Sperm motility in three coral reef fish species

A method of computer assisted sperm analysis (CASA) is used to determine the parameters of sperm motility in three fish species from the coral reefs of the Nha Trang Bay, South China Sea, Vietnam: Zebrasoma scopas (Acanthuridae), Abudefduf sexfasciatus, and Dascyllus trimaculatus (Pomacentridae). The representatives of the families are characterized by different reproductive tactics and possess pelagic and demersal eggs, respectively. The main morphological parameters of spermatozoa have been measured. The average curvilinear velocity of spermatozoa movement along the real trajectory (VCL) after 1 min of sperm activation ranges from 15.3 to 74.5 μm/s in Z. scopas, it is comparatively low (12.7–21.6 μm/s) in A. sexfasciatus, and high (58.4–92.2 μm/s) in D. trimaculatus. The duration of progressive movements in more than 50% of spermatozoa at 25°C is 3–20, 5–11, and 6–9 min after sperm activation, respectively. Following storage of the sperm of three species at 4.5°C for 7, 20, and 28 h, respectively, spermatozoa retain the ability of progressive movements. The results are discussed based on the available information on the activity of sperm in fish.     

Rapid and high quality DNA isolation from Origanum onites for RAPD and ISSR analysis

Origanum onites is an economically important medicinal plant with high essential oil content. Lack of an appropriate DNA isolation procedure is a limiting factor for any molecular study of this plant. We have used a protocol for genomic DNA isolation based on a hexadecyltrimethylammonium bromide (CTAB) method described for other plant species. The method involves mortar grinding of leaf tissue, modified CTAB extraction using high salt concentrations and polyvinyl pyrrolidone, and successive isoamyl alcohol/chloroform extractions. The yield was approx. 20 microg DNA per 200 mg of initial fresh plant material. The genomic DNA obtained by this method was suitable to be used in restriction digests, inter simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) reactions. This extraction method should facilitate the molecular analysis of Origanum chemotypes.     

Purification of CYP2B-like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization

In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.     

Proteomic profiling of HBV infected liver biopsies with different fibrotic stages

Background

Hepatitis B virus (HBV) is a global health problem, and infected patients if left untreated may develop cirrhosis and eventually hepatocellular carcinoma. This study aims to enlighten pathways associated with HBV related liver fibrosis for delineation of potential new therapeutic targets and biomarkers.

Methods

Tissue samples from 47 HBV infected patients with different fibrotic stages (F1 to F6) were enrolled for 2D-DIGE proteomic screening. Differentially expressed proteins were identified by mass spectrometry and verified by western blotting. Functional proteomic associations were analyzed by EnrichNet application.

Results

Fibrotic stage variations were observed for apolipoprotein A1 (APOA1), pyruvate kinase PKM (KPYM), glyceraldehyde 3-phospahate dehydrogenase (GAPDH), glutamate dehydrogenase (DHE3), aldehyde dehydrogenase (ALDH2), alcohol dehydrogenase (ALDH1A1), transferrin (TRFE), peroxiredoxin 3 (PRDX3), phenazine biosynthesis-like domain-containing protein (PBLD), immuglobulin kappa chain C region (IGKC), annexin A4 (ANXA4), keratin 5 (KRT5). Enrichment analysis with Reactome and Kegg databases highlighted the possible involvement of platelet release, glycolysis and HDL mediated lipid transport pathways. Moreover, string analysis revealed that HIF-1α (Hypoxia-inducible factor 1-alpha), one of the interacting partners of HBx (Hepatitis B X protein), may play a role in the altered glycolytic response and oxidative stress observed in liver fibrosis.

Conclusions

To our knowledge, this is the first protomic research that studies HBV infected fibrotic human liver tissues to investigate alterations in protein levels and affected pathways among different fibrotic stages. Observed changes in the glycolytic pathway caused by HBx presence and therefore its interactions with HIF-1α can be a target pathway for novel therapeutic purposes.

     

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.     

Physiological roles of the angiogenic factors during posthatching development period and adults in the quail lung

The bronchus and vasculature form an intrinsic functional component of the avian lung, and its growth must be tightly regulated and coordinated by lung epithelial and endothelial development. Vascular endothelial growth inhibitor (VEGI), vascular endothelial growth factor (VEGF) and its receptors (flk1/KDR, flt1/fms, flt4) are required for epithelial and endothelial cell survival and apoptosis. Especially, VEGF and its receptors are critical for the development of the lung and serve as a maintenance factor during adult life. To determine the function of VEGI, VEGF and its receptors in the posthatching lung development, we revealed its expression and localization using by immunohistochemical procedure. VEGI, VEGF and its receptors were observed in the structural components of the bronchi, atria and air capillaries, as well as in the pulmonary blood vessels throughout the posthatching development period. On the other hand, immunostaining for VEGI, VEGF and its receptors was faintly detected in the glands of the secondary bronchi. Furthermore, it was determined that the secondary bronchial and atrial muscles did not display VEGF immunoreactions. Our results showed that VEGF and its receptors (flt1/fms, flk1/KDR and flt4) and VEGI were expressed at varying intensity by different cell groups. Therefore, they are also required for the development of the lung component during posthatching period.     

