Differential Trk expression in explant and dissociated trigeminal ganglion cell cultures

During embryonic development, expression of neurotrophin receptor tyrosine kinases (Trks) by sensory ganglia is continuously and dynamically regulated. Neurotrophin signaling promotes selective survival and axonal differentiation of sensory neurons. In embryonic day (E) 15 rat trigeminal ganglion (TG), NGF receptor TrkA is expressed by small diameter neurons, NT-3 receptor TrkC and BDNF receptor TrkB are expressed by large diameter neurons. Organotypic explant and dissociated cell cultures of the TG (and dorsal root ganglia) are commonly used to assay neurotrophin effects on developing sensory neurons. In this study, we compared Trk expression in E15 rat TG explant and dissociated cell cultures with or without neurotrophin treatment. Only a subset of TG cells express each of the three Trk receptors in wholemount explant cultures as in vivo conditions. In contrast, all TG neurons co-express all three Trk receptors upon dissociation, regardless of neurotrophin treatment. Neurons cultured in low concentrations of one neurotrophin first, and switched to higher concentrations of another after 1 day, survive and display morphological characteristics of neurons cultured in a mixture of both neurotrophins for 3 days. Our results indicate that wholemount explant cultures of sensory ganglia represent in vivo conditions in terms of Trk expression patterns; whereas dissociation dramatically alters Trk expression by primary sensory neurons.     

Leptin ameliorates burn-induced multiple organ damage and modulates postburn immune response in rats

The present study was designed to determine whether exogenous leptin reduces remote organ injury in the rats with thermal burn trauma. Leptin (10 microg/kg) or saline was administered intraperitoneally after burn injury, and the rats were decapitated at either 6 or 24 h. Plasma samples of 24-h burn group were assayed for the determination of monocyte and neutrophil apoptosis. Thermal injury increased tissue-associated myeloperoxidase (MPO) activity and microscopic damage scores in the lung, liver, stomach, colon and kidney of both 6- and 24-h burn groups. In the 6-h burn group, leptin reduced microscopic damage score in the liver and kidney only, while damage scores in the 24-h burn group were reduced in all the tissues except the lung. Also, in both burn groups, leptin reduced elevated MPO activity in all tissues except the lung. The percentage of mononuclear cells was significantly reduced at the 24 h of burn injury, while the granulocyte percentage was increased. Leptin treatment, however, had no significant effect on burn-induced reversal of white blood cell ratios. On the other hand, burn-induced increase in the death of mononuclear cells and granulocytes was significantly reduced in leptin-treated rats. The results of the present study suggest that leptin may provide a therapeutic benefit in diminishing burn-induced inflammation and associated multiple organ failure.     

Effects of HA-1077 and Y-27632, two rho-kinase inhibitors, in the human umbilical artery

A role for the small G protein rho and rho-kinase has been shown in smooth muscle contraction regarding Ca⁺⁺ sensitivity. However, there are no data in the literature assessing how this system operates in human umbilical arteries (HUA). Therefore, we evaluated the effects of HA-1077 and Y-27632, two rho-kinase inhibitors, on agonist-(5-hydroxytryptamine [5-HT]) and depolarization-induced (KCl) contractions of HUA. HA-1077 and Y-27632 inhibited 5-HT-induced contractile responses at 10⁻⁴ M concentration but not at 10⁻⁵ M. HA-1077 at 10⁻⁴ M also significantly attenuated contractions induced by 20 mM KCl. In addition, HUA precontracted with 5-HT relaxed concentration dependently in response to HA-1077 and Y-27632. When precontracted with KCl, HUA also relaxed dose-dependently in response to HA-1077, but the maximal relaxation was significantly smaller than the response obtained when precontracted with 5-HT. To determine possible involvement of rho-kinase on agonist-induced intracellular calcium-mediated contractions, tissues were precontracted with 5-HT in Ca⁺⁺-free Krebs solution before cumulative addition of HA-1077 or Y-27632 (10⁻⁷ to 10⁻⁴ M). Both rho-kinase inhibitors relaxed HUA completely. Maximum relaxations of HUA to HA-1077 and Y-27632 were significantly larger than the responses seen in normal Krebs solution and were obtained with lower concentrations of the drugs considered to be more specific for rho-kinase inhibition. However, preincubation of HUA with HA-1077 or Y-27632 (10⁻⁵ M for both) did not affect the 5-HT-induced contractions in this medium. Finally, immunoblot experiments revealed the expression of rho-kinase isoform rockII protein in HUA. These results indicate that rhoA/rho-kinase pathway can contribute to agonist-induced contractions of HUA. However, this effect appears to be limited to intracellular calcium-induced contractions and may be more important in sustaining contractions rather than the initial phase of force development.     

