Association of thyroid hormones with the severity of chronic kidney disease: a cross-sectional observational study at Tabuk, Saudi Arabia.

Background: The interplay between chronic kidney disease (CKD) and thyroid dysfunction is becoming more evident in the biomedical community. However, the intricacies of their relationship warrant deeper investigation to understand the clinical implications fully. Objective: This study aims to systematically evaluate the correlation between thyroid hormone levels, including thyroid-stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4), and markers of renal disease severity. These markers include serum creatinine, urea, and parathyroid hormone (PTH) levels in individuals diagnosed with CK). Methods: We conducted a cross-sectional observational study involving a cohort of 86 participants with CKD recruited from the renal clinic at King Fahad Hospital in Tabuk. Biochemical parameters, encompassing plasma electrolytes and thyroid hormone concentrations, were quantitatively assessed. These measurements were performed with the aid of a Roche Cobas E411 analyzer. The Pearson correlation coefficient was employed to delineate the strength and direction of the associations between the thyroid function markers and renal disease indicators. Results: The statistical analysis highlighted a generally weak correlation between the concentrations of thyroid hormones and the indicators of renal disease severity, with Pearson correlation coefficients between -0.319 and 0.815. Critically, no significant correlation was found between creatinine and thyroid hormones (TSH, T3, T4), nor was any substantial correlation between urea and thyroid hormones. Conversely, a robust positive correlation was noted between the levels of parathyroid hormone and serum creatinine (r = 0.718, p < 0.001). Conclusion: The data suggests that thyroid hormone levels have a minimal correlation with the severity of renal disease markers. In contrast, the pronounced correlation between PTH and creatinine underscores the importance of considering PTH as a significant factor in managing and therapeutic intervention of CKD complications. These initial findings catalyze further research to thoroughly investigate the pathophysiological relationships and potential therapeutic targets concerning thyroid dysfunction in patients with renal impairment.

Impact of Various Washing Protocols on the Mitigation of Escherichia coli Contamination in Raw Salad Vegetables.

Vegetables are an essential component of a balanced diet. The consumption of ready-to-eat foods may lead to the risk of infections and illnesses due to microbial contamination. To mitigate the potential of microbial contamination risks, it is critical to promote safe handling practices among consumers. In this study, our research evaluated the efficacy of different vegetable washing methods, specifically with lettuce, tomato, and cucumber, to establish optimal practices for reducing microbial contamination. This study consisted of two phases. Initially, a survey was distributed to 150 volunteers using snowball sampling to assess everyday vegetable handling and washing methods. The survey's results identified four predominant methods: washing with a 5% vinegar solution for 3 min followed by tap water rinse (37.3% of participants), rinsing with tap water for 1 min (29.3%), washing with a 5% salt solution (vegetable soap) for 3 min followed by a tap water rinse (16.6%), and a 3 min tap water rinse (14%). A minor segment (3.33%) reported not washing their vegetables at all. The survey's findings guided the second phase, which tested the aforementioned washing protocols' effectiveness in reducing Escherichia coli (E. coli) levels on spiked contaminated salad vegetables. The tested vegetables were sterilized using UV light, inoculated with 0.5 McFarland E. coli, and then washed using the four identified methods. After that, E. coli enumeration after washing was performed using 3M™ Petrifilm and the comparison was analyzed via one-way ANOVA. During this study, it was revealed that the cucumbers had the highest E. coli contamination levels in comparison to the lettuce and tomato after washing. Interestingly, by comparing the three washing methods, it was found that washing the vegetables with vinegar proved to be the most effective solution for reducing microbial presence on both lettuce and cucumbers. Notably, the natural smoothness of tomato skin led to no significant differences in contamination levels across washing methods. In summary, vinegar washing effectively reduces microbial contamination from salad vegetables, highlighting the need for informed consumer practices to prevent foodborne outbreaks. This study emphasizes the importance of monitoring contamination sources and using safe washing techniques.

Exploring Therapeutic Potential of Catalase: Strategies in Disease Prevention and Management.

