Characterization of a polyclonal antibody to human pituitary tumor transforming gene 1 (PTTG1) protein.

Pituitary tumor transforming gene 1 (PTTG1), recently cloned from human testis, is a potent oncogene that is expressed in most tumors. However, assessment of its potential value as a prognostic marker is dependent on the development of a suitable antibody. We have developed a rabbit polyclonal antibody, SK601, that is highly specific for the PTTG1 gene product using recombinant PTTG1 protein (24 kD) containing an N-terminal His(6) tag as the immunogen. The antiserum is capable of detecting recombinant PTTG1 protein in ELISA assays at a titer of 1:100,000. Use of the antibody as the probe in Western blotting analyses revealed a single band with the anticipated relative molecular weights of 52 kD from E. coli expressing the GST-PTTG1 recombinant protein, and 56 kD from COS-7 cells transfected with the PTTG1-GFP chimeric construct. A single band with a relative molecular weight of 28 kD was observed in extract of COS-7 cells transfected with PTTG1 cDNA. The antiserum immunoprecipitated a protein of relative molecular weight of 56 kD from the extracts of COS-7 cells transfected with the PTTG1-GFP chimeric construct. Immunohistochemical analysis of COS-7 cells transfected with this construct confirmed that the antibody detected and was specific for expressing the PTTG1-GFP recombinant protein. Screening of various normal human tissues (testis, ovary, and breast) by immunohistochemistry indicated that these tissues did not exhibit staining with the exception of testis, a tissue that had previously been shown to express PTTG1 mRNA. In contrast all of the tumor tissues (testicular tumor, ovarian tumor, and breast tumor) that were assessed exhibited intense staining. The results suggest that antiserum SK601 is highly specific for the PTTG1 protein and therefore should prove useful in further analysis of the expression and interactions of this protein, including its potential application as an immunohistochemical marker of human tumors.     

Increasement of Heterologous Expression of Recombinant Vit v 1 in Pichia pastoris KM71 by Nonionic Detergents as a Cost-effective Approach

Applied Biochemistry and Microbiology - Vit v 1 as a lipid-transfer protein is a major allergen of grapes (Vitis vinifera) that elicits food allergy in many patients in Iran. Todays, recombinant...     

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp,Ctenopharyngodon idellus

Background  

Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs.     

GC-1 mRHBDD1 knockdown spermatogonia cells lose their spermatogenic capacity in mouse seminiferous tubules

Background  

Apoptosis is important for regulating spermatogenesis. The protein mRHBDD1 (mouse homolog of human RHBDD1)/rRHBDD1 (rat homolog of human RHBDD1) is highly expressed in the testis and is involved in apoptosis of spermatogonia. GC-1, a spermatogonia cell line, has the capacity to differentiate into spermatids within the seminiferous tubules. We constructed mRHBDD1 knockdown GC-1 cells and evaluated their capacity to differentiate into spermatids in mouse seminiferous tubules.     

An Alu repeat-mediated genomic GCNT2 deletion underlies congenital cataracts and adult i blood group

We performed homozygosity mapping in a consanguineous Pakistani family segregating autosomal-recessive congenital cataracts and identified linkage to a 3.03 Mb locus on chromosome 6p24 containing the GCNT2 gene. GCNT2 encodes glucosaminyl (N-acetyl) transferase 2, an enzyme responsible for the formation of the blood group I antigen. Rare biallelic GCNT2 mutations have been shown to cause the association of congenital cataracts and the adult i blood group, making GCNT2 the prime candidate gene for the observed phenotype. Indeed, we identified a homozygous deletion segregating with cataracts that encompasses exons 1B, 1C, 2 and 3 of GCNT2. Long-range polymerase chain reaction and breakpoint sequencing revealed that affected individuals in this and in a second, apparently unrelated Pakistani family segregating congenital cataracts are homozygous for the same 93 kb deletion. The deletion is flanked by Alu repeats of the AluS family on both sides and microsatellite genotyping suggested that its occurrence in the two families was the product of recurrent Alu-Alu repeat-mediated nonhomologous recombinations or an old founder effect. Subsequently, we showed that cataract-affected individuals in both families have the adult i blood group, whereas unaffected individuals have blood group I as the vast majority of the population. Because the GCNT2 locus is rich in Short INterspersed Elements (SINE repeats) and thus likely prone to genomic rearrangements, microdeletions or microduplications at this locus might cause a larger than currently anticipated fraction of apparently isolated autosomal-recessive cataracts.     

Withaferin A Synergizes the Therapeutic Effect of Doxorubicin through ROS-Mediated Autophagy in Ovarian Cancer

Application of doxorubicin (Dox) for the treatment of cancer is restricted due to its severe side effects. We used combination strategy by combining doxorubicin (Dox) with withaferin A (WFA) to minimize the ill effects of Dox. Treatment of various epithelial ovarian cancer cell lines (A2780, A2780/CP70 and CaOV3) with combination of WFA and Dox (WFA/DOX) showed a time- and dose-dependent synergistic effect on inhibition of cell proliferation and induction of cell death, thus reducing the dosage requirement of Dox. Combination treatment resulted in a significant enhancement of ROS production resulting in immense DNA damage, induction of autophagy analyzed by transmission electron microscope and increase in expression of autophagy marker LC3B, and culminated in cell death analyzed by cleaved caspase 3. We validated combination therapy on tumor growth using an in vitro 3Dimension (3D) tumor model and the more classic in vivo xenograft model of ovarian cancer. Both tumor models showed a 70 to 80% reduction in tumor growth compared to control or animals treated with WFA or Dox alone. Immunohistochemical analysis of the tumor tissues from animals treated with WFA/Dox combination showed a significant reduction in cell proliferation and formation of microvessels accompanied by increased in LC3B level, cleaved caspase 3, and DNA damage. Taken together, our data suggest that combining WFA with Dox decreases the dosage requirement of Dox, therefore, minimizing/eliminating the severe side effects associated with high doses of DOX, suggesting the application of this combination strategy for the treatment of ovarian and other cancers with no or minimum side effects.     

Transgenic expression of entire hepatitis B virus in mice induces hepatocarcinogenesis independent of chronic liver injury

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV ("preS2 mutant") that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged--2-year-old--mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines--expressing either mutant or wildtype HBV--therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC.