Heterologous Expression, Purification, and Characterization of a High-Temperature Requirement A (HtrA) Protease Homologue from Leptospira.

Leptospirosis is a zoonotic disease affecting humans in the tropical and temperate regions. Considerably high mortality rate (60 per 1000 adult) and associated morbidity necessitate the need for efficient diagnostic and therapeutic approaches for this disease. Proteins that play crucial roles in the invasion/pathogenesis are potential candidates for the diagnosis/therapeutics.

Cell cycle dysregulation in cancer.

Cancer is a systemic manifestation of aberrant cell cycle activity and dysregulated cell growth. Genetic mutations can determine tumor onset by either augmenting cell division rates or restraining normal controls such as cell cycle arrest or apoptosis. As a result, tumor cells not only undergo uncontrolled cell division but also become compromised in their ability to exit the cell cycle accurately.

Structural characterization of DNA-binding domain of essential mammalian protein TTF 1.

Transcription Termination Factor 1 (TTF1) is a multifunctional mammalian protein with vital roles in various cellular processes, including Pol I-mediated transcription initiation and termination, pre-rRNA processing, chromatin remodelling, DNA damage repair, and polar replication fork arrest. It comprises two distinct functional regions; the N-terminal regulatory region (1-445 aa), and the C-terminal catalytic region (445-859 aa). The Myb domain located at the C-terminal region is a conserved DNA binding domain spanning from 550 to 732 aa (183 residues).

miR-17 ~ 92 suppresses proliferation and invasion of cervical cancer cells by inhibiting cell cycle regulator Cdt2.

Cervical cancer (CC) is the 4th most leading cause of death among women worldwide, and if diagnosed in late stages the treatment options are almost negligible. 99% of CC is caused by high-risk human papilloma viruses (HR-HPV). Upon integration into human genome, the encoded viral proteins mis-regulate various onco-suppressors and checkpoint factors including cell cycle regulators.

Purification and Structural Characterization of N-Terminal 190 Amino Acid Deleted Essential Mammalian Protein; Transcription Termination Factor 1.

The mammalian transcription termination factor 1 (TTF1) is an essential protein that plays diverse cellular physiological functions like transcription regulation (both initiation and termination), replication fork blockage, chromatin remodeling, and DNA damage repair. Hence, understanding the structure and mechanism conferred by its variable conformations is important. However, so far, almost nothing is known about the structure of either the full-length protein or any of its domains in isolation.

A study for evaluating clinical relevance of circulating cell-free DNA in cervical cancer.

Introduction: Recent techniques available for the detection of cervical cancer (CC) are highly invasive and costly, which makes it a rate-limiting step toward early diagnosis of this fatal disease. Evaluation of circulating cell-free DNA (ccfDNA) through liquid biopsy is a minimally invasive and cost-effective method that may serve as a unique tumor marker for early detection, treatment monitoring, the status of residual disease, and distant tumor metastasis in CC patients.

Materials And Methods: In this study, initially, ccfDNA was measured in serum samples from 11 histopathologically proven cervix carcinoma patients and 8 controls.

modelling of an essential mammalian protein: Transcription Termination Factor 1 (TTF1).

Transcription Termination Factor 1 (TTF1) is an essential mammalian protein that regulates transcription, replication fork arrest, DNA damage repair, chromatin remodelling etc. TTF1 interacts with numerous cellular proteins to regulate various cellular phenomena which play a crucial role in maintaining normal cellular physiology, and dysregulation of this protein has been reported to induce oncogenic transformation of the cells. However, despite its key role in many cellular processes, the complete structure of human TTF1 has not been elucidated to date, neither experimentally nor computationally.

miR-34a negatively regulates cell cycle factor Cdt2/DTL in HPV infected cervical cancer cells.

MicroRNAs have emerged as an important regulator of cell cycle and various other cellular processes. Aberration in microRNAs has been linked with development of several cancers and other diseases but still very little is known about the mechanism by which they regulate these cellular events. High risk human papilloma virus (HR HPV) is the causative agent of 99% of cervical cancer cases which attenuates multiple tumor suppressors and checkpoint factors of the host cell.