The antioxidant defense mechanisms play a critical role in mitigating the deleterious effects of reactive oxygen species (ROS). Catalase stands out as a paramount enzymatic antioxidant. It efficiently catalyzes the decomposition of hydrogen peroxide (H2O2) into water and oxygen, a potentially harmful byproduct of cellular metabolism. This reaction detoxifies H2O2 and prevents oxidative damage. Catalase has been extensively studied as a therapeutic antioxidant. Its applications range from direct supplementation in conditions characterized by oxidative stress to gene therapy approaches to enhance endogenous catalase activity. The enzyme's stability, bioavailability, and the specificity of its delivery to target tissues are significant hurdles. Furthermore, studies employing conventional catalase formulations often face issues related to enzyme purity, activity, and longevity in the biological milieu. Addressing these challenges necessitates rigorous scientific inquiry and well-designed clinical trials. Such trials must be underpinned by sound experimental designs, incorporating advanced catalase formulations or novel delivery systems that can overcome existing limitations. Enhancing catalase's stability, specificity, and longevity in vivo could unlock its full therapeutic potential. It is necessary to understand the role of catalase in disease-specific contexts, paving the way for precision antioxidant therapy that could significantly impact the treatment of diseases associated with oxidative stress.

Hydroxychloroquine interaction with phosphoinositide 3-kinase modulates prostate cancer growth in bone microenvironment: In vitro and molecular dynamics based approach.

Patients with advanced prostate cancer (PCa) are more likely to develop bone metastases. Tumor cells thrive in the bone microenvironment, interacting with osteoblasts and osteoclasts. Given the PI3K/AKT pathway's metastatic potential and signal integration's ability to modulate cell fates in PCa development, drugs targeting this system have great therapeutic promise. Hydroxychloroquine (HCQ) is an anti-malarial medication commonly used to treat clinical conditions such as rheumatology and infectious disorders. We explored the anti-neoplastic effect of HCQ on PC3 and C4-2B cell lines in the bone microenvironment. Interestingly, HCQ treatment substantially decreases the viability, proliferation, and migration potential of PCa cells in the bone microenvironment. HCQ induces apoptosis and cell cycle arrest, even in the presence of osteoblast-secreted factors. Mechanistically, HCQ inhibited the activity of the PI3K/AKT signaling pathway, which ultimately regulates the proliferation and migration of PCa cells in the bone. The binding energy for docking HCQ with PI3K was -6.7 kcal/mol, and the complex was stabilized by hydrogen bonds, hydrophobic forces, and van der Waals forces. Molecular simulations further validated the structural integrity of the HCQ-PI3K complex without altering PI3K's secondary structure. Our findings underscore the efficacy of HCQ as a potential therapeutic agent in treating PCa.

Nizatidine interacts with ct-DNA causing genotoxicity and cytotoxicity: an assessment by in vitro, in vivo, and in silico studies.

H2 receptor antagonists are the medication given for treating stomach ulcers, but lately, reports have shown their role in healing several malignant ulcers. The present work entails the interaction of H2 blocker nizatidine with calf thymus (ct)-DNA for determining the binding mode and energetics of the interaction. Multi-spectroscopic, calorimetric, viscometric and bioinformatic analysis revealed that nizatidine interacted with ct-DNA via groove-binding mode and is characterised by exothermic reaction. Moreover, assessment of genotoxic potential of nizatidine in vitro was carried out in peripheral human lymphocytes by alkaline comet assay. DNA damage occurred at high concentrations of nizatidine. Genotoxicity of nizatidine was also evaluated in vivo by assessing cytogenetic biomarkers viz. micronuclei formation and chromosomal aberration test. Nizatidine was able to induce micronuclei formation and chromosomal damage at high dose. Additionally, cytotoxic activity of nizatidine was determined in cancer cell lines, namely HeLa and HCT-116 and compared with the normal human cell line HEK-293 employing MTT assay. It was observed that nizatidine was more toxic towards HeLa and HCT-116 than HEK-293. Cell morphology analysis by compound inverted microscopy further strengthens the finding obtained through MTT assay.

