Cloning and identification of a novel C18-Δ9 polyunsaturated fatty acid specific elongase gene from DHA-producing <Emphasis Type="Italic">Isochrysis galbana</Emphasis> H29

Isochrysis galbana, a marine prymnesiophyte microalga, is able to produce a high level of long chain polyunsaturated fatty acids such as docosahexaenoic acid (DHA, C22:6n-3). In this article, a novel gene (IgASE2) that encoded a C18-Δ9 polyunsaturase fatty acids specific (C18-Δ9-PUFAs-specific) elongase was isolated and characterized from DHA-rich microalga, I. galbana H29. A full-length cDNA of 1653 bp was cloned by rapidamplification of cDNA ends (RACE) PCR techniques. The IgASE2 contained a 786 bp ORF encoding a protein of 261 amino acids that shared 87% identity with the reported Δ9-elongase IgASE1, a 44 bp 5′ untranslated region and an 823 bp 3′ untranslated region. The function of IgASE2 was demonstrated by its heterologous expression in Saccharomyces cerevisiae. In S. cerevisiae, IgASE2 elongated linoleic acid (LA, C18:2n-6), α-linolenic (ALA, C18:3n-3) to eicosadienoic acid (EDA, C20:2n-6) and eicosatrienoic acid (ETrA, C20:3n-3). The conversion ratios of LA to EDA and ALA to ETrA were 60.47 and 58.36%, respectively. However, IgASE2 could not catalyze the elongation reactions of oleic acid (OA, C18:1n-9) and other fatty acids. These results confirmed that IgASE2 had C18-Δ9-PUFAs-specific elongase activity.     

The roles of parathyroid hormone-like hormone during mouse preimplantation embryonic development

Parathyroid hormone-like hormone (PTHLH) was first identified as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcemia in malignancies in the 1980s. Previous studies demonstrated that PTHLH is expressed in multiple tissues and is an important regulator of cellular and organ growth, development, migration, differentiation, and survival. However, there is a lack of data on the expression and function of PTHLH during preimplantation embryonic development. In this study, we investigated the expression characteristics and functions of PTHLH during mouse preimplantation embryonic development. The results show that Pthlh is expressed in mouse oocytes and preimplantation embryos at all developmental stages, with the highest expression at the MII stage of the oocytes and the lowest expression at the blastocyst stage of the preimplantation embryos. The siRNA-mediated depletion of Pthlh at the MII stage oocytes or the 1-cell stage embryos significantly decreased the blastocyst formation rate, while this effect could be corrected by culturing the Pthlh depleted embryos in the medium containing PTHLH protein. Moreover, expression of the pluripotency-related genes Nanog and Pou5f1 was significantly reduced in Pthlh-depleted embryos at the morula stage. Additionally, histone acetylation patterns were altered by Pthlh depletion. These results suggest that PTHLH plays important roles during mouse preimplantation embryonic development.     

Novel HRPT2/CDC73 Gene Mutations and Loss of Expression of Parafibromin in Chinese Patients with Clinically Sporadic Parathyroid Carcinomas

Objective

It is widely recognized that the diagnosis of parathyroid carcinoma (PC) is often difficult because of the overlap of characteristics between malignant and benign parathyroid tumors, especially at an early stage. Based on the identification of tumor suppressor gene HRPT2/CDC73 and its association with hereditary and sporadic PC, screening of gene mutations and detection of parafibromin immunoreactivity have been suggested as diagnostic instruments of PC in Whites. There is little information about HRPT2/CDC73 mutations and its corresponding protein expression in patients with sporadic PC in Chinese population, and the long-term follow-up data is scarce.

Methods

Paraffin-embedded tissues were obtained from 13 patients with PC, 13 patients with parathyroid adenoma (PA) and 7 patients with parathyroid hyperplasia(PH), and 6 normal parathyroid (NP) tissues as controls. Peripheral blood from 11 patients with PC was collected. PCR products using Genomic DNA extracted from tumor tissues or blood as template was sequenced for HRPT2/CDC73 gene. Expression of parafibromin in tumor tissues was evaluated by immunohistochemical analysis.

Results

Six mutations in 6 of 13 patients with PC were identified, with three being novel. Four of them were germ-line mutations. Patients with mutations were susceptible to recurrence of the PC. Complete (8/13, 61.5%) or partial (5/13, 38.5%) loss of parafibromin expression was observed in PC tissues. All of tissue samples from normal parathyroid or benign parathyroid tumors displayed positive immunostaining of parafibromin except one adenoma.

Conclusions

The present study supplies information on the mutations and protein expression of HRPT2/CDC73 gene and phenotypes of parathyroid carcinoma in Chinese population. And the expanded mutation database of this gene may benefit patients in the diagnosis and treatment of this disease.     

