Jasmonic acid affects changes in the growth and some components content in alga Chlorella vulgaris

The present study was undertaken to test the influence of exogenous applied jasmonic acid upon the growth and changes in some metabolites levels in the cells of green alga Chlorella vulgaris Beijerinck (Chlorophyceae). It was found, that JA in algal cells acted in a concentration-dependent manner. Treatment with JA at high concentrations range of 10⁻⁵–10⁻⁴ M, resulted in the decrease in cell number and reduction of major photosynthetic pigments, monosaccharides, soluble cellular and extracellular proteins levels as well as decrease in pH of the medium. In contrast to 10⁻⁵ and 10⁻⁴ M JA, this phytohormone applied at 10⁻⁸–10⁻⁶ M induced the increase in cell number, photosynthetic pigments and monosaccharides contents, significant accumulation and extracellular secretion of soluble proteins over control and neutralization of the medium. Quantitative changes in polypeptide pattern of total cellular proteins after treatment with the optimal concentration of 10⁻⁷ M JA on the 7^th day of cultivation as analyzed by SDS-PAGE, was also observed. JA induced synthesis de novo of 15 specific polypeptides with molecular weight 334-16 kDa which were’t detected in the control. The data suggest that JA plays a important role in algal growth and development.     

The construction of bifunctional fusion proteins consisting of MutS and GFP

MutS as a mismatch binding protein is a promising tool for SNP detection. Green fluorescent protein (GFP) is known as an excellent reporter domain. We constructed chimeric proteins consisting of MutS from Thermus thermophilus and GFPuv from Aequorea victoria by cloning the GFPuv gene into the plasmid vectors carrying the mutS gene. The GFPuv domain fused to the N-terminus of MutS (histag-GFP-MutS) exhibited the same level of green fluorescence as free GFPuv. To obtain the fluorescing histag-GFP-MutS protein the expression at 30 degrees C was required, while free GFPuv fluoresces when expressed both at 30 and 37 degrees C. The chimeric protein where the GFPuv domain was fused to the C-terminus of MutS exhibited much weaker green fluorescence (20-25% compared with those of histag-GFP-MutS or free GFPuv). The insertion of (ProGly)5 peptide linker between the MutS and GFP domains resulted in no significant improvement in GFP fluorescence. No shifts in the excitation and emission spectra have been observed for the GFP domain in the fusion proteins. The fusion proteins with GFP at the N- and C-terminus of MutS recognised DNA mismatches similarly like T. thermophilus MutS. The fluorescent proteins recognising DNA mismatches could be useful for SNP scanning or intracellular DNA analysis. The fusion proteins around 125 kDa were efficiently expressed in E. coli and purified in milligram amounts using metal chellate affinity chromatography.     

The neuroendocrine events during the ovine growth-promoted maturation: the developmental importance of hypophysiotrophic action of somatostatin in ewes