Interaction of memantine with calf thymus DNA: an in-vitro and in-silico approach and cytotoxic effect on the cancerous cell lines.

Memantine belongs to the class of cognition enhancers that functions as NMDA receptor antagonist, used to treat Alzheimer's disease. The interaction of memantine with DNA was not investigated. In the present study, the interaction of memantine with ct-DNA, as well as its cytotoxicity on cancer cells, was evaluated. UV-visible spectroscopy, steady-state fluorescence spectroscopic studies revealed the interaction between memantine and ct-DNA. The quenching studies, chemical denaturation, (CD), and DNA melting studies showed the groove binding mode of memantine with ct-DNA. The thermodynamic parameters revealed that the interaction between memantine and ct-DNA is enthalpically driven, and the stabilizing forces involved were hydrogen bonding and van der Waals interaction. The groove-binding was also observed by molecular docking studies, which corroborated the findings of spectroscopic investigations. Density function theory calculations confirmed the existence of electron donor and recipient groups. The stability of memantine and DNA interaction, as well as the critical residues involved in the interaction, was identified by molecular dynamics simulations. Memantine showed cytotoxicity towards the cancer cells as compared to normal cells, as observed by MTT assay. Inverted compound microscopy analysis of memantine treated cancer cell lines further confirmed the results obtained by MTT assay.Communicated by Ramaswamy H. Sarma.

Glyburide inhibits non-enzymatic glycation of HSA: An approach for the management of AGEs associated diabetic complications.

Advanced glycation endproducts (AGEs) are the final product of glycation, highly reactive in nature and contribute directly or indirectly to numerous complications related to diabetes. In this study, the antiglycation activity of glyburide was investigated using HSA as model protein, both against glucose and methylglyoxal mediated glycation. The possible mechanism of action was also deciphered using biophysical and computational tools. Approximately 70% inhibition of both early and advanced glycation end products were recorded in the presence of glyburide. Free lysine modification was reduced by glyburide treatment and improvement in biochemical markers such as free thiol groups and carbonyl content was observed. Interaction studies revealed that glyburide showed moderate to strong binding affinity towards HSA with binding constant in the order of 106 M-1. The interaction of glyburide with HSA was entropically favourable and spontaneous in nature. Molecular dynamics simulation deciphered that glyburide-HSA complex was quite stable where RMSD, RMSF, Rg, SASA, and secondary structure of HSA remained approximately same over the entire simulation period. The average binding energy of the MD simulation for glyburide-HSA complex was found to be -15.386 kJ mol-1. The findings demonstrate the antiglycation potential of glyburide and its possible mechanism of action.

Identification of a novel alternatively spliced isoform of antithrombin containing an additional RCL-like loop.

Antithrombin (AT3) is one of the most important inhibitors of blood coagulation proteases that belong to the serpin family of protease inhibitors. In this study, a novel alternatively spliced isoform of AT3 was identified, both at transcript and protein level. This novel transcript contains an additional region in the continuation of exon 3b that was included in the transcript due to use of an alternate 5' splice site. The existence of the novel transcript was confirmed in human brain and liver through RT-PCR. An analysis of the complete transcript indicated that the native reactive centre loop (RCL) of AT3 is maintained; however the novel amino acid sequence projects out as an additional loop as evident from MD simulation studies. A unique amino acid sequence present in the novel isoform was used for the development of polyclonal antibody. The expression of novel isoform was confirmed in human brain and liver tissue using Western blot analysis. Interestingly an alignment of RCL like domain with other inhibitory serpins showed significant similarity with the neuroserpin RCL. To the best of our knowledge, this is the first evidence of alternatively spliced AT3 sequence containing an additional loop and could have physiological relevance.

Synergistic Interaction of Rutin and Silibinin on Human Colon Cancer Cell Line.

AIM OF THE STUDY: Rutin and Silibinin are active flavonoid compounds, well-known for possessing multiple biological activities. We have studied how Rutin and Silibinin in combination modulate wide range intracellular signaling cascades as evidenced by in-vitro research. Data obtained from preclinical studies provide evidence to be supportive to bridge basic and translational studies. METHODS: In this study, cytotoxic effect of Rutin and Silibinin individually and in combination on the viability of colon cancer cell line (HT-29) was revealed using the MTT assay. Mechanism involved in the cytotoxic effect were then investigated in terms of apoptosis using comet assay, DNA fragmentation and fluorescent microscopy analyses. The apoptosis associated proteins viz; Caspase-3, 8, 9, Bax, Bcl-2, p53, inflammation associated proteins viz; NFκB, IKK-α IKK-ß and MAPK pathway associated proteins viz; p38 and MK-2 were determined by western-blot and Real Time-PCR analysis. RESULTS: Results suggest that Rutin and Silibinin produce anticancer effects via induction of apoptosis as well as regulating the expressions of genes related to apoptosis, inflammation and MAPK pathway proteins more effectively in combination than individually. CONCLUSION: Our study supports the viability of developing Rutin and Silibinin in combination as a novel therapeutic prodrug for colon cancer treatment and may have a promising role in the development of new anticancer drugs in the future.

Identification and expression of alternatively spliced novel isoforms of cancer associated MYD88 lacking death domain in mouse.

MYD88 is an adaptor protein known to involve in activation of NF-κB through IL-1 receptor and TLR stimulation. It consists of N-terminal death domain and C-terminal Toll/IL-R homology domain that mediates its interaction with IL-1R associated kinase and IL-1R/TLR, respectively. MYD88 contributes to various types of carcinogenesis due to its involvement in oncogene induced inflammation. In the present study, we have recognized two new alternatively spliced variants of MyD88 gene in mouse using bioinformatics tools and molecular biology techniques in combination. The newly identified non-coding exon (NE-1) from 5' upstream region alternatively splices with either exon E-2 or exon E-5 to produce two novel transcript variants MyD88N1 and MyD88N2 respectively. The transcript variant MyD88N1 was expressed in several tissues studied while the variant MyD88N2 was found to be expressed only in the brain. The analysis of the upstream region of novel exon by in silico approach revealed new promoter region PN, which possess potential signature sequences for diverse transcription factors, suggesting complex gene regulation. Studies of post translational modifications of conceptualized amino acid sequences of these isoforms revealed diversity in properties. Western blot analysis further confirmed the expression of protein isoform MYD88N1.

Differentially expressed novel alternatively spliced transcript variant of tumor suppressor Stk11 gene in mouse.

Serine/threonine kinase 11 (STK11) is a protein kinase that is encoded by Stk11 gene located on chromosome 19 and 10 in humans and mouse respectively. It acts as a master kinase of adenine monophosphate-activated protein kinase (AMPK) pathway that coordinates the regulation of cellular energy metabolism and cell division. STK11 exerts effect by activating more than 14 kinases including AMPK and AMPK-related kinases. It is also known to regulate cell polarity and acts as tumor suppressor. Alternative splicing of pre-mRNA is a mechanism which results in multiple transcript variants of a single gene. In human, two STK11 isoforms have been reported, an alternatively spliced isoform which has variation at its C-terminal and mostly expressed in testis (LKB1S). Another isoform exhibiting oncogenic properties lacks few residues at its N-terminal (ΔN-LKB1). In the present study, we report the identification of a new transcript variant Stk11N which is generated through alternative splicing. The new variant was found to have differential and tissue specific expression at Postnatal-7 and adult stages of mouse. As compared to the known variant Stk11C, the conceptually translated amino acid sequences of the new variant differ from exon-E2 onwards. In silico post translational studies of the new and published variant show similarity in some of the properties while differ in properties like nuclear export signals, phosphorylation, glycosylation, etc. Thus, alternative splicing of Stk11 gene generating new variant with heterogeneous properties suggests for complex regulation of these variants in controlling the AMPK pathway and other functions.

Unravelling the interaction of pirenzepine, a gastrointestinal disorder drug, with calf thymus DNA: An in vitro and molecular modelling study.

Pirenzepine is an anti-ulcer agent which belongs to the anti-cholinergic group of gastrointestinal disorder drugs and functions as an M1 receptor selective antagonist. Drug-DNA interaction studies are of great significance as it helps in the development of new therapeutic drugs. It provides a deeper understanding into the mechanism through which therapeutic drugs control gene expression. Interaction of pirenzepine with calf-thymus DNA (Ct-DNA) was determined via a series of biophysical techniques. UV-visible absorption and fluorescence spectroscopy confirmed the formation of pirenzepine-Ct-DNA complex. The values of binding constant from various experiments were calculated to be in the order of 103 M-1 which is consistent with the groove binding mode. Various spectrofluorimetric experiments like competitive displacement of well known dyes with drug, iodide quenching experiments and the effect of Ct-DNA denaturation in presence of drug confirmed the binding of pirenzepine to the groove of Ct-DNA. The binding mode was further established by viscometric, circular dichroic and molecular modelling studies. Thermodynamic parameters obtained from isothermal titration calorimetric studies suggest that the interaction of pirenzepine with Ct-DNA is enthalpically driven. The value of TΔS and ΔH calculated from calorimetric studies were found to be 4.3 kcal mol-1 and -2.54 kcal mol-1 respectively, indicating that pirenzepine-Ct-DNA complex is mainly stabilized by hydrophobic interaction and hydrogen bonding. The binding energy calculated was -7.5 kcal mol-1 from modelling studies which was approximately similar to that obtained by isothermal titration calorimetric studies. Moreover, the role of electrostatic interaction in the binding of pirenzepine to Ct-DNA cannot be precluded.

Molecular spectroscopic and thermodynamic studies on the interaction of anti-platelet drug ticlopidine with calf thymus DNA.

Ticlopidine is an anti-platelet drug which belongs to the thienopyridine structural family and exerts its effect by functioning as an ADP receptor inhibitor. Ticlopidine inhibits the expression of TarO gene in S. aureus and may provide protection against MRSA. Groove binding agents are known to disrupt the transcription factor DNA complex and consequently inhibit gene expression. Understanding the mechanism of interaction of ticlopidine with DNA can prove useful in the development of a rational drug designing system. At present, there is no such study on the interaction of anti-platelet drugs with nucleic acids. A series of biophysical experiments were performed to ascertain the binding mode between ticlopidine and calf thymus DNA. UV-visible and fluorescence spectroscopic experiments confirmed the formation of a complex between ticlopidine and calf thymus DNA. Moreover, the values of binding constant were found to be in the range of 103M-1, which is indicative of groove binding between ticlopidine and calf thymus DNA. These results were further confirmed by studying the effect of denaturation on double stranded DNA, iodide quenching, viscometric studies, thermal melting profile as well as CD spectral analysis. The thermodynamic profile of the interaction was also determined using isothermal titration calorimetric studies. The reaction was found to be endothermic and the parameters obtained were found to be consistent with those of known groove binders. In silico molecular docking studies further corroborated well with the experimental results.

Identification and expression analysis of alternatively spliced new transcript isoform of Bax gene in mouse.

Bax, a pro-apoptotic member of Bcl-2 family regulates apoptosis through homodimerization/heterodimerization with Bcl-2. Bax-α is the only product of the Bax gene that has been extensively studied. Bax-α exists in inactive form and several conformational changes are required during apoptosis to activate it. Here, we have identified a novel transcript variant of Bax gene in mouse which contains alternatively spliced new first exon that is different from the first exon of previously reported transcript. Conceptual translation of new transcript encodes a protein (Bax-α1), having different N-terminus. The existence of the new transcript variant was confirmed by reverse transcriptase-PCR, semi-nested PCR using primers designed for the newly identified transcript variant. The identity of PCR product obtained after semi-nested PCR was confirmed by DNA sequencing. Relative expression of new transcript variant with respect to reported transcript was also studied with the help of real time PCR. The existence of new transcript variant was further supported by the presence of clusters of overlapping ESTs from the database. Bax-α1 possibly displays heterogeneous properties as predicted by post-translational modification analysis tools. The differences in post-translational modifications might play important roles in divergent function of the new isoform. The three dimensional structure was generated by homology modelling to visualize the differences at N termini of known and newly identified variant.