Considering alternative life history modes and genetic divergence in conservation: a case study of the Oklahoma salamander

Alternative life history strategies can provide important variation for the long-term persistence of a lineage. However, conservation of such lineages can be complicated because each life history mode may have different habitat requirements and may be vulnerable to different environmental perturbations. The Oklahoma salamander (Eurycea tynerensis) is endemic to the Ozark Plateau of North America, and has two discrete life history modes, biphasic (metamorphic) and aquatic (paedomorphic). Until recently, these modes were considered separate species and conservation attention focused only on paedomorphic populations. We perform phylogenetic analyses of the mitochondrial gene cytochrome b (Cytb) and nuclear gene proopiomelanocortin (POMC) to assess patterns of historical isolation in E. tynerensis, and test whether life history mode is randomly distributed with respect to the phylogeny and geography. We find three divergent Cytb lineages and significant shifts in POMC allele frequencies between the eastern, western, and southwestern portions of the distribution. Life history mode varies extensively, but paedomorphosis is largely restricted to the widespread western clade. Therefore, the two most divergent and narrowly distributed clades (southwestern and eastern) were previously overlooked due to their metamorphic life history. Paedomorphosis has allowed E. tynerensis to drastically increase its niche breadth and distribution size. Nevertheless, metamorphosis is also an important attribute, and metamorphic populations are the ultimate source for paedomorphic evolution. Preservation of divergent genetic lineages, and regions that include adjacent habitat for both life history modes, may be the most effective way to maintain historical and adaptive variation and provide gateways for ongoing life history evolution.     

HER2 Ile655Val and PTEN IVS4 polymorphisms in patients with breast cancer

Although HER2/PTEN pathway is commonly disrupted in cancer, association of HER2 and PTEN polymorphisms with breast cancer (BC) remains controversial. We investigated the HER2 Ile655 Val and PTEN IVS4 polymorphisms in patients with BC in Turkish population. HER2 Ile655Val (rs 1136201) and PTEN IVS4 (rs 3830675) polymorphisms were determined using polymerase chain reaction-based restriction fragment length polymorphism (PCR–RFLP) in blood samples of 118 BC patients and 118 age-matched healthy controls. We found that the frequency of the Ile/Val genotype of HER2 Ile655Val gene was significantly higher in BC patients (p < 0.009; OR: 1,983 95 % CI: 1.181—3.328). The presence of ATCTT insertion (+/+) genotype at downstream of exon 4 in intron 4 of PTEN IVS4 gene was also associated with 1.83 fold decreased risk of BC development (p < 0.033; OR: 1.83, 95 % CI: 1.11—3.03). Analysis on clinico-pathological parameters showed neither HER2 Ile655Val nor PTEN IVS4 genotypes were not associated with any of the variables (p > 0.05).In conclusion, our findings suggest that the Ile/Val genotype of HER2 and ATCTT insertion (+/+) genotype of PTEN IVS4 gene may play an important role as genetic markers for breast cancer risk, but both genes genotypes may not be useful for predicting tumor prognosis in Turkish population.     

