ItemLocation and characterization of autonomously replicating sequences from chromosome VI of Saccharomyces cerevisiae(Mol Cell Biol, 1993) Rashid, BazlurWe have reported the isolation of linking clones of HindIII and EcoRI fragments, altogether spanning a 230-kb continuous stretch of chromosome VI. The presence or absence of autonomously replicating sequence (ARS) activities in all of these fragments has been determined by using ARS searching vectors containing CEN4. Nine ARS fragments were identified, and their positions were mapped on the chromosome. Structures essential for and/or stimulative to ARS activity were determined for the ARS fragments by deletions and mutations. The organization of functional elements composed of core and stimulative sequences was found to be variable. Single core sequences were identified in eight of nine ARSs. The remaining ARS (ARS603) essential element is composed of two core-like sequences. The lengths of 3'- and 5'-flanking stimulative sequences required for the full activity of ARSs varied from ARS to ARS. Five ARSs required more than 100 bp of the 3'-flanking sequence as stimulative sequences, while not more than 79 bp of the 3' sequence was required by the other three ARSs. In addition, five ARSs had stimulative sequences varying from 127 to 312 bp in the 5'-flanking region of the core sequence. In general, these stimulative activities were correlated with low local delta Gs of unwinding, suggesting that the low local delta G of an ARS is an important element for determining the efficiency of initiation of replication of ARS plasmids. ItemAnatomy of the stimulative sequences flanking the ARS consensus sequence of chromosome VI in S. cerevisiae(Gene, 1994) Rashid, Bazlur ItemEffect of short 5' UTR on protein synthesis in two biological kingdoms(Gene, 1998) Rashid, BazlurEfficient ribosomal protein synthesis is dependent on cis-acting elements in the 5′ untranslated region (UTR) of mRNAs. Between prokaryotes and eukaryotes, the sequence and location of these elements differ to the extent of not being functionally interchangeable. We explored the possibility of constructing bifunctional UTRs that could direct translation in both prokaryotes and eukaryotes. A variant of a UTR from ner of phage Mu (ner-ACC) enhanced protein synthesis in a rabbit reticulocyte lysate, and it was compared to a lacZ-CTA, containing the λ cro RBS and the Escherichia coli lacZ spacer. Several mutants in the −3 to −1 regions of both lacZ-CTA and ner-ACC were tested in rabbit reticulocyte lysate and E. coli to select UTRs that were optimized simultaneously for both biological kingdoms. The lacZ-ATC proved 217-fold more effective than ner-ACC in this cross-species ability to enhance translation. The lacZ-ACC and ner-ATC were 83- and 78-fold, respectively, better than ner-ACC. We conclude that short UTRs (12–15 nt in length) can be fine-tuned in the −9 to −1 regions to enhance protein synthesis concurrently in prokaryotes and eukaryotes. In related studies, we show that nt at the −3 to −1 region of mRNAs exert an enormous impact on synthesis of proteins in E. coli. ItemRoles of Gln81 and Cys80 in Catalysis by GPI-Phospholipase C from Trypanosoma brucei(Eur J Biochem., 1999-09-15) Rashid, BazlurGlycosylphosphatidylinositol-specific phospholipase C (GPtdIns-PLC) is found in the protozoan parasite Trypanosoma brucei. A region of protein sequence similarity exists between the protozoan enzyme and eubacterial phosphatidylinositol-phospholipases C. The functional relevance of Cys80 and Gln81 of GPtdIns-PLC, both in this region, was tested with a panel of mutations at each position. Gln81Glu, Gln81Ala, Gln81Gly, Gln81Lys and Gln81Leu mutants were inactive. Cleavage of GPtdIns was detectable in Gln81Asn, although the specific activity decreased 500-fold, and kcat was reduced 50-fold. Thus an amide side-chain at residue 81 is essential for catalysis by GPtdIns-PLC. Sulfhydryl reagents inactivate GPtdIns-PLC, suggesting that a Cys could be close to the enzyme active site. Surprisingly, p-chloromercuriphenyl sulfonate (p-CMPS) is significantly more potent than N-ethylmaleimide, the less bulky compound. This knowledge prompted us to test whether replacement of Cys80 with an amino acid possessing a bulky side-chain would inactivate GPtdIns-PLC: Cys80Ala, Cys80Thr, Cys80Phe, Cys184Ala, and Cys269-270-273Ser were constructed for that purpose. Cys80Phe lacked enzyme activity, while Cys80Ala, Cys80Thr and Cys269-270-273Ser retained 33 to 100% of wild-type activity. Interestingly, the Cys80Ala and Cys80Thr mutants became resistant to p-CMPS, as predicted if the sulfhydryl reagent reacted with Cys80 in the wild-type enzyme to form a cysteinyl mercurylphenylsulfonate moiety, a bulky adduct that inactivated GPtdIns-PLC, similar to the Cys80Phe mutation. We conclude that a bulky side-chain (or adduct) at position 80 of GPtdIns-PLC abolishes enzyme activity. Together, these observations place Cys80 and Gln81 at, or close to, the active site of GPtdIns-PLC from T. brucei. ItemA novel kanamycin/neomycin phosphotransferase cassette increases transformation efficiency of E. coli(Biotechniques, 2000-01) Rashid, BazlurStable transformation depends on the efficient delivery of DNA into cells and the robust expression of genes that encode proteins which provide resistance to selective (cytotoxic) compounds. We have examined the possibility that altering the 5'untranslated region (UTR) of a selectable marker may increase transformation efficiency. A 15-nucleotide synthetic UTR (the so-called universal translational enhancer [UTE]) was placed upstream of a kanamycin/neomycin phosphotransferase (kanaR) gene to create a novel expression cassette, UTE-kanaR. In comparison to a wild-type version of kanaR, UTE-kanaR produced up to 30-fold more transformants in E. coli. The superior performance of UTE-kanaR was independent of the promoter strength, indicating that the gene may find general use in routine transformation experiments. ItemCharacterization of key residues in the subdomain encoded by exons 8 and 9 of human inducible nitric oxide synthase: a critical role for Asp-280 in substrate binding and subunit interactions(Proc Natl Acad Sci U S A, 2001-08-28) Rashid, BazlurHuman inducible nitric oxide synthase (iNOS) is active as a dimer of two identical subunits. Each subunit has an amino-terminal oxygenase domain that binds the substrate l-Arg and the cofactors heme and tetrahydrobiopterin and a carboxyl-terminal reductase domain that binds FMN, FAD, and NADPH. We previously demonstrated that a subdomain in the oxygenase domain encoded by exons 8 and 9 is important for dimer formation and NO synthesis. Further, we identified Trp-260, Asn-261, Tyr-267, and Asp-280 as key residues in that subdomain. In this study, using an Escherichia coli expression system, we produced, purified, and characterized wild-type iNOS and iNOS-Ala mutants. Using H2O2-supported oxidation of Nω-hydroxy-l-Arg, we demonstrate that the iNOS mutants' inabilities to synthesize NO are due to selective defects in the oxygenase domain activity. Detailed characterization of the Asp-280–Ala mutant revealed that it retains a functional reductase domain, as measured by its ability to reduce cytochrome c. Gel permeation chromatography confirmed that the Asp-280–Ala mutant exists as a dimer, but, in contrast to wild-type iNOS, urea-generated monomers of the mutant fail to reassociate into dimers when incubated with l-Arg and tetrahydrobiopterin, suggesting inadequate subunit interaction. Spectral analysis reveals that the Asp-280–Ala mutant does not bind l-Arg. This indicates that, in addition to dimerization, proper subunit interaction is required for substrate binding. These data, by defining a critical role for Asp-280 in substrate binding and subunit interactions, give insights into the mechanisms of regulation of iNOS activity. ItemCysteine-less GPI-phospholipase C is Inhibited Competitively by a Sulfhydryl Reagent: Glyco-mimicry by para-Chloromercuriphenylsulfonate(Biochem J., 2002-08-15) Rashid, BazlurGlycosylphosphatidylinositol (GPI)-specific phospholipases are highly valuable for studying the structure and function of GPIs. GPI-specific phospholipase C (GPI-PLC) from Trypanosoma brucei and phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus are the most widely studied of this class of phospholipases C. Inhibition of protein activity by thiol reagents is indicative of the participation of cysteine residues in biochemical events. The thiol reagent p-chloromercuriphenylsulphonate (pCMPS) inhibits T. brucei GPI-PLC, which has eight cysteine residues. Surprisingly, we found that the activity of B. cereus PI-PLC is also blocked by pCMPS, although the protein does not contain cysteine residues. Inhibition of B. cereus PI-PLC was reversed when pCMPS was size-separated from a preformed pCMPS·PI-PLC complex. In contrast, no activity was recovered when T. brucei GPI-PLC was subjected to a similar protocol. Equimolar β-mercaptoethanol (β-ME) reversed the inhibition of PI-PLC activity in a pCMPS·PI-PLC complex. For T. brucei GPI-PLC, however, ultrafiltration of the pCMPS·GI-PLC complex and addition of a large excess of β-ME was necessary for partial recovery of enzyme activity. Thus T. brucei GPI-PLC is susceptible to inactivation by covalent modification with pCMPS, whereas PI-PLC is not. Kinetic analysis indicated that pCMPS was a competitive inhibitor of PI-PLC when a GPI was a substrate. Curiously, with phosphatidylinositol as substrate, inhibition was no longer competitive. These data suggest that pCMPS is a glyco-mimetic that occupies the glycan binding site of PI-PLC, from where, depending on the substrate, it inhibits catalysis allosterically or competitively. ItemIntracellular formation of "undisruptable" dimers of inducible nitric oxide synthase(Proc Natl Acad Sci U S A, 2003-11-25) Rashid, BazlurOverproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of many diseases. iNOS is active only as a homodimer. Dimerization of iNOS represents a potentially critical target for therapeutic intervention. In this study, we show that intracellular iNOS forms dimers that are ”undisruptable” by boiling, denaturants, or reducing agents. Undisruptable (UD) dimers are clearly distinguishable from the easily dissociated dimers formed by iNOS in vitro. UD dimers do not form in Escherichia coli-expressed iNOS and could not be assembled in vitro, which suggests that an in vivo cellular process is required for their formation. iNOS UD dimers are not affected by intracellular depletion of H4B. However, the mutation of Cys-115 (critical for zinc binding) greatly affects the formation of UD dimers. This study reveals insight into the mechanisms of in vivo iNOS dimer formation. UD dimers represent a class of iNOS dimers that had not been suspected. This unanticipated finding revises our understanding of the mechanisms of iNOS dimerization and lays the groundwork for future studies aimed at modulating iNOS activity in vivo. Nitric oxide (NO) is an important signaling and cytotoxic molecule that is synthesized from l-Arg by isoforms of NO synthase (NOS) (1-3). As a signaling molecule, NO is produced by two constitutive calcium (Ca2+)-dependent isoforms: neuronal NOS and endothelial NOS. Ca2+-activated calmodulin binds to and transiently activates constitutive NOS dimers. Due to the transient nature of elevated Ca2+ levels, the activity of produced NO is short-lived. As an agent of inflammation and cell-mediated immunity, NO is generated by a Ca2+-independent cytokine-inducible NOS (iNOS) that is widely expressed in diverse cell types under transcriptional regulation by inflammatory mediators (2-4). Even at basal Ca2+ levels, calmodulin is tightly bound to iNOS. For this reason, iNOS is notably distinguished from the constitutive isoforms for its production of relatively large amounts of NO (5). iNOS has been implicated in the pathogenesis of many diseases, some of which include Alzheimer's disease, tuberculosis, asthma, glaucoma, inflammatory bowel disease, arthritis, stroke, and septic shock (6, 7). Such wide-based implication has in turn produced an interest in understanding the regulation of NO synthesis by iNOS with the intrinsic goal of developing therapeutic strategies aimed at selective modulation of iNOS activity (6-8). The human iNOS gene contains 26 exons and encodes a protein of 131 kDa (9, 10). Human iNOS has three domains: (i) an amino-terminal oxygenase domain that binds heme, tetrahydrobiopterin (H4B) and l-Arg; (ii) a carboxyl-terminal reductase domain that binds FMN, FAD, and NADPH; and (iii) an intervening calmodulin-binding domain that regulates electron transfer between the oxygenase and reductase domains (3, 9, 11). iNOS, similar to other NOSs, is active only as a homodimer in which the subunits align in a head-to-head manner, the aminoterminal oxygenase domains forming the dimer interface (12). Posttranslational subunit dimerization of iNOS represents a potentially critical locus for therapeutic interventions aimed at regulating its activity. There have been a large number of studies addressing the mechanisms of iNOS dimerization (3, 12-15), but most of these studies were performed in vitro under dictated experimental conditions and by using either recombinant protein or partial domains. Very few studies have addressed iNOS dimerization in vivo using cultured or primary cells. In this study, we show that intracellular iNOS forms dimers that are ”undisruptable” by heat, SDS, strong denaturants, and/or reducing agents. These dimers are clearly distinguishable from the easily dissociated dimers formed by iNOS in vitro. This unexpected finding revises our understanding of the mechanisms of iNOS dimerization. ItemProspects of molecular biotechnology for crop cultivation in low-lying coastal areas(Emir. J. Food Agric., 2012) Rashid, Bazlur ItemMicroRNA Expression Patterns Responsible for Inducing Chemo-Resistance in Ovarian Cancer(J. Biomed, 2018) Rashid, BazlurDespite significant progress in our understanding of the pathophysiology of cancer as a whole, ovarian cancer is still remains as one of the most intractable forms of the disease with only 30% cure rate with the conventional therapy. It is also one of most common gynecologic cancer among women. One histologic variety is the epithelial ovarian cancer that constitutes more than 90% of the cases; also develop resistance to chemotherapeutic agents. Recent studies have demonstrated the role of micro RNA (miRNA) in the evolution and progression of ovarian cancer. Furthermore, aberrant expression of miRNA has been implicated in the development of resistance to chemotherapy in such cases. This review compiles recent advances in the role of miRNA in chemoresistant ovarian cancer and approaches to tackle the problem and design therapy that is more effective.