Caffeic acid binds to the minor groove of calf thymus DNA: A multi-spectroscopic, thermodynamics and molecular modelling study.

Caffeic acid (CA) is a plant polyphenol which acts as an antioxidant and has various pharmacological effects. DNA is one of the major cellular targets of therapeutic molecules. Thus, studying the interaction of small molecules with DNA is of great importance. In the current article, we have studied the mode of binding of CA with calf thymus DNA (Ct-DNA) using a series of biophysical techniques. Formation of complex between CA and Ct-DNA is ascertained by analyzing the UV-vis absorbance and fluorescence emission spectra of CA upon successive addition of Ct-DNA. Binding constants of CA with Ct-DNA obtained using multiple experiments was in the order of 103 M-1 which is consistent with known groove binders. Analysis of thermodynamic parameters suggest that hydrogen bonding and van der Waal's forces played major role in the binding process. Competitive displacement studies confirmed that CA binds to the minor groove of Ct-DNA. These observations were further validated by KI quenching experiment, DNA melting studies, CD and viscosity measurements. In silico molecular docking further provided insight into the mode of binding of CA with Ct-DNA. Through in vitro experiments and in silico molecular docking studies, it was concluded that CA binds to the minor groove of Ct-DNA.

Identification of two novel isoforms of mouse NUR77 lacking N-terminal domains.

Nur77 is a member of nuclear receptor superfamily that acts as a transcription factor and regulates expression of multiple genes. Subcellular localization of Nur77 protein plays an important role in the survival and cell death. In this study, we have predicted and confirmed alternatively spliced two new transcripts of Nur77 gene in mouse. The newly identified transcripts have their alternatively spliced first exon located upstream of published 5'-UTR of the gene. Transcription factor binding sites in the possible promoter regions of these transcripts were also analyzed. Expression of novel transcript variants was found to be significantly lower than the already published transcript. New transcript variants encode for NUR77 protein isoforms which are significantly smaller in size due to lack of transactivation domain and a part of DNA binding domain. Western blot analysis using NUR77 specific antibody confirmed the existence of these smaller variants in mouse. Localization of these new isoforms was predicted to be majorly outside the nucleus. In silico analysis of the conceptually translated proteins was performed using different bioinformatics tools. The results obtained in this study offer further insight into novel area of research on extensively studied Nur77. © 2017 IUBMB Life, 69(2):106-114, 2017.

NSAIDs Induced Regulation of Alternatively Spliced Transcript Isoforms: Possible Role in Cancer and Alzheimer Disease.

BACKGROUND: Alternative splicing is one of the post transcriptional modifications through which multiple mRNA isoforms are produced from any gene, also known as splice variants. These are expressed in tissue and developmental stage specific manner that are important during the development. Most human genes undergo alternative splicing, thus contributing to the diversity of proteins. However, many abnormal splicing processes may result in human diseases. Non-steroidal antiinflammatory drugs (NSAIDs) are medications that act as analgesics, anti-pyretics and antiinflammatory by affecting Cox genes and their products. Usually NSAIDs cause gastrotoxicity however, isozyme-specific NSAIDs exhibit a comparatively reduced gastrotoxic effect. Such NSAIDs have a broader range of application particularly as chemo-preventive drugs. It is known that changes at the active site of an enzyme may illicit a diverse range of responses. Such changes might explain the underlying reason as to why patients appear to respond differently to different NSAIDs. METHODS: An extensive literature search has been carried out using Pubmed and web of science databases considering the papers in last 10 years mainly on alternative splicing and NSAIDs. CONCLUSION: We have reviewed in detail the insight into the action of NSAIDs targeting specific isoforms of different genes. In future, the complete understanding of NSAIDs associated genes and their expression studies may be helpful in generating drugs with increased specificity.

Interaction of indomethacin with calf thymus DNA: a multi-spectroscopic, thermodynamic and molecular modelling approach.