Shoot proliferation and HPLC-determination of iridoid glycosides in clones of <Emphasis Type="Italic">Gentiana cruciata</Emphasis> L

Gentiana cruciata L. (Cross gentian) is a medicinal and ornamental plant, threatened in its natural habitats. The wild root extracts of this species are known to exhibit many curative properties. In the present study, an efficient protocol for in vitro propagation of G. cruciata L. was developed from node culture. A semi-solidified Murashige and Skoog (MS) basal medium supplemented with 2.22 μM 6-benzyladenine (BA), 2.46 μM indole-3-butyric acid (IBA) and sucrose (3% w/v) improved the production of multiple shoots directly from nodal segments, providing 3.9 shoots per explants on average. The highest rooting (81.7%) was observed with half-strength MS medium supplemented with 2.46 μM IBA. Plants with well-developed roots were transferred to pots containing turf/vermiculite mixtures and acclimatized in plant growth chamber conditions. Acclimatized plants showed 100% survival and remained healthy. The content of secondary metabolites in the clones was determined by HPLC, and the presence of gentiopicroside, loganic acid, swertiamarin, and sweroside in the samples was confirmed. Gentiopicroside was found to be the major compound.     

Poly(hydroxyethyl methacrylate) based affinity cryogel for plasmid DNA purification

The aim of this study is to prepare supermacroporous pseudospecific cryogel which can be used for the purification of plasmid DNA (pDNA) from bacterial lysate. N-methacryloyl-(l)-histidine methyl ester (MAH) was chosen as the pseudospecific ligand and/or comonomer. Poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-histidine methyl ester) [PHEMAH] cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Compared with the PHEMA cryogel (50 μg/g polymer), the pDNA adsorption capacity of the PHEMAH cryogel (13,350 μg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The amount of pDNA bound onto the PHEMAH cryogel disks first increased and then reached a saturation value (i.e., 13,350μg/g) at around 300 μg/ml pDNA concentration. pDNA adsorption amount decreased from 1137 μg/g to 160 μg/g with the increasing NaCl concentration. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 90%. The PHEMAH cryogel could be used 3 times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH cryogel disks promise high selectivity for pDNA.     

Evaluation of Antifungal Susceptibility Testing with Microdilution and Etest Methods of <Emphasis Type="Italic">Candida</Emphasis> Blood Isolates

Candida species that show an increasing number of clinical and/or microbiological resistance to several antifungals and are the most common agents of invasive fungal infections. The aim of this study was to investigate the in vitro susceptibility of Candida blood isolates to antifungal agents (amphotericin B, fluconazole, itraconazole, and voriconazole) by comparative use of the CLSI reference microdilution method and Etest. Four hundred Candida blood isolates (215 Candida albicans, 185 non-albicans Candida strains) were included in the study. The broth microdilution test was performed according to the CLSI M27 A2 document. Etest was carried out according to the manufacturer’s instructions. The MIC results obtained with reference microdilution were compared with those obtained with the Etest by using percent and categorical agreements. According to MIK₉₀ values, voriconazole was the most active and itraconazole was the least active drug in vitro against all Candida species. Other than voriconazole, statistically significant differences were found when the susceptibility of Candida albicans and non-albicans Candida spp. to amphotericin B, fluconazole, and itraconazole were compared. These antifungal agents were found to be more active to C. albicans. Among the non-albicans Candida species, the lowest MIC values were obtained for Candida parapsilosis isolates. When the standard method was compared with Etest, the total agreement was higher for C. albicans than for non-albicans species, especially for fluconazole and voriconazole. In view of the findings, it was concluded that itraconazole showed the lowest activity against all Candida species. Etest could be an alternative method in assessing the in vitro antifungal susceptibility of Candida spp., but it is more convenient to use the microdilution method for studying in vitro susceptibility of non-albicans species, in particular for those possessing high MIC values against azoles.