Validation of Arsenic Resistance in <Emphasis Type="Italic">Bacillus cereus</Emphasis> Strain AG27 by Comparative Protein Modeling of <Emphasis Type="Italic">ars</Emphasis>C Gene Product

The ars gene system provides arsenic resistance to a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. Therefore, arsC gene from Bacillus cereus strain AG27 isolated from soil was amplified, cloned and sequenced. The strain exhibited a minimum inhibitory concentration of 40 and 35 mM to sodium arsenate and sodium arsenite, respectively. Homology of the sequence, when compared with available database using BLASTn search showed that 300 bp amplicons obtained possess partial arsC gene sequence which codes for arsenate reductase, an enzyme involved in the reduction of arsenate to arsenite which is then effluxed out of the cell, thereby indicating the presence of efflux mechanism of resistance in strain. The efflux mechanism was further confirmed by atomic absorption spectroscopy and scanning electron microscopy studies. Moreover, three dimensional structure of modeled arsC from Bacillus cereus strain shares significant structural similarity with arsenate reductase protein of B.subtilis, consisting of, highly similar overall fold with single α/β domain containing a central four stranded, parallel, open-twisted β-sheet flanked by α-helices on both sides. The structure harbors the arsenic binding motif AB loop or P-loop that is highly conserved in arsenate reductase family.     

7, 8-diacetoxy-4-methylcoumarin induced cell death in human tumor cells is influenced by calreticulin

Calreticulin (CRT), an endoplasmic reticulum resident protein demonstrates transacetylase activity in presence of 7, 8 diacetoxy-4-methyl coumarin (DAMC) in vitro. To investigate the possible role of CRT and DAMC mediated protein acetylation in cells, we investigated the effects of DAMC in tumor cells with different levels of CRT. DAMC was more toxic (clonogenicity, metabolic viability and proliferation) to human glioma cells (BMG-1) expressing low endogenous CRT level as compared to head and neck carcinoma cells (KB) with a high CRT level. The cytotoxicity was accompanied by loss of mitochondrial membrane potential in both the cells, which correlated with corresponding changes in the levels of pro-apoptotic (Bax) and anti-apoptotic (NFkB) regulators. Manipulation of CRT protein level in KB cells by application of small RNA interference enhanced the sensitivity by four folds while over expression of CRT in BMG-1 cells reduced their sensitivity to DAMC by ∼20% strongly suggesting the influence of CRT on DAMC induced cytotoxicity. The partial rescue of CROE cells from DAMC induced toxicity was accompanied by changes in NFkB levels and over all protein acetylation status, besides increase in the NADPH-cytochrome c reductase activity related to its well known antioxidant property. Since CRT is over-expressed in cancer cells, which are generally resistant to radio- and chemotherapy; targeting CRT transacetylase system, may be an attractive approach for increasing the efficacy of anticancer therapies.     

Characterization of protein transacetylase from human placenta as a signaling molecule calreticulin using polyphenolic peracetates as the acetyl group donors

We have earlier shown that a unique membrane-bound enzyme mediates the transfer of acetyl group(s) from polyphenolic peracetates (PA) to functional proteins, which was termed acetoxy drug: protein transacetylase (TAase) because it acted upon several classes of PA. Here, we report the purification of TAase from human placental microsomes to homogeneity with molecular mass of 60 kDa, exhibiting varying degrees of specificity to several classes of PA confirming the structure-activity relationship for the microsome-bound TAase. The TAase catalyzed protein acetylation by a model acetoxy drug, 7,8-diacetoxy-4-methyl coumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with anti-acetyl lysine antibody. TAase activity was severely inhibited in calcium-aggregated microsomes as well as when Ca2+ was added to purified TAase, suggesting that TAase could be a calcium binding protein. Furthermore, the N-terminal sequence analysis of purified TAase (EPAVYFKEQFLD) using Swiss Prot Database perfectly matched with calreticulin (CRT), a major microsomal calcium binding protein of the endoplasmic reticulum (ER). The identity of TAase with CRT was substantiated by the observation that the purified TAase avidly reacted with commercially available antibody raised against the C-terminus of human CRT (13 residues peptide, DEEDATGQAKDEL). Purified TAase also showed Ca2+ binding and acted as a substrate for phosphorylation catalyzed by protein kinase C (PKC), which are hallmark characteristics of CRT. Further, purified placental CRT as well as the commercially procured pure CRT yielded significant TAase catalytic activity and were also found effective in mediating the acetylation of the target protein NADPH cytochrome P-450 reductase by DAMC as detected by Western blot using anti-acetyl lysine antibody. These observations for the first time convincingly attribute the transacetylase function to CRT. Hence, this transacetylase function of CRT is designated calreticulin transacetylase (CRTAase). We envisage that CRTAase plays an important role in protein modification by way of acetylation independent of Acetyl CoA.     

