The mutation that makes Escherichia coli resistant to lambda P gene-mediated host lethality is located within the DNA initiator Gene dnaA of the bacterium

Earlier, we reported that the bacteriophage lambda P gene product is lethal to Escherichia coli, and the E. coli rpl mutants are resistant to this lambda P gene-mediated lethality. In this paper, we show that under the lambda P gene-mediated lethal condition, the host DNA synthesis is inhibited at the initiation step. The rpl8 mutation maps around the 83 min position in the E. coli chromosome and is 94 % linked with the dnaA gene. The rpl8 mutant gene has been cloned in a plasmid. This plasmid clone can protect the wild-type E. coli from lambda P gene-mediated killing and complements E. coli dnaAts46 at 42 degrees C. Also, starting with the wild-type dnaA gene in a plasmid, the rpl-like mutations have been isolated by in vitro mutagenesis. DNA sequencing data show that each of the rpl8, rpl12 and rpl14 mutations has changed a single base in the dnaA gene, which translates into the amino acid changes N313T, Y200N, and S246T respectively within the DnaA protein. These results have led us to conclude that the rpl mutations, which make E. coli resistant to lambda P gene-mediated host lethality, are located within the DNA initiator gene dnaA of the host.     

Production and partial characterization of a novel capsular polysaccharide KP-EPS produced by <Emphasis Type="Italic">Paenibacillus pabuli</Emphasis> strain ATSKP

The aerobic nitrogen fixing xylanolytic bacterium Paenibacillus pabuli strain ATSKP produces loosely attached capsular polysaccharide KP-EPS. On 0.5% birchwood xylan 70 ± 5.02 mg of KP-EPS was produced per gram dry weight of cells by the fourth day of growth in the absence of combined nitrogen source at 30°C. It was separated and purified using centrifugation, cold acetone precipitation and dialysis and is a sulfate containing heteropolymer as revealed by FT-IR spectrometry and elemental analysis. CHN analysis revealed the presence of 37.50% carbon, 5.90% hydrogen and 8.28% nitrogen in KP-EPS. Absence of phosphorus was confirmed by ³¹P NMR. ICP-OES analysis showed the presence of various metals in small concentrations. Specific binding with aniline blue suggested the presence of (1,3)-β-d-glucan. Thermal gravimetric analysis and differential scanning calorimetric analysis confirmed its thermal stability as high as 200°C. The EPS was not pseudo plastic and the viscosity was less than xanthan. The intrinsic viscosity did not reduce drastically when dissolved in 0.1 M NaCl.     

Generation and characterization of anti-MUC4 monoclonal antibodies reactive with normal and cancer cells in humans.

We have previously cloned the full-length cDNA (approximately 28 Kb) and established the complete genomic organization (25 exons/introns over 100 kb) of the human MUC4 mucin. This large molecule is predicted to protrude over 2 microm above the cell surface, in which MUC4alpha is an extracellular mucin-type glycoprotein subunit and MUC4beta is the transmembrane subunit. Over two thirds of the encoded protein sequence consists of 16-amino-acid tandem repeats (TR), which are flanked by unique sequences. In this study we generated and characterized monoclonal antibodies (MAbs) directed against the TR region of MUC4. Mice were immunized with a KLH-conjugated MUC4 TR peptide, STGDTTPLPVTDTSSV. Several clones were purified by three rounds of limited dilutions and stable clones presenting a sustained antibody production were selected for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the MUC4 peptide and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the MAbs (8G7) was strongly reactive against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis. Anti-MUC4 MAb may represent a powerful tool for the study of MUC4 function under normal and pathological conditions and for diagnosis of solid tumors including those in the breast, pancreas, lungs, and ovaries.     

