy (SEM). Almost all sperm were located in the isthmus and then only on ciliated cells and showed without exception fully matured, intact morphology. Secretory activity of noninseminated oviduct epithelia was induced after ovulation which was most predominant in the pockets of the ipsi-lateral ampulla compared to the contra-lateral ampulla (P < 0.01). Second, ex vivo, explants dissected from oviducts of the noniseminated cows were incubated with sperm. In all cases, the sperm bound to the explants in a similar pattern as observed in vivo and this binding was strictly fucose-dependent. The main difference with in vivo experiments was the high numbers of sperm bound at any site of the oviduct ( approximately 3,000 cells per mm(2)) indicating the high sperm binding capacity of the oviduct epithelia. Ovulation induced a striking drop in sperm binding capacity in the oviducts and was most pronounced in the isthmus ( approximately 1,300 cells per mm(2); P < 0.001) and to a lesser extent in the ampulla ( approximately 2,000 cells per mm(2), P < 0.01). Third, in vitro, pieces of tissue dissected from oviducts of the noninseminated cows were cultured to mono-layers. Culturing epithelial cells resulted in loss of their normal morphological appearance. In all cases, the sperm binding capacity in monolayers was very low (<50 cells per mm(2)) when compared to corresponding explants (P < 0.0001). Sperm binding to monolayers originating from the isthmus (<25 cells per mm(2)) was lower than in those from the ampulla (40-50 cells per mm(2); P < 0.01) and remained similar after ovulation. In all three approaches, no significant differences were found in sperm-oviduct binding characteristics and sperm-distribution in the ipsi- versus contra-lateral oviducts. This indicates, that systemic endocrine changes around ovulation rather than specific oviduct changes at the ipsi-lateral oviduct induce secretion in oviduct epithelial cells, and thus induce sperm release.     

Oviductal cell proteome alterations during the reproductive cycle in pigs

The mammalian oviduct plays a crucial role in events leading to the establishment of pregnancy. During the reproductive cycle, the reproductive system undergoes various changes, including alterations in the number of different cell types in the oviductal epithelium and changes in the height of oviductal cells. Maintaining the unique oviductal environment required for the fertilization and early embryonic development comes with an energy cost to the organism. Therefore, it is hypothesized that structural and functional changes to the oviduct during the reproductive cycle represent vital preparations for the development of suitable environments for conception and embryo support. Here, we aimed to identify the changes in protein expression profile that occur during the follicular and luteal stages of the reproductive cycle in oviductal epithelial cells. The porcine oviductal epithelial cell proteomes from the follicular and luteal stages of the reproductive cycle were contrasted after separation by 2-D gel electrophoresis. Several oviductal epithelial cell proteins were up- or down-regulated during the reproductive cycle. We checked the quantitative changes of two of these molecules during different stages of the reproductive cycle using Western blot analysis. Finally, a number of these proteins were identified using tandem mass spectrometry. The results demonstrated distinctive differences in the proteomic profiles of the oviduct between follicular and luteal phases of the reproductive cycle.     

Blood sampling from the cranial vena cava in the Norway rat (Rattus norvegicus)

This paper describes blood sampling from the cranial vena cava (CVC) in the Norway rat. In order to limit stress, the blood sampling should be done under short-term inhalation anaesthesia, for example, an oxygen/isoflurane mixture. The injection site is just cranial to the first rib, 0.3-0.8 cm lateral to the manubrium when the animal is in dorsal recumbency. The needle, attached to a syringe, is inserted at 30 degrees in the direction of the opposite femoral head. After penetration of the skin, negative pressure is developed in the syringe and the insertion of the needle is continued for another 0.2-1 cm in the given direction until blood begins to flow. The amount of blood sampled ranges from 0.8 to 2.5 mL depending on the body weight of the patient. A trial on 50 rats aged 5-24 months included 25 rats sampled once, eight rats sampled twice with an interval of seven days, 11 rats sampled twice with an interval of three weeks and four rats sampled four times with intervals of four weeks--a total of 87 blood samplings. The serious complications quoted in association with blood sampling from the CVC in other experimental animals (vascular lacerations, heart puncture, serious haemorrhage, tracheal and throat trauma) were not observed in our study. There were only four blood samplings (4.5%) with mild haemorrhage from the injection site, due to erroneous sampling from the jugular vein.     

Intrinsic Thermodynamics and Structure Correlation of Benzenesulfonamides with a Pyrimidine Moiety Binding to Carbonic Anhydrases I,II, VII,XII, and XIII

The early stage of drug discovery is often based on selecting the highest affinity lead compound. To this end the structural and energetic characterization of the binding reaction is important. The binding energetics can be resolved into enthalpic and entropic contributions to the binding Gibbs free energy. Most compound binding reactions are coupled to the absorption or release of protons by the protein or the compound. A distinction between the observed and intrinsic parameters of the binding energetics requires the dissection of the protonation/deprotonation processes. Since only the intrinsic parameters can be correlated with molecular structural perturbations associated with complex formation, it is these parameters that are required for rational drug design. Carbonic anhydrase (CA) isoforms are important therapeutic targets to treat a range of disorders including glaucoma, obesity, epilepsy, and cancer. For effective treatment isoform-specific inhibitors are needed. In this work we investigated the binding and protonation energetics of sixteen [(2-pyrimidinylthio)acetyl]benzenesulfonamide CA inhibitors using isothermal titration calorimetry and fluorescent thermal shift assay. The compounds were built by combining four sulfonamide headgroups with four tailgroups yielding 16 compounds. Their intrinsic binding thermodynamics showed the limitations of the functional group energetic additivity approach used in fragment-based drug design, especially at the level of enthalpies and entropies of binding. Combined with high resolution crystal structural data correlations were drawn between the chemical functional groups on selected inhibitors and intrinsic thermodynamic parameters of CA-inhibitor complex formation.     