The Deubiquitinase USP46 Is Essential for Proliferation and Tumor Growth of HPV-Transformed Cancers.

High-risk human papilloma viruses (HPVs) cause cervical, anal, and oropharyngeal cancers, unlike the low-risk HPVs, which cause benign lesions. E6 oncoproteins from the high-risk strains are essential for cell proliferation and transformation in HPV-induced cancers. We report that a cellular deubiquitinase, USP46, is selectively recruited by the E6 of high-risk, but not low-risk, HPV to deubiqutinate and stabilize Cdt2/DTL.

Role of RAG1 autoubiquitination in V(D)J recombination.

The variable domains of Ig and T-cell receptor genes in vertebrates are assembled from gene fragments by the V(D)J recombination process. The RAG1-RAG2 recombinase (RAG1/2) initiates this recombination by cutting DNA at the borders of recombination signal sequences (RSS) and their neighboring gene segments. The RAG1 protein is also known to contain a ubiquitin E3 ligase activity, located in an N-terminal region that is not strictly required for the basic recombination reaction but helps to regulate recombination.

Crystallization and preliminary X-ray characterization of the eukaryotic replication terminator Reb1-Ter DNA complex.

The Reb1 protein from Schizosaccharomyces pombe is a member of a family of proteins that control programmed replication termination and/or transcription termination in eukaryotic cells. These events occur at naturally occurring replication fork barriers (RFBs), where Reb1 binds to termination (Ter) DNA sites and coordinates the polar arrest of replication forks and transcription approaching in opposite directions. The Reb1 DNA-binding and replication-termination domain was expressed in Escherichia coli, purified and crystallized in complex with a 26-mer DNA Ter site.

Metabolic sensor AMPK directly phosphorylates RAG1 protein and regulates V(D)J recombination.

The ability to sense metabolic stress is critical for successful cellular adaptation. In eukaryotes, the AMP-activated protein kinase (AMPK), a highly conserved serine/threonine kinase, functions as a critical metabolic sensor. AMPK is activated by the rising ADP/ATP and AMP/ATP ratios during conditions of energy depletion and also by increasing intracellular Ca(2+).

"Chromosome kissing" and modulation of replication termination.

Previously, inter-chromosomal interactions called "chromosome kissing" have been reported to control tissue-specific transcription and cell fate determination. Using the fission yeast as a model system we have shown that physiologically programmed replication termination is also modulated by chromosome kissing. The published report reviewed here shows that a myb-like replication terminator protein Reb1 of S.

Regulation of replication termination by Reb1 protein-mediated action at a distance.

DNA transactions driven by long-range protein-mediated inter- and intrachromosomal interactions have been reported to influence gene expression. Here, we report that site-specific replication termination in Schizosaccharomyces pombe is modulated by protein-mediated interactions between pairs of Ter sites located either on the same or on different chromosomes. The dimeric Reb1 protein catalyzes termination and mediates interaction between Ter sites.

Plasmid encoded AcrAB-TolC tripartite multidrug-efflux system in Acidiphilium symbioticum H8.

Acidophilic bacterium, Acidiphilium symbioticum H8, is resistant to high levels of several heavy metals, hydrophobic agents, and organic solvents. The approximately 9.6 kb plasmid pASH8, was purified, digested with HindIII, and sub-cloned in pUC19 at the respective site.

Nucleotide sequence analysis of cryptic plasmid pAM5 from Acidiphilium multivorum.

Plasmid pAM5 of Acidiphilium multivorum JCM-8867 has been completely sequenced by initial cloning of HindIII-PstI fragments followed by primer walking. It has a size of 5161bp and single site for several restriction enzymes as revealed by DNA sequencing. Sequence analysis predicts five putative open reading frames.

High-yielding plasmid extraction method from acidophilic heterotrophic bacteria of the genus Acidiphilium.

Plasmid yield from Acidiphilium strains always had been poor following various standard methods. We adopted some simple modifications in the alkaline lysis procedure to get a better yield of plasmid from these bacteria. An approximately 10- to 20-fold increase in the plasmid yield was achieved when harvested Acidiphilium cells were preincubated 16-20 h at pH 6 in nitrogen-free medium.