Robust expression of LINE-1 retrotransposon encoded proteins in oral squamous cell carcinoma.

BACKGROUND: Oral Squamous Cell Carcinoma (OSCC) results from a series of genetic alteration in squamous cells. This particular type of cancer considers one of the most aggressive malignancies to control because of its frequent local invasions to the regional lymph node. Although several biomarkers have been reported, the key marker used to predict the behavior of the disease is largely unknown. Here we report Long INterpersed Element-1 (LINE1 or L1) retrotransposon activity in post-operative oral cancer samples. L1 is the only active retrotransposon occupying around 17% of the human genome with an estimated 500,000 copies. An active L1 encodes two proteins (L1ORF1p and L1ORF2p); both of which are critical in the process of retrotransposition. Several studies report that the L1 retrotransposon is highly active in many cancers. L1 activity is generally determined by assaying L1ORF1p because of its high expression and availability of the antibody. However, due to its lower expression and unavailability of a robust antibody, detection of L1ORF2p has been limited. L1ORF2p is the crucial protein in the process of retrotransposition as it provides endonuclease and reverse transcriptase (RT) activity. METHODS: Immunohistochemistry and Western blotting were performed on the post-operative oral cancer samples and murine tissues. RESULTS: Using in house novel antibodies against both the L1 proteins (L1ORF1p and L1ORF2p), we found L1 retrotransposon is extremely active in post-operative oral cancer tissues. Here, we report a novel human L1ORF2p antibody generated using an 80-amino-acid stretch from the RT domain, which is highly conserved among different species. The antibody detects significant L1ORF2p expression in human oral squamous cell carcinoma (OSCC) samples and murine germ tissues. CONCLUSIONS: We report exceptionally high L1ORF1p and L1ORF2p expression in post-operative oral cancer samples. The novel L1ORF2p antibody reported in this study will serve as a useful tool to understand why L1 activity is deregulated in OSCC and how it contributes to the progression of this particular cancer. Cross-species reactivity of L1ORF2p antibody due to the conserved epitope will be useful to study the retrotransposon biology in mice and rat germ tissues.

Transcriptomic analysis of Entamoeba histolytica reveals domain-specific sense strand expression of LINE-encoded ORFs with massive antisense expression of RT domain.

LINEs are retrotransposable elements found in diverse organisms. Their activity is kept in check by several mechanisms, including transcriptional silencing. Here we have analyzed the transcription status of LINE1 copies in the early-branching parasitic protist Entamoeba histolytica. Full-length EhLINE1 encodes ORF1, and ORF2 with reverse transcriptase (RT) and endonuclease (EN) domains. RNA-Seq analysis of EhLINE1 copies (both truncated and full-length) showed unique features. Firstly, although 20/41 transcribed copies were full-length, we failed to detect any full-length transcripts. Rather, sense-strand transcripts mapped to the functional domains- ORF1, RT and EN. Secondly, there was strong antisense transcription specifically from RT domain. No antisense transcripts were seen from ORF1. Antisense RT transcripts did not encode known functional peptides. They could possibly be involved in attenuating translation of RT domain, as we failed to detect ORF2p, whereas ORF1p was detectable. Lack of full-length transcripts and strong antisense RT expression may serve to limit EhLINE1 retrotransposition.

Beneficial dietary effect of turmeric and sulphur on weight gain, fat deposition and lipid profile of serum and liver in rats.

This study was designed to investigate the effects of turmeric powder and processed sulphur on the weight gain, body fat deposition and lipid profile of serum and liver in Wistar rats. Twenty-five rats of 6 weeks old were divided into five groups with 5 rats in each group. Each group was fed different diets as follows I. common diet (CON); II. high fat diet (HFD); III. 10% turmeric powder with HFD (T); IV. 10% turmeric powder and 0.19% processed sulphur with HFD (TS); and V. 0.38% processed sulphur with HFD (S). The experimental feeding was continued for 6 weeks. The body weight gain and feed efficiency ratio (FER) in the T and TS group rats were significantly (p < 0.05) lower than that of the HFD group rats. The retroperitoneal fat weights in the rats belong to T, TS and S groups were lower than that of the HFD group rats and the TS group had significant (p < 0.05) reduction in retroperitoneal fat compared to the HFD group rats. The epididymal fat weights in rats of the T, TS and S groups also showed a lowering tendency compared to that of the HFD group rats. The hepatic total lipid levels in the T and TS group rats were significantly (p < 0.05) lower than that of the HFD group rats. The hepatic triglyceride level in the rats of TS group was significantly (p < 0.05) lower than that of the HFD group rats. The serum total cholesterol, high-density lipoprotein (HDL) and low density lipoprotein (LDL) associated cholesterol contents in rats of the T and TS group were significantly (p < 0.05) higher than that of the HFD group rats, however, there was no significant difference in serum triglyceride. The results suggest that turmeric powder along with sulphur can reduce the weight gain, body fat deposition and improve serum and liver lipid profile in rats fed with a high fat diet.

