Cryopreservation of Protoplast-Derived Cell Suspensions of Rice (Oryza sativa L.)

The protoplast-derived cell suspensions of rice (Oryza sativa L. cv. A 58 MS) were frozen in liquid nitrogen (LN) in the presence of 10–20% dimethylsulfoxide (DMSO) and 10–20% sucrose in combination, the survival reached 40-50% of the control. The retrieved cells were proliferated in Linsmier-Skoog (LS, 1965) medium supplemented with 2×10-5 mol/ l 2,4-D. Shoots and roots were induced from calli formed from frozen cells in LS medium with l×l0 mol/l NAA plus 4×10-6 mol/l kinetin and lxl0-6mol/l 2 IP. The results are discussed.     

Molecular cloning and expression analysis of the mevalonate kinase gene from Arabidopsis thaliana

Mevalonate kinase (MVK), the enzyme that catalyzes the phosphorylation of mevalonate to produce mevalonate 5-phosphate, is considered as a potential regulatory enzyme of the isoprenoid biosynthetic pathway. The Arabidopsis thaliana MVK gene corresponding to the MVK cDNA previously isolated has been cloned and characterized. RNAse protection analysis indicated that the expression of the MVK gene generates three mRNA populations with 558m8j01g528h871/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> ends mapping 203, 254 and 355 nt upstream of the MVK ATG start codon. Northern blot analysis showed that the MVK mRNA accumulates preferentially in roots and inflorescences. Histochemical analysis, with transgenic A. thaliana plants containing a translational fusion of a 1.8 kb fragment of the 558m8j01g528h871/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> region of the MVK gene to the 58m8j01g528h871/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase (GUS) reporter gene, indicated that the MVK 558m8j01g528h871/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-flanking region directs widespread expression of the GUS gene throughout development, although the highest levels of GUS activity are detected in roots (meristematic region) and flowers (sepals, petals, anthers, style and stigmatic papillae). The expression pattern of the MVK gene suggests that the role of the encoded MVK is the production of a general pool of mevalonate-5-phosphate for the synthesis of different classes of isoprenoids involved in both basic and specialized plant cell functions. Functional promoter deletion analysis in transfected A. thaliana protoplasts indicated that regulatory elements between positions –295 and –194 of the MVK 558m8j01g528h871/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-flanking region are crucial for high-level MVK gene expression.     

Functional IFNG polymorphism in intron 1 in association with an increased risk to promote sporadic breast cancer

Interferon (IFN)-58m215205365/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0"> is an important Th1 cytokine, which plays a role in immune surveillance and anti-tumor activity. A case-control study involving 54 sporadic breast cancer patients and 144 healthy controls was carried out to explore if the genotype variation of a proposed non-specific enhancer element with a dinucleotide (CA)_n repeat in intron 1 has a role in the susceptibility to promote sporadic breast cancer. Genotype analysis carried out by single-strand length polymorphism and confirmed by sequencing showed an increased frequency of (CA)₁₂ allele (P<0.001) and decreased frequencies of (CA)₁₅ (P<0.01) and (CA)_>15 (p<0.001) alleles in sporadic breast cancer patients as compared to controls. Further, in vitro reporter assays for (CA)₁₂ and (CA)₁₅ alleles suggested these to be associated with decreased and increased expressions, respectively, suggesting the (CA)₁₂/(CA)₁₂ background to act as one of the factors that could lead to low production of IFN-58m215205365/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">. The study concludes that such genetic background for a proposed non-specific enhancer element with (CA)_n repeat within intron 1 of the IFNG gene might put the individuals with this genotype at higher risk to promote the development of sporadic breast cancer due to a resultant compromised immune surveillance.Anjana Saha and Ashish Dhir contributed equally.     

Increased DNA breaks and up-regulation of both G1 and G2 checkpoint genes p21WAF1/CIP1 and 14-3-3 σ in circulating leukocytes of glaucoma patients and vasospastic individuals