Determination of the Effects of Bee Venom on Triple Negative Breast Cancer Cells in Vitro

Honeybees provide multiple products such as bee venom (BV) which are used for various nutritional and medicinal purposes. BV has received great attention due to its wide range of bioactive components with potential anti-cancer effects on different cancers. Triple negative breast cancer (TNBC) is defined as an aggressive type of breast cancer and new therapeutic targets are required for its treatment. In the current literature information is varied about the composition and quantity of BV bioactive compounds as well as the origin of BV and its significance. In this context, the cytotoxic and apoptotic effects of BV with a higher rate of mellitin from Apis mellifera anatoliaca (Muğla ecotype) on MDA-MB-231 cells was evaluated, in vitro. The cytotoxic, apoptotic and morphological effects of BV were determined by WST-1, Annexin V, cell cycle analysis and Acridine Orange staining. The results showed that BV caused apoptotic cell death in TNBC cells at a lower dose (0.47 μg/mL, p<0.01). This study suggests that BV could be developed as a potential therapeutic agent for cancer treatment. However, the mechanism of BV-induced apoptosis death should be clarified at the molecular level.     

Elevated interleukin-12 and CXCL10 in subacute sclerosing panencephalitis

Immunosuppression associated with measles virus (MV) can be demonstrated by cytokine production failure in subacute sclerosing panencephalitis (SSPE) and may have implications on the pathogenesis of the disease. Cytokines (IL-12, IL-10, IL-4, IL-17, IL-18, IFN-alpha, IFN-gamma) and chemokines (CXCL8, CXCL10, CCL2 and CCL5) were measured in the cerebrospinal fluid (CSF) and serum samples from 60 patients with SSPE, 36 patients with infectious and/or inflammatory (IN) and 28 with other non-inflammatory (NIN) neurological diseases by ELISA. IL-12 p70+p40 was elevated in CSF and sera of SSPE when compared to the NIN group. However, the CSF levels of IL-12 p70 alone were not increased, indicating an increase of p40. The CSF of SSPE patients also showed relatively higher levels of IL-10 than that of the NIN group. CXCL10 levels in CSF were significantly higher in SSPE, whereas CXCL8 was increased in sera compared to NIN. No difference was detected in IFN-gamma, IFN-alpha, IL-17, IL-18, IL-4 or CCL2 and CCL5 levels. These results demonstrate that immune response against MV in SSPE may be impaired, although some T cell/Th1 inducing stimulations are present.     

Study of thermoregulatory activity of a fragment of natural dermorphin precursor Arg-Dermorphin and its analogs

We studied the effect of a fragment of natural dermorphin (DM) precursor Arg-DM and its analogs (Pro-DM, 4Amino-Pro-DM, Met-DM, Kre-DM, Arg-[DArg²]-DM, and Arg-[DAla⁴]-DM) after intraperitoneal administration on the functional status of the thermoregulation system in rats. The obtained data demonstrated that the hypothermic and vasomotor effects of Arg-DM were temperature-dependent and had the same pattern as DM (Emelyanova et al., 1996). At termoneutral and room temperatures, the peptide induced a two-phase vascular response. The first phase, vasodilation, was twice as strong as for DM and was not removed by naloxone preadministration. The second phase, vasoconstriction, was blocked by naloxone. Replacement of Arg with 4Amino-Pro, Met, and Kre as well as DAla² to DArg² or Gly⁴ to DAla⁴ replacements in the Arg-DM molecule affected the thermoregulatory activity of the peptide. For instance, only the vasodilatory response was observed for Arg-[DAla²]-DM and Arg-[DAla⁴]-DM while only the vasoconstrictive response was observed for 4Amino-Pro-DM.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 1, 2005, pp. 47–54.Original Russian Text Copyright © 2005 by Emelyanova, Guzevatykh, Goryacheva, Andreeva, Alfeeva, Myasoedov.     

Is There a Possible Relation Between Atrophic Gastritis and Premature Atherosclerosis?

BACKGROUND: In this study, we have aimed to show the possible relation between atrophic gastritis and premature atherosclerosis via hyperhomocysteinemia. MATERIALS AND METHODS: Thirty-four patients with atrophic gastritis were enrolled to the study. The control group consisted of 35 patients with non-atrophic gastritis. Classical cardiovascular disease risk factors did not significantly differ between atrophic gastritis and control subjects. The presence and degree of atrophic gastritis were assessed histologically and Helicobacter pylori infection was determined by both histologic and serologic methods. Body mass index was measured by standard technique blood fasting glucose, serum creatinine, total cholesterol, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, vitamin B12, folic acid, and homocysteine levels were measured by biochemical methods. Carotid intima-media thickness was measured by B-mode ultrasonography to examine the premature atherosclerosis. RESULTS: Plasma vitamin B12 levels were significantly lower (p = .00) and homocysteine levels were significantly higher (p = .01) in the atrophic gastritis group. There was no statistically significant difference in plasma folic acid levels between the two groups (p = .728). Carotid intima-media thickness was higher in the atrophic gastritis group than in the control group (0.516 mm versus 0.465 mm), but this difference did not show any statistical significance (p = .062). CONCLUSION: Our results showed that atrophic gastritis may cause hyperhomocysteinemia, which is an independent risk factor for atherosclerosis and cardiovascular diseases. However, when compared with controls, carotid intima-media thickness of the atrophic gastritis patients was found to be higher but did not reach statistically significant levels.