Enhanced charge-independent mitochondrial free Ca2 + and attenuated ADP-induced NADH oxidation by isoflurane: Implications for cardioprotection

Modulation of mitochondrial free Ca^2 + ([Ca^2 +]_m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca^2 +]_m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2 mM) on the magnitudes and time-courses of [Ca^2 +]_m and mitochondrial redox state (NADH), membrane potential (ΔΨ_m), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10 mM Na⁺- or K⁺-pyruvate/malate (NaPM or KPM) or Na⁺-succinate (NaSuc) followed by additions of isoflurane, 0.5 mM CaCl₂ (≈ 200 nM free Ca^2 + with 1 mM EGTA buffer), and 250 μM ADP. Isoflurane stepwise: (a) increased [Ca^2 +]_m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨ_m and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨ_m, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨ_m, and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca^2 + uptake by inhibiting the Na⁺/Ca^2 + exchanger (NCE), independent of changes in ΔΨ_m and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca^2 +]_m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level.     

Establishment of glutamine synthetase of Mycobacterium smegmatis as a protein acetyltransferase utilizing polyphenolic acetates as the acetyl group donors

Acetoxy Drug: Protein Transacetylase (TAase) mediating the transfer of acetyl group(s) from polyphenolic acetates (PA) to certain functional proteins in mammalian cells was identified by our earlier investigations. TAase activity was characterized in the cell lysates of Mycobacterium smegmatis and the purified protein was found to have M(r) 58,000. TAase catalysed protein acetylation by a model acetoxy drug 7,8-diacetoxy-4-methylcoumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with an anti-acetyllysine antibody. The specificity of the TAase of M. smegmatis (MTAase) to various acetoxycoumarins was found to be in the order DAMC > 7-AMC > 6-AMC > 4-AC > 3-AC > ABP. Also, the N-terminal sequence of purified MTAase was found to perfectly match with glutamine synthetase (GS) of M. smegmatis. The identity of MTAase with GS was confirmed by the observation that the purified MTAase as well as the purified recombinant GS exhibited all the properties of GS. The finding that purified Escherichia coli GS was found to have substantial TAase activity highlighted the TAase function of GS in other bacteria. These results conclusively established for the first time the protein acetyltransferase function of GS of M. smegmatis.     

Candida ruelliae sp. nov., a novel yeast species isolated from flowers of Ruellia sp. (Acanthaceae)

Two novel yeast strains designated as 16Q1 and 16Q3 were isolated from flowers of the Ruellia species of the Acanthaceae family. The D1/D2 domain and ITS sequences of these two strains were identical. Sequence analysis of the D1/D2 domain of large-subunit rRNA gene indicated their relationship to species of the Candida haemulonii cluster. However, they differ from C. haemulonii by 14% nucleotide sequence divergence, from Candida pseudohaemulonii by 16.1% and from C. haemulonii type II by 16.5%. These strains also differ in 18 physiological tests from the type strain of C. haemulonii, and 12 and 16 tests, respectively, from C. pseudohaemulonii and C. haemulonii type II. They also differ from C. haemulonii and other related species by more than 13% sequence divergence in the internal transcribed spacer region. In the SSU rRNA gene sequences, strain 16Q1 differs by 1.7% nucleotide divergence from C. haemulonii. Sporulation was not observed in pure or mixed cultures on several media examined. All these data support the assignment of these strains to a novel species; we have named them as Candida ruelliae sp. nov., and designate strain 16Q1(T)=MTCC 7739(T)=CBS10815(T) as type strain of the novel species.