Role of p38 mitogen-activated protein kinase phosphorylation and Fas-Fas ligand interaction in morphine-induced macrophage apoptosis

In this study, we evaluated the molecular mechanisms involved in morphine-induced macrophage apoptosis. Both morphine and TGF-beta promoted P38 mitogen-activated protein kinase (MAPK) phosphorylation, and this phosphorylation was inhibited by SB 202190 as well as by SB 203580. Anti-TGF-beta Ab as well as naltrexone (an opiate receptor antagonist) inhibited morphine-induced macrophage P38 MAPK phosphorylation. Anti-TGF-beta Ab also attenuated morphine-induced p53 as well as inducible NO synthase expression; in contrast, N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase, inhibited morphine-induced P38 MAPK phosphorylation and Bax expression. Morphine also enhanced the expression of both Fas and Fas ligand (FasL), whereas anti-FasL Ab prevented morphine-induced macrophage apoptosis. Moreover, naltrexone inhibited morphine-induced FasL expression. In addition, macrophages either deficient in FasL or lacking p53 showed resistance to the effect of morphine. Inhibitors of both caspase-8 and caspase-9 partially prevented the apoptotic effect of morphine on macrophages. In addition, caspase-3 inhibitor prevented morphine-induced macrophage apoptosis. These findings suggest that morphine-induced macrophage apoptosis proceeds through opiate receptors via P38 MAPK phosphorylation. Both TGF-beta and inducible NO synthase play an important role in morphine-induced downstream signaling, which seems to activate proteins involved in both extrinsic (Fas and FasL) and intrinsic (p53 and Bax) cell death pathways.     

An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani

DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes.     

A five‐step risk management process for geriatric dental practice during SARS‐CoV‐2 pandemic

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is an RNA virus that causes coronavirus infection (COVID‐19). COVID‐19 is a highly contagious disease transmitted through respiratory droplets, saliva and other contact routes. Within 10 months of its outbreak, SARS‐CoV‐2 has infected more than 23 million people around the world. Evidence suggests that older adults are the most vulnerable to infection and have an increased risk of mortality. Reduced immunity and underlying medical conditions make them risk‐prone and vulnerable to critical care. Older adults affected with the SARS‐CoV‐2 virus present with distinct clinical manifestations necessitating specific treatment needs and management protocols. While it is crucial to prevent the spread of novel coronavirus (2019‐nCoV), the role of oral healthcare workers in addressing the specific needs of ageing adult patients by adopting specific guidelines and appropriate infection control protocols is timely. This paper aims to develop specific guidelines and protocols for the dental management of geriatric patients during the COVID‐19 pandemic.     

Specific ratio and survival of Pseudomonas CF600 as co-culture for phenol degradation in continuous cultivation

Studies were carried out to understand parallel survival of two strains when cultivated as co-culture on a single carbon source in continuous cultivation. Strains used were Pseudomonas sp. strain CF600 that is reported for degradation of phenol; and HKR1 a lab strain, which was isolated from a site contaminated with phenol. In continuous cultivation Pseudomonas sp. CF600 showed an accumulation of colored intermediate, 2-hydroxy muconic semialdehyde (HMS), when fed with phenol as a sole source of carbon under dissolved oxygen limiting condition (40% saturation level). Under the same cultivation condition when it was co-cultured with strain HKR1, complete degradation of phenol was observed with no accumulation of intermediate. Different dilution rates (0.03, 0.15, and 0.30) were set in the bioreactor during cultivation. It was also observed that both the strains follow a typical cell density ratio of 1:18 as strain HKR1: Pseudomonas sp. CF600 irrespective of the dilution rates used in the study to favor degradation of phenol. Pseudomonas sp. CF600 is reported to degrade phenol via a plasmid-encoded pathway (pVI150). The enzymes for this meta-cleavage pathway are clustered on 15 genes encoded by a single operon, the dmp operon. PCR using primers from the different catabolic loci of dmp operon, demonstrated that the strain HKR1 follows a different metabolic pathway for intermediate utilization.     

Soluble production and function of vascular endothelial growth factor/basic fibroblast growth factor complex peptide