Indomethacin belongs to the acetic acid derivative class of non-steroidal anti-inflammatory drugs with diverse pharmacological and biological activities. Understanding the mechanism of interaction of drugs with possible target and off-target biomolecules can prove useful in the development of a rational drug designing system. In this paper, we have attempted to ascertain the mode of binding of indomethacin with calf thymus DNA (Ct-DNA) through various biophysical techniques and in silico molecular docking. Analysis of the UV-visible absorbance spectra and fluorescence emission profile of indomethacin upon addition of Ct-DNA indicates the formation of a drug-DNA complex. UV-visible absorbance and steady state fluorescence experiments revealed a binding constant on the order of 103 L mol-1, which is consistent with those of well-known groove binders. Competitive displacement studies with ethidium bromide, acridine orange and Hoechst 33258 further suggested that indomethacin binds to the minor groove of the Ct-DNA. The above observations were further confirmed by KI induced quenching experiments, DNA melting studies, CD spectral analysis and viscosity measurements. The thermodynamic parameters like spontaneous free energy (ΔG < 0) and large favourable enthalpy (ΔH < 0) obtained from isothermal calorimetry indicated the involvement of hydrogen bonding and van der Waals forces in the binding process. Molecular docking further corroborated the experimental results.

A novel exon generates ubiquitously expressed alternatively spliced new transcript of mouse Abcc4 gene.

Abcc4 gene codes for a protein (ABCC4) involved in the transportation of different classes of drugs outside the cells. Various important drugs transported by ABCC4 include antiviral and anticancer drugs as well as endogenous molecules such as bile acids, cyclic nucleotides, folates, prostaglandins and steroids. Alternative splicing generates multiple mRNAs that encode protein isoforms having diverse functions. In this study, we have identified a novel transcript of mouse Abcc4 gene using a combination of bioinformatics and molecular biology techniques. This transcript was found to be different from the reported transcript in having a different first exon that was found to be located on previously identified first intron. Newly identified transcript was found to be expressed across different tissues we studied and in different developmental stages. Expression level of novel and reported transcripts was studied using quantitative real-time PCR. After conceptually translating the novel transcript, various post-translational modifications were studied. Translation efficiency and predicted half life of encoded protein isoforms were analysed in silico. Molecular modelling was performed to compare the structural differences in both isoforms. The diversity at N-termini in these protein isoforms explains the diverse function of ABCC4 in mouse.

Identification of differentially expressed three novel transcript variants of mouse ARNT gene.

The aryl hydrocarbon receptor nuclear translocator (ARNT/HIF1-ß) is an obligatory transcriptional partner of the aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor-1α (HIF-1α). It has a basic helix-loop-helix domain that belongs to period-ARNT-single-minded (PAS) protein family. PAS proteins act as heterodimeric transcription factors with ARNT being master dimerization partner. The ARNT-HIF-1α complex is an important transcriptional regulator of the hypoxic response of the tumor cells. Previous studies have reported two transcript variants of the gene produced by alternative splicing in mouse. One transcript variant contains all 22 exons while the other variant lacks exon-E5. In our study, using combinatorial approach comprising bioinformatics tools and molecular biology techniques involving RT-PCR, semi-nested PCR, sequencing and qPCR, we have identified three novel transcript variants of Arnt gene in mouse. All three new transcripts arise as a result of alternative splicing of newly identified exons with exon-E2, replacing reported exon-E1. These transcripts encode for three protein isofoms having different N-termini. The expression of these transcripts was found to be different in different tissues of adult mice. In silico analysis of the upstream region of the new exons revealed three distinct promoter regions designated as PA, PB and PC present upstream of newly identified exons. These promoters possess potential signature sequences for common as well as different transcription factors suggesting complex regulation of Arnt gene. In silico post translational studies of the conceptually translated amino acid sequences of these transcripts show similarity in some of the properties while differ in others. The diversity at N-termini of protein isoforms suggests the possibility of forming different complexes in different tissues and may also be important for unique interactions with partner molecules.