Proteasomal dysfunction and ER stress triggers 2′-hydroxy-retrochalcone-induced paraptosis in cancer cells

Chalcones are biologically active class of compounds, known for their anticancer activities. Here we show for the first time that out of the six synthetic derivatives of chalcone tested, 2′-hydroxy-retrochalcone (HRC) was the most effective in inducing extensive cytoplasmic vacuolation mediated death called paraptosis in malignant breast and cervical cancer cells. The cell death by HRC is found to be nonapoptotic in nature due to the absence of DNA fragmentation, PARP cleavage, and phosphatidylserine externalization. It was also found to be nonautophagic as there was an increase in the levels of autophagic markers LC3I, LC3II and p62. Immunofluorescence with the endoplasmic reticulum (ER) marker protein calreticulin showed that the cytoplasmic vacuoles formed were derived from the ER. This ER dilation was due to ER stress as evidenced from the increase in polyubiquitinated proteins, Bip and CHOP. Docking studies revealed that HRC could bind to the Thr1 residue on the active site of the chymotrypsin-like subunit of the proteasome. The inhibition of proteasomal activity was further confirmed by the fluorescence based assay of the chymotrypsin-like subunit of the 26S proteasome. The cell death by HRC was also triggered by the collapse of mitochondrial membrane potential and depletion of ATP. Pretreatment with thiol antioxidants and cycloheximide were able to inhibit this programmed cell death. Thus our data suggest that HRC can effectively kill cancer cells via paraptosis, an alternative death pathway and can be a potential lead molecule for anticancer therapy.     

Melanoma central nervous system metastases: An update to approaches,challenges, and opportunities

In February 2018, the Melanoma Research Foundation and the Moffitt Cancer Center hosted the Second Summit on Melanoma Central Nervous System (CNS) Metastases in Tampa, Florida. In this white paper, we outline the current status of basic science, translational, and clinical research into melanoma brain metastasis development and therapeutic management. We further outline the important challenges that remain for the field and the critical barriers that need to be overcome for continued progress to be made in this clinically difficult area.     

Fine Mapping QTL for Drought Resistance Traits in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) Using Bulk Segregant Analysis

Drought stress is a major limitation to rice (Oryza sativa L.) yields and its stability, especially in rainfed conditions. Developing rice cultivars with inherent capacity to withstand drought stress would improve rainfed rice production. Mapping quantitative trait loci (QTLs) linked to drought resistance traits will help to develop rice cultivars suitable for water-limited environments through molecular marker-assisted selection (MAS) strategy. However, QTL mapping is usually carried out by genotyping large number of progenies, which is labour-intensive, time-consuming and cost-ineffective. Bulk segregant analysis (BSA) serves as an affordable strategy for mapping large effect QTLs by genotyping only the extreme phenotypes instead of the entire mapping population. We have previously mapped a QTL linked to leaf rolling and leaf drying in recombinant inbred (RI) lines derived from two locally adapted indica rice ecotypes viz., IR20/Nootripathu using BSA. Fine mapping the QTL will facilitate its application in MAS. BSA was done by bulking DNA of 10 drought-resistant and 12 drought-sensitive RI lines. Out of 343 rice microsatellites markers genotyped, RM8085 co-segregated among the RI lines constituting the respective bulks. RM8085 was mapped in the middle of the QTL region on chromosome 1 previously identified in these RI lines thus reducing the QTL interval from 7.9 to 3.8 cM. Further, the study showed that the region, RM212–RM302–RM8085–RM3825 on chromosome 1, harbours large effect QTLs for drought-resistance traits across several genetic backgrounds in rice. Thus, the QTL may be useful for drought resistance improvement in rice through MAS and map-based cloning.     

Cysteine and serine protease-mediated proteolysis in body homogenate of a zooplankter, Moina macrocopa, is inhibited by the toxic cyanobacterium, Microcystis aeruginosa PCC7806

The paper describes the characterization of proteases in the whole body homogenate of Moina macrocopa, which can possibly be inhibited by the extracts of Microcystis aeruginosa PCC7806. With the use of oligopeptide substrates and specific inhibitors, we detected the activities of trypsin, chymotrypsin, elastase and cysteine protease. Cysteine protease, the predominant enzyme behind proteolysis of a natural substrate, casein, was partially purified by gel filtration. The substrate SDS-polyacrylamide gel electrophoresis of body homogenate revealed the presence of nine bands of proteases (17-72 kDa). The apparent molecular mass of an exclusive cysteine protease was 60 kDa, whereas of trypsin, it was 17-24 kDa. An extract of M. aeruginosa PCC7806 significantly inhibited the activities of trypsin, chymotrypsin and cysteine protease in M. macrocopa body homogenate at estimated IC(50) of 6- to 79-microg dry mass mL(-1). Upon fractionation by C-18 solid-phase extraction, 60% methanolic elute contained all the protease inhibitors, and these metabolites could be further separated by reverse-phase liquid chromatography. The metabolites inhibitory to M. macrocopa proteases also inhibited the corresponding class of proteases of mammalian/plant origin. The study suggests that protease inhibition may contribute to chemical interaction of cyanobacteria and crustacean zooplankton.     

Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents

Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency whereas a reverse trend was observed with apicidin. Together, these finding provide a new strategy for differential mitochondrial targeting in cancer therapy.Cancer cells favor glycolysis over oxidative phosphorylation (OXPHOS) to meet their energy demand,¹ suggesting that they have adapted to survive and proliferate in the absence of fully functional mitochondria. Research in the last two decades demonstrates that, in addition to generation of energy, mitochondria including cancer cell mitochondria regulate multiple cellular signaling pathways encompassing cell death, proliferation, cellular redox balance, and metabolism.^{2, 3} As cancer cells possess defects in these pathways that provide an opportunity to target this organelle for therapeutic purposes. Subsequently, several agents have been developed that target cancer cell mitochondria to induce apoptosis, a cell death pathway, and eradicate cancer cells.^{4, 5} Cancer cell mitochondria harbor several proapoptotic proteins including cytochrome c, which is released from mitochondria in response to anticancer agents and activates caspases to execute apoptosis.^{5, 6} Thus, anticancer agents that induce cytochrome c release from mitochondria will be beneficial for induction of apoptosis in cancer cells. Indeed, several such agents have been developed, which include inhibitors targeting prosurvival Bcl-2 family members including Bcl-2, Bcl-xL, and Mcl-1.^{7, 8, 9} Unfortunately, cancer cells have developed multiple mechanisms to resist or overcome cytochrome c release and evade apoptosis.Although underlying mechanisms of cancer cell resistance to apoptosis are still undefined, the OXPHOS defect is known to be one of the key reasons for the attenuation of apoptosis in cancer cells.^{10, 11} Multiple lines of evidence support the notion that cancer cell survival and proliferation commonly associate with an OXPHOS defect in cancer.^{1, 12} Active OXPHOS is an efficient form of respiration but also regulates apoptosis through the OXPHOS complexes. The OXPHOS system consists of five multimeric protein complexes (I, II, III, IV, and V). The components of these complexes (except complex-II) are encoded by both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA).^{12, 13} Thus mutations, deletions, and translocations in either mtDNA or nDNA can potentially result in OXPHOS deficiency. MtDNA mutations associate with inhibition of apoptosis, induction of angiogenesis, invasion and metastasis of various types of cancer.^{3, 12, 14} Thus, mtDNA could potentially be an important target to restore cell death in cancer and attenuate cancer growth. Therefore, there is an urgent need to investigate the role of OXPHOS in the molecular mechanisms underlying cancer cell death.We investigated the effects of several anticancer agents of different classes including DNA-damaging agents (etoposide and doxorubicin), protein kinase inhibitors (staurosporine and sorafenib), mitotic inhibitor (taxol), ER stressor/inhibitor of Ca²⁺-ATPases (thapsigargin), and histone deacetylase (HDAC) inhibitor (apicidin) on mtDNA. We also determined the impact of OXPHOS defects on apoptosis induction by these agents. Although most anticancer agents induced caspase activation and apoptosis, the mtDNA level was elevated maximally by etoposide and it was not modulated by a caspase inhibitor but reduced by an autophagy inhibitor. Induction of mtDNA is associated with increased reactive oxygen species (ROS) production and elevated mitochondrial mass. Pharmacologic inhibition of OXPHOS complexes reduced the etoposide-induced elevation in mtDNA, suggesting the involvement of these complexes in etoposide-induced apoptosis. Together, we define the impact of mtDNA and OXPHOS function on mitochondrial apoptosis, which has significance in restoring cancer cell apoptosis for therapeutic purposes.     

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.