Aberrant Promoter Methylation Profile of Niemann-Pick Type C1 Gene in Cardiovascular Disease

Background: The protein of Niemann-pick type C1 (NPC1) gene promotes the egress of cholesterol from late endosomes and lysosomes to other cellular compartments and contributes to a process known as reverse cholesterol transport. This study aimed to examine whether promoter methylation of NPC1 is associated with risk of cardiovascular disease (CVD). Methods: Fifty CVD patients and 50 healthy subjects as the control group were recruited in this study. Promoter methylation of NPC1 gene was defined using a nested-methylation specific polymerase chain reaction method. Statistical analyses were done using the chi-square, t-test or ANOVA tests. Results: Our study showed that the frequency of semi-methylated promoter (methylated/unmethylated status) was significantly higher in CVD patients than that in controls (OR = 6.521, 95% CI = 2.211-19.215, P = 0.008). However, a completely methylated promoter (methylated/methylated status) was not detected in any subjects in either of the two groups tested. Additionally, the analysis of clinical data according to the methylation status of NPC1 gene demonstrated that serum levels of total cholesterol, total triglycerides, high low-density lipoprotein cholesterol (LDL-C) and low high-density lipoprotein cholesterol (HDL-C) are influenced by NPC1 methylation, so that subjects with a completely unmethylated promoter (Unmethylated/unmethylated status) held lower levels of total triglycerides, total cholesterol, LDL-C and higher levels of HDL-C. Conclusion: Our findings propose that the NPC1 promoter methylation is a probable mechanism that can result in reduced/impaired NPC1 expression/activity and may thus contribute to progression of CVD. Key Words: Niemann-pick type C1 (NPC1), Promoter methylation, cardiovascular disease     

Evaluating Beech Tree Phenology in a Deciduous Broadleaf Forest in Northern Iran Using Ground Observation and MODIS Images

We selected 15 beech trees (Fagus orientalis) from the study area in a mountainous region from 520 to 1310 m above sea level. Ground observations of the beech tree growth process from January to December 2004 in 7- to 15-day intervals were performed both visually and by measuring leaf chlorophyll concentration (chlorophyll meter SPAD-502). Results for the regression analysis showed that the leaved period positively correlated with the air temperature (r?=?.894). Anthesis and SPAD measurements have a negative relationship with air temperature and as temperature increases they appear earlier in the year. Anthesis and SPAD measurements had correlation coefficients of r?=??.883 and r?=??.855, respectively. The anthesis and bud bursting and leaf tip appearance have a negative relationship with increasing altitude from sea level and as altitude increases they appear later in the year. To make phenological events in deciduous broadleaf forests recognizable, we used a seasonal 8-day composite Moderate Resolution Imaging Spectroradiometer/Normalized Difference Vegetation Index (MODIS/NDVI). Prior to leaf expansion, NDVI increased from the 109th day of the year or earlier. Existence of the evergreen plants (Ruscus hyrcanus) on the ground and snow-melting exert an influence on inconsistency of phenology monitoring of MODIS/NDVI images. Regression analysis showed that there is a positive relationship between NDVI and SPAD measurements (chlorophyll content) in beech trees.     

Evaluation of Methods to Determine Sperm Density for the European eel,Anguilla anguilla