Recombinant SINEs are formed at high frequency during induced retrotransposition in vivo.

Non-long terminal repeat Retrotransposons are referred to as long interspersed nuclear elements (LINEs) and their non-autonomous partners are short interspersed nuclear elements (SINEs). It is believed that an active SINE copy, upon retrotransposition, generates near identical copies of itself, which subsequently accumulate mutations resulting in sequence polymorphism. Here we show that when a retrotransposition-competent cell line of the parasitic protist Entamoeba histolytica, transfected with a marked SINE copy, is induced to retrotranspose, >20% of the newly retrotransposed copies are neither identical to the marked SINE nor to the mobilized resident SINEs. Rather they are recombinants of resident SINEs and the marked SINE. They are a consequence of retrotransposition and not DNA recombination, as they are absent in cells not expressing the retrotransposition functions. This high-frequency recombination provides a new explanation for the existence of mosaic SINEs, which may impact on genetic analysis of SINE lineages, and measurement of phylogenetic distances.

Development of restructured chicken block utilizing gizzard and its refrigerated storage stability.

A study was planned to develop restructured chicken product by incorporating optimum level of gizzard and fat and evaluate their quality attributes under refrigerated storage condition (4 ± 10 C). Incorporation of gizzard did not show any change in pH, but cooking yield reduced significantly (P < 0.05) at 40% level. There was a significant (P < 0.05) decrease in fat content and increase in protein and ash content with increase in the level of gizzard in the formulation. Significant (P < 0.05) improvement in the sensory attributes of chicken blocks was noticed by addition of gizzard. Incorporation of fat resulted in no change in pH, significant (P < 0.05) reduction in cooking yield, moisture and protein content and increase in fat and total ash content. Products incorporated with fat rated better than control for various sensory attributes. Significant (P < 0.05) increase in thiobarbituric acid reactive substance (TBARS), tyrosine value, stand plate count and psychrophilic count was observed during refrigerated storage (4 ± 10 C) in both control (no gizzard and fat) and test chicken blocks incorporated with gizzard and fat. The restructured chicken products were found acceptable up to 10 days under refrigerated storage condition (4 ± 10 C).

Quality of low-fat pork sausages with tomato powder as colour and functional additive during refrigerated storage.

Low fat pork sausages were formulated with tomato powder at 0% (C), 0.8% (T1), 1.2% (T2) and 1.5% (T3) levels in basic formula. With the increase in tomato powder concentration the lightness of the sausage decreased but the redness and yellowness increased significantly (p < 0.05). The pH values of T2 and T3 were significantly (p < 0.05) lower than the others, whereas, water holding capacity of T2 and T3 was significantly (p < 0.05) higher. Thiobarbituric acid reactive substances, cohesiveness and springiness values of treated groups were significantly (p < 0.05) lower than those of control samples, however, hardness values of sausages with tomato powder were significantly (p < 0.05) higher. The scores of overall acceptability in tested groups were significantly (p < 0.05) higher than those of control samples after 30 days of storage. The low fat pork sausage with tomato powder up to 1.5% was found to be well acceptable up to 30 days at refrigerated storage. This new product will have special value due to the functional additive lycopene in tomato powder.

Characterization of the restriction enzyme-like endonuclease encoded by the Entamoeba histolytica non-long terminal repeat retrotransposon EhLINE1.

The genome of the human pathogen Entamoeba histolytica, a primitive protist, contains non-long terminal repeat retrotransposable elements called EhLINEs. These encode reverse transcriptase and endonuclease required for retrotransposition. The endonuclease shows sequence similarity with bacterial restriction endonucleases. Here we report the salient enzymatic features of one such endonuclease. The kinetics of an EhLINE1-encoded endonuclease catalyzed reaction, determined under steady-state and single-turnover conditions, revealed a significant burst phase followed by a slower steady-state phase, indicating that release of product could be the slower step in this reaction. For circular supercoiled DNA the K(m) was 2.6 x 10(-8) M and the k(cat) was 1.6 x 10(-2) sec(-1). For linear E. histolytica DNA substrate the K(m) and k(cat) values were 1.3 x 10(-8) M and 2.2 x 10(-4) sec(-1) respectively. Single-turnover reaction kinetics suggested a noncooperative mode of hydrolysis. The enzyme behaved as a monomer. While Mg(2+) was required for activity, 60% activity was seen with Mn(2+) and none with other divalent metal ions. Substitution of PDX(12-14)D (a metal-binding motif) with PAX(12-14)D caused local conformational change in the protein tertiary structure, which could contribute to reduced enzyme activity in the mutated protein. The protein underwent conformational change upon the addition of DNA, which is consistent with the known behavior of restriction endonucleases. The similarities with bacterial restriction endonucleases suggest that the EhLINE1-encoded endonuclease was possibly acquired from bacteria through horizontal gene transfer. The loss of strict sequence specificity for nicking may have been subsequently selected to facilitate spread of the retrotransposon to intergenic regions of the E. histolytica genome.