The comparison of hypothalamic somatostatin (SRIH)-neuronal systems, hypophyseal somatotroph populations and growth hormone (GH) blood plasma patterns among developmental stages, from infancy until puberty, may help to describe the nature of the hypothalamo-hypophyseal mechanisms underlying the changes in GH on the systemic level leading to the somatic, that is growth and sexual maturity in sheep. The aim of this study was to elucidate (i) developmental importance of hypophysiotrophic action of SRIH, (ii) precise time of maturation of this action and (iii) photoperiodic regulation of the postnatal ontogeny in ewes. The central and peripheral activity of the SRIH-GH axis is described through a sequence of histomorphological and functional changes in Merino ewes born after the summer solstice. The actual time of puberty of these animals was delayed until the following breeding season, when the sheep were 14-month old. Histomorphometric examinations have been made in 21 infantile (preweanling, 12-week old), prepubertal (15- and 22-week old), peripubertal (30- and 52-week old) and pubertal (63-week old) ovary-intact sheep. Functional examinations of the GH plasma levels were determined every 1-2 weeks during the period from the 12th to 63rd week of age. The highest GH level was observed at the 13th week of age, on the beginning of the breeding season. The fluctuations in the GH level just after the winter and summer solstice were detected as the one and only deviation from a rule of uniformly low GH concentrations observed until puberty. The age of the fall in serum GH levels corresponded with the postweaning period and the beginning of the phase of the lower daily live-weight gains (growth rate). Thus, the development of GH secretion was finished before the 15th week of age, that is together with the ending of the transitional infantile/prepubertal period, whereas the maturational processing within the hypothalamo-hypophyseal unit prolonged after the 15th week of age until 22 weeks of age and concerned the role of SRIH as the hypophysiotrophic factor regulating somatic maturation, i.e. attenuating growth. Altogether, the pattern of GH secretion during weaning is important for the shift between infancy and prepuberty depended upon an intensive growth and defined as growth maturation. The maturation of the SRIH-GH axis is finished by 22 weeks of age, independently of photoperiodic influences, whereas the neuroendocrine mechanisms to integrate somatic, that is growth and sexual maturation, are seasonal in nature in the ewe. Our observations confirm the hypothesis of the inherent endogenous rhythm controlling somatic maturation in the sheep.     

Elevation of circulating interleukin-8 is related to lymph node and distant metastases in esophageal squamous cell carcinomas--implication for clinical evaluation of cancer patient

The presence of lymph node metastasis (LNM) is an important factor in clinical evaluation of esophageal cancer patients. Biological markers able to support detection of metastatic lymph nodes are sought after. Interleukin-8 (IL-8) is overexpressed by many cancers and involved in cancer dissemination. We investigated the relationship between circulating IL-8 and clinicopathological features of esophageal squamous cell carcinoma (ESCC), and evaluated the diagnostic potential of IL-8, with reference to the key angiogenic and lymphangiogenic factors: vascular endothelial growth factors A and C (VEGF-A and VEGF-C). We found elevated IL-8 levels in ESCC patients, correlated with tumor size and cancer dissemination, especially LNM. Circulating IL-8 correlated with lymphangiogenic VEGF-C rather then angiogenic VEGF-A. The association weakened in metastatic cancers, suggesting divergent mechanism of IL-8 involvement in the dissemination process. The cytokine levels correlated with platelets and neutrophils, pointing at these cells as possible sources of circulating IL-8. We demonstrated IL-8 that positively correlated with inflammation status of ESCC patients. Circulating IL-8 was a better indicator of ESCC dissemination than VEGF-A or VEGF-C. Yet, the detection rates were not satisfactory enough to allow for the recommendation of IL-8 determination as an adjunct to the clinical evaluation of lymph node involvement in ESCC patients.     

Overexpression, purification and characterization of functional calf purine nucleoside phosphorylase (PNP)

Calf PNP is a ubiquitous enzyme of the salvage metabolic pathway. The procedure for this enzyme production in large quantities is described. The coding sequence of bovine PNP was amplified from the calf spleen cDNA library and was inserted into an expression vector pET28a(+). The construct was transformed into Escherichia coli BL21(DE3) strain. The protein expression efficiencies in the presence and the absence of IPTG were compared. It was found that IPTG is not necessary for obtaining a large quantity of recombinant calf PNP: 35 mg from 1 L cell culture. The enzyme was purified to 92% homogeneity by a two-step procedure consisting of gel filtration and ion exchange chromatography. The purity of recombinant enzyme is sufficient to form well diffracting single crystals.The basic kinetic parameters of recombinant PNP were determined and compared with the parameters of commercially available PNP from calf spleen. The specific activity in 50 mM phosphate buffer with inosine as a variable substrate (30.7 μmol min⁻¹ mg⁻¹) and other kinetic parameters: Michaelis constants, maximal velocities, dissociation and inhibition constants, determined for several typical PNP ligands, are similar to the values published previously for non-recombinant calf spleen PNP. As expected for mammalian PNP, recombinant calf PNP was found to have no substrate activity vs adenosine. The overexpression and purification method of the recombinant calf PNP provides significant amounts of the enzyme, which can successfully replace the non-recombinant PNP.     