European eel, Anguilla anguilla, is a target species for future captive breeding, yet best methodology to estimate sperm density for application in in vitro fertilization is not established. Thus, our objectives were to evaluate methods to estimate European eel sperm density including spermatocrit, computer‐assisted sperm analysis (CASA) and flow cytometry (FCM), using Neubauer Improved haemocytometer as benchmark. Initially, relationships between spermatocrit, haemocytometer counts and sperm motility were analysed, as well as the effect of sperm dilution on haemocytometer counts. Furthermore, accuracy and precision of spermatocrit, applying a range of G‐forces, were tested and the best G‐force used in method comparisons. We found no effect of dilution on haemocytometer sperm density estimates, whereas motility associated positively with haemocytometer counts, but not with spermatocrit. Results from all techniques, spermatocrit, CASA and FCM, showed significant positive correlations with haemocytometer counts. The best correlation between spermatocrit and haemocytometer counts was obtained at 6000 ×  g (r = 0.68). Of two CASA variants, one or three photographic fields (CASA‐1 and CASA‐2), CASA‐2 showed a very high accuracy to haemocytometer counts (r  =  0.93), but low precision (CV: CASA‐2 = 28.4%). FCM was tested with and without microfluorospheres (FCM‐1 and FCM‐2), and relationships to haemocytometer counts were highly accurate (FCM‐1: r  =  0.94; FCM‐2: r  =  0.88) and precise (CV: FCM‐1 = 2.5; FCM‐2 = 2.7%). Overall, CASA‐2 and FCM‐1 feature reliable methods for quantification of European eel sperm, but FCM‐1 has a clear advantage featuring highest precision and accuracy. Together, these results provide a useful basis for gamete management in fertilization protocols.     

Expression and localization of angiopoietin family in corpus luteum during different stages of oestrous cycle and modulatory role of angiopoietins on steroidogenesis,angiogenesis and survivability of cultured buffalo luteal cells

The objective of this study was to document the expression and localization of angiopoietin (ANGPT) family members comprising of angiopoietin (ANGPT1 and ANGPT2), and their receptors (Tie1 and Tie2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle, and the modulatory role of ANGPT1 and ANGPT2 alone or in combinations on progesterone (P₄) secretion and mRNA expression of phosphotidylinositide‐3kinase‐protein kinase B (PI3K‐AKT), phosphoinositide‐dependent kinase (PDK), protein kinase B (AKT), Bcl2 associated death promoter (BAD), caspase 3 and von willebrand factor (vWF) in luteal cells obtained from midluteal phase (MLP) of oestrous cycle in buffalo. Real‐time RT‐PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors whereas, the P₄ secretion was assessed by RIA. The mRNA and protein expression of ANGPT1 and Tie2 was maximum (p < .05) in mid luteal phase (MLP) of oestrous cycle. The ANGPT2 mRNA and protein expression was maximum (p < .05) in early luteal phase, decreased in MLP and again increased in late luteal phase of oestrous cycle. ANGPT family members were localized in luteal cells and endothelial cells with a stage specific immunoreactivity. P₄ secretion was highest (p < .05) with 100 ng/ml at 72 hr when luteal cells were treated with either protein alone. The mRNA expression of PDK, AKT and vWF was highest (p < .05) and BAD along with caspase 3 were lowest (p < .05) at 100 ng/ml at 72 hr of incubation period, when cultured luteal cells were treated with either protein alone or in combination. To conclude, our study explores the steroidogenic potential of angiopoietins to promote P₄ secretion, luteal cell survival and angiogenesis through an autocrine and paracrine actions in buffalo CL.     

Induction of humoral and cellular immunity against latent HSV-1 infections by DNA immunization in BALB/c mice

Previously, we have reported that the injection of an expression vector containing Herpes simplex virus (HSV) Glycoprotein D-1 (gD-1) generated a significant antibody response in mice and protected them against HSV lethal challenge. We tested its potential to induce antibody and cell mediated immune responses in latently infected mice. Positive control group (KOS) and HSV gD-1 vaccinated mice demonstrated protection against a lethal ocularly challenge of 10(5.5) plaque-forming units (pfu)/eye of wild HSV-1 versus negative control groups. For neutralizing antibody titers, delayed-type hypersensitivity (DTH), lymphocyte proliferation responses, clinical evaluation and survival following lethal challenge, no considerable difference was observed between mice vaccinated with DNA plasmid and those vaccinated with KOS. KOS-vaccinated mice demonstrated the ability to completely prevent latency whereas DNA vaccinated group showed some degree of protection and displayed less latency than negative control groups and had considerably high levels of IFN-gamma and strong CTL responses versus negative control groups. It can be concluded that although immunization with the DNA vaccine is more effective in both protecting mice and induction of immune response, however it could not completely block the latent infection in sensory nerves.