Different associations of apoE gene polymorphism with metabolic syndrome in the Vojvodina Province (Serbia)

The metabolic syndrome (MetS) is a polygenic multifactorial metabolic disorder with strong socioeconomic influence. MetS has became a worldwide epidemic, that directly increases the risk of cardiovascular diseases and type 2 diabetes mellitus. The human apoE gene, coding Apolipoprotein E, has three common polymorphisms in human population: e2, e3 and e4, which are proved to be associated with impaired lipid metabolism. The contribution of apoE polymorphism to MetS disorders has not been investigated previously in Vojvodina Province, region with the highest number of obese people in Serbia. The aim of this study was to evaluate apoE gene polymorphism in relation to MetS disorders. The healthy control group of 30 individuals and 63 MetS patients were examined for apoE variants in relation to biochemical and anthropometric parameters. The genotypes were determined by PCR–RFLP. Regarding all parameters, significantly higher values were detected in MetS group compared to control. The MetS group of patients had significantly higher frequency of e4 allele. In addition, positive relation was revealed between e4 allele presence and all measured parameters. It was found that the e4 allele was related with a significantly increased OR of MetS disorders according to the International Diabetes Federation definition. These results suggested that e4 allele may act as a one of determinants for development of metabolic syndrome.     

Acid response of exponentially growing Escherichia coli K-12

Induction of acid tolerance response (ATR) of exponential-phase Escherichia coli K-12 cells grown and adapted at different conditions was examined. The highest level of protection against pH 2.5 challenges was obtained after adaptation at pH 4.5-4.9 for 60 min. To study the genetic systems, which could be involved in the development of log-phase ATR, we investigated the acid response of E. coli acid resistance (AR) mutants. The activity of the glutamate-dependent system was observed in exponential cells grown at pH 7.0 and acid adapted at pH 4.5 in minimal medium. Importantly, log-phase cells exhibited significant AR when grown in minimal medium pH 7.0 and challenged at pH 2.5 for 2 h without adaptation. This AR required the glutamate-dependent AR system. Acid protection was largely dependent on RpoS in unadapted and adapted cells grown in minimal medium. RpoS-dependent oxidative, glutamate and arginine-dependent decarboxylase AR systems were not involved in triggering log-phase ATR in cells grown in rich medium. Cells adapted at pH 4.5 in rich medium showed a higher proton accumulation rate than unadapted cells as determined by proton flux assay. It is clear from our study that highly efficient mechanisms of protection are induced, operate and play the main role during log-phase ATR.     

Characterization of Escherichia coli dinJ-yafQ toxin-antitoxin system using insights from mutagenesis data

Escherichia coli dinJ-yafQ operon codes for a functional toxin-antitoxin (TA) system. YafQ toxin is an RNase which, upon overproduction, specifically inhibits the translation process by cleaving cellular mRNA at specific sequences. DinJ is an antitoxin and counteracts YafQ-mediated toxicity by forming a strong protein complex. In the present study we used site-directed mutagenesis of YafQ to determine the amino acids important for its catalytic activity. His50Ala, His63Ala, Asp67Ala, Trp68Ala, Trp68Phe, Arg83Ala, His87Ala, and Phe91Ala substitutions of the predicted active-site residues of YafQ abolished mRNA cleavage in vivo, whereas Asp61Ala and Phe91Tyr mutations inhibited YafQ RNase activity only moderately. We show that YafQ, upon overexpression, cleaved mRNAs preferably 5' to A between the second and third nucleotides in the codon in vivo. YafQ also showed RNase activity against mRNA, tRNA, and 5S rRNA molecules in vitro, albeit with no strong specificity. The endoribonuclease activity of YafQ was inhibited in the complex with DinJ antitoxin in vitro. DinJ-YafQ protein complex and DinJ antitoxin alone selectively bind to one of the two palindromic sequences present in the intergenic region upstream of the dinJ-yafQ operon, suggesting the autoregulation mode of this TA system.     

Variation in the contents of pseudohypericin and hypericin in Hypericum perforatum from Lithuania

Hypericin and hypericin-like substances are considered the main active compounds in Hypericum perforatum L. (Hypericaceae). In this work pseudohypericin and hypericin of H. perforatum collected in Lithuania were quantified. Studies on accumulation dynamics and between-accession variation of the contents of these secondary metabolites were carried out by high performance liquid chromatography (HPLC). The data were statistically processed with ANOVA and PCA. Significant difference between pseudohypericin and hypericin content in floral budding and full flowering stages was detected. The highest amounts of the secondary metabolites were observed in the flowering stage. The study revealed evident within population variations in H. perforatum. Mean concentrations of pseudohypericin and hypericin among accessions varied from 3.45 to 6.82 mg/g and from 1.17 to 2.59 mg/g, respectively. Accessions of H. perforatum showed remarkable differences in chemical composition depending on the provenance of plants.