Expression, purification and crystallization of cysteine-rich human protein muskelin in Escherichia coli

The increasing interest in the structural arrangements and functional interdependencies of individual modules within large multidomain proteins requires the development of new methods allowing efficient production and purification of large human proteins. Heterologous expression in bacteria is still the most convenient and widely-used approach. However, most of the existing tools are not well suited to expression of cysteine-rich proteins in a native-like soluble form, and with the increasing protein size refolding may result in obtaining non-native conformations or improper disulfide bridging pattern. Here, we present an efficient method of expression and purification of muskelin, a large, multidomain, cysteine-rich eukaryotic protein involved in cell adhesion and regulation of cytoskeleton dynamics. Using a broad range of purification and solubility tags, expression strains and conditions we optimized the procedure to acquire a natively folded protein of crystallization-scale quantity and purity. The correct protein conformation and disulfide bonding were anticipated from the results of circular dichroism spectra and Ellman’s assay. Successful crystallization trials are a step towards muskelin crystal-structure determination, while the optimized expression and purification procedure can easily be applied to produce other eukaryotic proteins in the bacterial expression system.     

Cholesterol sulphate sulphohydrolase of human placenta lysosomal membrane

In this paper we report that the activity of cholesterol sulphate sulphohydrolase (CHS-ase) is associated with the lysosomal membranes. The procedure of purification of CHS-ase from human placenta lysosomes was elaborated. The purified enzyme is highly specific to cholesterol sulphate (specific activity 2126.60+/-940.90 nmol min(-1) mg protein(-1)) and acts optimally at pH 3.4. The K(M) value for the hydrolysis of cholesterol sulphate is 3.6+/-0.95 x 10(-5)mol/l. The isoelectric point (pI) has the value 5.7, molecular weight estimated by SDS-PAGE electrophoresis is 38 kDa. The described enzyme may be involved in a regulation of cholesterol and cholesterol sulphate levels in the lysosomal membrane.     

Genistein, a plant-derived isoflavone, counteracts the antilipolytic action of insulin in isolated rat adipocytes

Genistein is a phytoestrogen exerting numerous biological effects. Its direct influence on adipocyte metabolism and leptin secretion was previously demonstrated. This study aimed to determine whether genistein antagonizes the antilipolytic action of insulin in rat adipocytes. Freshly isolated adipose cells were incubated for 90 min with epinephrine, epinephrine with insulin and epinephrine with a specific inhibitor of protein kinase A (H-89) at different concentrations of genistein (0, 6.25, 12.5, 25, 50 and 100 μM). Genistein failed to affect epinephrine-induced glycerol release, however, the inhibitory action of insulin on epinephrine-induced lipolysis was significantly abrogated in cells exposed to the phytoestrogen (12.5–100 μM). The increase in insulin concentration did not suppress the genistein effect. Its inhibitory influence on the antilipolytic action of insulin was accompanied by a substantial rise in cAMP in adipocytes. This rise appeared despite the presence of 10 nM insulin in the incubation medium. Further experiments, in which insulin was replaced by H-89, revealed that the antilipolytic action of protein kinase A inhibitor on epinephrine-induced lipolysis was not affected by genistein. This means that genistein counteracted the antilipolytic action of insulin due to the increase in cAMP levels and activation of protein kinase A in adipocytes. The observed attenuation of the inhibitory effect of insulin on triglyceride breakdown evoked by genistein was not related to its estrogenic activities, as evidenced in experiments employing the intracellular estrogen receptor blocker, ICI 182,780. Moreover, it was found that genistein-induced impairment of the antilipolytic action of insulin was not accompanied by changes in the proportion between fatty acids and glycerol released from adipocytes. The ability of genistein to counteract the antilipolytic action of insulin may contribute to the decreased triglyceride accumulation in adipose tissue.