Summary. Objective: Vascular disorder leading to local ischemia/reperfusion has been shown to play an important role in the glaucomatous damage. A decreased expression level of XPGC-gene has been found in circulating leukocytes of normal-tension glaucoma patients. Although decreased activity of XPGC-gene leads to insufficient DNA-repair, no leukopenia has been observed in glaucoma. Molecular mechanisms ensuring cell survival have not been elucidated yet for glaucoma with vascular disorder.Material and methods: Using the ex vivo optical imaging method of alkaline 58ec1u7rjk/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">comet assay58ec1u7rjk/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> comparative quantification of DNA breaks was performed in circulating leukocytes of non-glaucomatous non-vasospastic and vasospastic individuals as well as both normal-tension and high-tension glaucoma patients. Relative expression levels of the anti-apoptotic factors P21^WAF1/CIP1 and 14-3-3 58ec1u7rjk/xxlarge963.gif" alt="sgr" align="BASELINE" BORDER="0"> were investigated in all groups tested.Results and conclusions: The quantification of P21^WAF1/CIP1 showed the highest expression rates in high-tension glaucoma patients which were significantly higher than those in all other groups tested. The highest expression rates of 14-3-3 58ec1u7rjk/xxlarge963.gif" alt="sgr" align="BASELINE" BORDER="0"> were found in both groups of glaucoma patients. These expression levels correlated well with DNA breaks measured. Since the expression of P21^WAF1/CIP1 in leukocytes was shown to be crucial for their survival under stress conditions, we suppose further that the up-regulation of this gene is the key event in the survival mechanisms of leukocytes in glaucoma accompanied with vascular disorder. The p21^WAF1/CIP1 gene should be further taken into consideration as a potential marker, the up-regulation of which in circulating leukocytes of vasospastic individuals may indicate an increased risk for the developing glaucoma.     

Changes in cell wall polysaccharides in developing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) coleoptiles

Cell wall polysaccharides in developing barley coleoptiles were examined using acetic acid–nitric acid extraction, alditol acetate and methylation analyses and enzymatic digestion. The coleoptile cell wall from imbibed grain was rich in pectic polysaccharides (30 mol%), arabinoxylan (25 mol%), cellulose (25 mol%) and xyloglucan (6 mol%), but contained only low levels of (158p24v/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">3,158p24v/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">4)-58p24v/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-D-glucan (1 mol%). During 5 days of coleoptile growth, pectic polysaccharides decreased steadily to about 9 mol%, while (158p24v/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">3,158p24v/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">4)-58p24v/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-D-glucan increased to 10 mol%. Following the cessation of growth of the coleoptiles at about 5 days, (158p24v/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">3,158p24v/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">4)-58p24v/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-D-glucan content rapidly decreased to 1 mol%. The cellulose content of the walls remained at about 35–40 mol% throughout coleoptile growth. Similarly, arabinoxylan content remained essentially constant at 25–30 mol% during growth, although the ratio of substituted to unsubstituted 4-linked xylosyl units decreased from about 4:1 to 1:1. Xyloglucan content ranged from 6 mol% to 10 mol% and the oligosaccharide profile determined using a xyloglucan-specific endoglucanase and MALDI-TOF mass spectrometry indicated that the oligosaccharides XXGG and XXGGG were the principal components, with one and two acetyl groups, respectively, Thus, dramatic changes in wall composition were detected during the growth of barley coleoptiles, both with respect to the relative abundance of individual wall constituents and to the fine structure of the arabinoxylans.     

Sonic hedgehog pathway activation regulates cervical cancer stem cell characteristics during epithelial to mesenchymal transition

Resistance to therapy and metastasis remains one of the leading causes of mortality due to cervical cancer despite advances in detection and treatment. The mechanism of epithelial to mesenchymal transition (EMT) provides conceptual explanation to the invasiveness and metastatic spread of cancer but it has not been fully understood in cervical cancer. This study aims to investigate the mechanism by which silencing of E-cadherin gene regulates EMT leading to proliferation, invasion, and chemoresistance of cervical cancer cells through the Hedgehog (Hh) signaling pathway. We developed an in vitro EMT model by the knockdown of E-cadherin expression in cervical cancer cell lines. To understand the role of developmental pathway like Hh in the progression of cervical cancer, we investigated the expression of Hh pathway mediators by array in E-cadherin low cervical cancer cells and observed upregulation of Hh pathway. This was further validated on low passage patient-derived cell lines and cervical carcinoma tissue sections from cervical cancer patients. Further, we evaluated the role of two inhibitors (cyclopamine and GANT58) of the Hh pathway on invasiveness and apoptosis in E-cadherin low cervical cancer cells. In conclusion, we observed that inhibition of Hh pathway with GANT58 along with current therapeutic procedures could be more effective in targeting drug-resistant EMT cells and bulk tumor cells in cervical cancer.     

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall ^1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients ^3,4.In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients ³. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.