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are important proangiogenic factors in tumor procession. The autocrine and paracrine bFGF and the VEGF in tumor tissue can promote tumor angiogenesis, tumor growth, and metastasis. A VEGF/bFGF Complex Peptide (VBP3) was designed on the basis of epitope peptides from both VEGF and bFGF to elicit in vivo production of anti‐bFGF and anti‐VEGF antibodies. In this study, we reported on the production of recombinant VBP3 using high cell density fermentation. Fed‐batch fermentation for recombinant VBP3 production was conducted, and the production procedure was optimized in a 10‐L fermentor. The fraction of soluble VBP3 protein obtained reached 78% of total recombinant protein output under fed‐batch fermentation. Purified recombinant VBP3 could inhibit tumor cell proliferation in vitro and stimulate C57BL/6 mice to produce high titer anti‐VEGF and anti‐bFGF antibodies in vivo. A melanoma‐grafted mouse model and an immunohistochemistry assay showed that tumor growth and tumor angiogenesis were significantly inhibited in VBP3‐vaccinated mice. These results demonstrated that soluble recombinant VBP3 could be produced by large‐scale fermentation, and the product, with good immunogenicity, elicited production of high‐titer anti‐bFGF and anti‐VEGF antibodies, which could be used as a therapeutic tumor vaccine to inhibit tumor angiogenesis and tumor growth. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:194–203, 2015     

Broad-host-range plasmid-mediated metabolic perturbations in Pseudomonas fluorescens 13525

Genetic engineering of fluorescent pseudomonads for various industrially, agriculturally and environmentally important bioprocesses often involves the use of suitable plasmids. Plasmid-mediated alterations in host physiology and metabolism are poorly understood for this group of organisms. Thus, we investigated the metabolic perturbations in Pseudomonas fluorescens 13525 due to the independent and combined presence of broad-host-range plasmids, pBBR1MCS-2 (copy number 30) and pUCPM18 derived pAB4 and pAB8 (copy number 14-16). Presence of pAB4 and pAB8 not only significantly increased the growth rate and glucose utilization of P. fluorescens 13525, but also increased glucose dehydrogenase activity and gluconic acid production indicating enhanced direct oxidative pathway for glucose catabolism. Additionally, increased secretion of pyruvic, acetic, and citric acids caused faster media acidification in presence of pAB4 and pAB8. Simultaneous presence of pAB4/pAB8 in Pf (pAB48) and pAB4/pBBR1MCS-2 in Pf (pAB4BBR1MCS-2) reduced their respective copy numbers to nearly half. Pf (pAB48) demonstrated further increase in direct oxidation pathway without altering growth and glucose depletion rates, as compared with single transformants. Conversely, pBBR1MCS-2 plasmid did not greatly alter P. fluorescens 13525 metabolism when present independently but masked the effects imposed by pAB4 when present in its combination. In conclusion, P. fluorescens 13525 redesigns its metabolism in response to the presence of plasmids irrespective of their nature, by enhancing anaplerosis with a simultaneous reduction in catabolism as indicated by increased pyruvate carboxylase and decreased citrate synthase activities, respectively. Such information will be helpful for vector designing during genetic engineering of fluorescent pseudomonads.     

Semi-automated high-throughput fluorescent intercalator displacement-based discovery of cytotoxic DNA binding agents from a large compound library

High-throughput fluorescent intercalator displacement (HT–FID) was adapted to the semi-automated screening of a commercial compound library containing 60,000 molecules resulting in the discovery of cytotoxic DNA-targeted agents. Although commercial libraries are routinely screened in drug discovery efforts, the DNA binding potential of the compounds they contain has largely been overlooked. HT–FID led to the rapid identification of a number of compounds for which DNA binding properties were validated through demonstration of concentration-dependent DNA binding and increased thermal melting of A/T- or G/C-rich DNA sequences. Selected compounds were assayed further for cell proliferation inhibition in glioblastoma cells. Seven distinct compounds emerged from this screening procedure that represent structures unknown previously to be capable of targeting DNA leading to cell death. These agents may represent structures worthy of further modification to optimally explore their potential as cytotoxic anti-cancer agents. In addition, the general screening strategy described may find broader impact toward the rapid discovery of DNA targeted agents with biological activity.     

Crystal structure of the BARD1 BRCT domains

The interaction of the breast tumor suppressor BRCA1 with the protein BARD1 results in the formation of a heterodimeric complex that has ubiquitin ligase activity and plays central roles in cell cycle checkpoint control and DNA repair. Both BRCA1 and BARD1 possess a pair of tandem BRCT domains that interact in a phosphorylation-dependent manner with target proteins. We determined the crystal structure of the human BARD1 BRCT repeats (residues 568-777) at 1.9 A resolution. The composition and structure of the BARD1 phosphoserine-binding pocket P1 are strikingly similar to those of the BRCA1 and MDC1 BRCT domains, suggesting a similar mode of interaction with the phosphate group of the ligand. By contrast, the BARD1 BRCT selectivity pocket P2 exhibits distinct structural features, including two prominent histidine residues, His685 and His686, which may be important for ligand binding. The protonation state of these histidines has a marked effect on the calculated electrostatic potential in the vicinity of P2, raising the possibility that ligand recognition may be regulated by changes in pH. Importantly, the BARD1 BRCT structure provides insights into the mechanisms by which the cancer-associated missense mutations C645R, V695L, and S761N may adversely affect the structure and function of BARD1.