GroEL1, from Chlamydia pneumoniae, Induces Vascular Adhesion Molecule 1 Expression by p37(AUF1) in Endothelial Cells and Hypercholesterolemic Rabbit

The expression of vascular adhesion molecule-1 (VCAM-1) by endothelial cells may play a major role in atherogenesis. The actual mechanisms of chlamydia pneumoniae (C. pneumoniae) relate to atherogenesis are unclear. We investigate the influence of VCAM-1 expression in the GroEL1 from C. pneumoniae-administered human coronary artery endothelial cells (HCAECs) and hypercholesterolemic rabbits. In this study, we constructed the recombinant GroEL1 from C. pneumoniae. The HCAECs/THP-1 adhesion assay, tube formation assay, western blotting, enzyme-linked immunosorbent assay, actinomycin D chase experiment, luciferase reporter assay, and immunohistochemical stainings were performed. The results show that GroEL1 increased both VCAM-1expression and THP-1 cell adhesives, and impaired tube-formation capacity in the HCAECs. GroEL1 significantly increased the VCAM-1 mRNA stability and cytosolic AU-binding factor 1 (AUF1) level. Overexpression of the p37(AUF1) significantly increased VCAM-1 gene expression in GroEL1-induced bovine aortic endothelial cells (BAECs). GroEL1 prolonged the stability of VCAM-1 mRNA by increasing both p37(AUF1) and the regulation of the 5' untranslated region (UTR) of the VCAM-1 mRNA in BAECs. In hypercholesterolemic rabbits, GroEL1 administration enhanced fatty-streak and macrophage infiltration in atherosclerotic lesions, which may be mediated by elevated VCAM-1 expression. In conclusion, GroEL1 induces VCAM-1 expression by p37(AUF1) in endothelial cells and enhances atherogenesis in hypercholesterolemic rabbits.     

Association of Fcγ Receptor IIB Polymorphism with Cryptococcal Meningitis in HIV-Uninfected Chinese Patients

Background

As important regulators of the immune system, the human Fcγ receptors (FcγRs) have been demonstrated to play important roles in the pathogenesis of various infectious diseases. The aim of the present study was to identify the association between FCGR polymorphisms and cryptococcal meningitis.

Methodology/Principal Findings

In this case control genetic association study, we genotyped four functional polymorphisms in low-affinity FcγRs, including FCGR2A 131H/R, FCGR3A 158F/V, FCGR3B NA1/NA2, and FCGR2B 232I/T, in 117 patients with cryptococcal meningitis and 190 healthy controls by multiplex SNaPshot technology. Among the 117 patients with cryptococcal meningitis, 59 had predisposing factors. In patients with cryptococcal meningitis, the FCGR2B 232I/I genotype was over-presented (OR = 1.652, 95% CI [1.02–2.67]; P = 0.039) and the FCGR2B 232I/T genotype was under-presented (OR = 0.542, 95% CI [0.33–0.90]; P = 0.016) in comparison with control group. In cryptococcal meningitis patients without predisposing factors, FCGR2B 232I/I genotype was also more frequently detected (OR = 1.958, 95% CI [1.05–3.66]; P = 0.033), and the FCGR2B 232I/T genotype was also less frequently detected (OR = 0.467, 95% CI [0.24–0.91]; P = 0.023) than in controls. No significant difference was found among FCGR2A 131H/R, FCGR3A 158F/V, and FCGR3B NA1/NA2 genotype frequencies between patients and controls.

Conclusion/Significance

We found for the first time associations between cryptococcal meningitis and FCGR2B 232I/T genotypes, which suggested that FcγRIIB might play an important role in the central nervous system infection by Cryptococcus in HIV-uninfected individuals.     

Changes in DNA methylation and transgenerational mobilization of a transposable element (mPing) by the topoisomerase II inhibitor, etoposide, in rice

ABSTRACT: BACKGROUND: Etoposide (epipodophyllotoxin) is a chemical commonly used as an anti-cancer drug which inhibits DNA synthesis by blocking topoisomerase II activity. Previous studies in animal cells have demonstrated that etoposide constitutes a genotoxic stress which may induce genomic instability including mobilization of normally quiescent transposable elements (TEs). However, it remained unknown whether similar genetically mutagenic effects could be imposed by etoposide in plant cells. Also, no information is available with regard to whether the drug may cause a perturbation of epigenetic stability in any organism. RESULTS: To investigate whether etoposide could generate genetic and/or epigenetic instability in plant cells, we applied etoposide to germinating seeds of six cultivated rice (Oryza sativa L.) genotypes including both subspecies, japonica and indica. Based on the methylation-sensitive gel-blotting results, epigenetic changes in DNA methylation of three TEs (Tos17, Osr23 and Osr36) and two protein-encoding genes (Homeobox and CDPK-related genes) were detected in the etoposide-treated plants (S0 generation) in four of the six studied japonica cultivars, Nipponbare, RZ1, RZ2, and RZ35, but not in the rest japonica cultivar (Matsumae) and the indica cultivar (93-11). DNA methylation changes in the etoposide-treated S0 rice plants were validated by bisulfite sequencing at both of two analyzed loci (Tos17 and Osr36). Transpositional activity was tested for eight TEs endogenous to the rice genome in both the S0 plants and their selfed progenies (S1 and S2) of one of the cultivars, RZ1, which manifested heritable phenotypic variations. Results indicated that no transposition occurred in the etoposide-treated S0 plants for any of the TEs. Nonetheless, a MITE transposon, mPing, showed rampant mobilization in the S1 and S2 progenies descended from the drug-treated S0 plants. CONCLUSIONS: Our results demonstrate that etoposide imposes a similar genotoxic stress on plant cells as it does on animal and human cells, which may induce transgenerational genomic instability by instigating transpositional activation of otherwise dormant TEs. In addition, we show for the first time that etoposide may induce epigenetic instability in the form of altered DNA methylation patterns in eukaryotes. However, penetration of the genotoxic effects of etoposide on plant cells, as being reflected as genetic and epigenetic instability, appears to be in a strictly genotype- and/or generation-dependent manner.