Utility of anion and cation combinations for phasing of protein structures

We report the use of anionic (I(-)), cationic (Ba(2+), Cd(2+)) and ionic mixtures (I(-) plus Ba(2+)) for derivatizing liver fatty acid binding protein (LFABP) crystals. Use of cationic and anionic salts in phasing experiments revealed distinct non-overlapping sites for these ions, suggesting exclusive binding regions on LFABP. Interestingly, cations of identical charge and valency (like Ba(2+) and Cd(2+)) bound to distinct pockets on the protein surface. Furthermore, a mixture of salts containing both I(-) and Ba(2+) was very useful in phasing experiments as these oppositely charged ions bound to different regions of LFABP. Our data therefore suggest that cationic and anionic salt mixtures like BaCl(2) with NH(4)I or salts like CdI, BaI where each ion has a significant anomalous signal for a given X-ray wavelength may be valuable reagents for phasing during structure determination.     

A novel disintegrin protein from Naja naja venom induces cytotoxicity and apoptosis in human cancer cell lines in vitro

Animal venoms and toxins are potential bioresources that have been known to mankind as a therapeutic tool for more than a century through folk and traditional medicine. The purified “disintegrin protein” (64 kDa) from the venom of the Indian cobra snake (Naja naja) exhibited cytotoxic effects of various types of human cancer cell lines such as breast cancer (MCF-7), lung cancer (A549) and liver cancer (HepG2). In vitro cytotoxicity, DNA fragmentation, an apoptotic assay and a cell cycle analysis were performed to evaluate the anticancer activity of disintegrin against the above cell lines. The IC₅₀ value of disintegrin was determined to be 2.5 ± 0.5 μg/mL, 3.5 ± 0.5 μg/mL, and 3 ± 0.5 μg/mL for the MCF-7, A549 and HepG2 cell lines respectively. Moreover, the increased distribution of G0/G1 and S phase led to decreased populations of cells in the G2/M phase of MCF-7, HepG2 and A549 cells.     

Comparison of individual and composite field analysis using array detector for Intensity Modulated Radiotherapy dose verification

AimTo compare the measured and calculated individual and composite field planar dose distribution of Intensity Modulated Radiotherapy plans.Materials and methodsThe measurements were performed in Clinac DHX linear accelerator with 6 MV photons using Matrixx device and a solid water phantom. The 20 brain tumor patients were selected for this study. The IMRT plan was carried out for all the patients using Eclipse treatment planning system. The verification plan was produced for every original plan using CT scan of Matrixx embedded in the phantom. Every verification field was measured by the Matrixx. The TPS calculated and measured dose distributions were compared for individual and composite fields.Results and discussionThe percentage of gamma pixel match for the dose distribution patterns were evaluated using gamma histogram. The gamma pixel match was 95–98% for 41 fields (39%) and 98% for 59 fields (61%) with individual fields. The percentage of gamma pixel match was 95–98% for 5 patients and 98% for other 12 patients with composite fields. Three patients showed a gamma pixel match of less than 95%. The comparison of percentage gamma pixel match for individual and composite fields showed more than 2.5% variation for 6 patients, more than 1% variation for 4 patients, while the remaining 10 patients showed less than 1% variation.ConclusionThe individual and composite field measurements showed good agreement with TPS calculated dose distribution for the studied patients. The measurement and data analysis for individual fields is a time consuming process, the composite field analysis may be sufficient enough for smaller field dose distribution analysis with array detectors.     

Structural analyses of the malaria parasite aminoacyl‐tRNA synthetases provide new avenues for antimalarial drug discovery

Malaria is a parasitic illness caused by the genus Plasmodium from the apicomplexan phylum. Five plasmodial species of P. falciparum (Pf), P. knowlesi, P. malariae, P. ovale, and P. vivax (Pv) are responsible for causing malaria in humans. According to the World Malaria Report 2020, there were 229 million cases and ~ 0.04 million deaths of which 67% were in children below 5 years of age. While more than 3 billion people are at risk of malaria infection globally, antimalarial drugs are their only option for treatment. Antimalarial drug resistance keeps arising periodically and thus threatens the main line of malaria treatment, emphasizing the need to find new alternatives. The availability of whole genomes of P. falciparum and P. vivax has allowed targeting their unexplored plasmodial enzymes for inhibitor development with a focus on multistage targets that are crucial for parasite viability in both the blood and liver stages. Over the past decades, aminoacyl‐tRNA synthetases (aaRSs) have been explored as anti‐bacterial and anti‐fungal drug targets, and more recently (since 2009) aaRSs are also the focus of antimalarial drug targeting. Here, we dissect the structure‐based knowledge of the most advanced three aaRSs—lysyl‐ (KRS), prolyl‐ (PRS), and phenylalanyl‐ (FRS) synthetases in terms of development of antimalarial drugs. These examples showcase the promising potential of this family of enzymes to provide druggable targets that stall protein synthesis upon inhibition and thereby kill malaria parasites selectively.     

Comparative biochemistry of eumelanogenesis and the protective roles of phenoloxidase and melanin in insects

The phenolic biopolymer eumelanin is an important skin pigment found throughout the animal kingdom. The enzyme, tyrosinase, initiates melanogenesis in mammals. The biogenesis is assisted by a number of mammalian protein factors including dopachrome tautomerase and 5,6-dihydroxyindole-2-carboxylate oxidase. Invertebrates, such as insects, employ phenoloxidase and dopachrome (decarboxylating) isomerase for melanin biosynthesis. Recently generated molecular biological and biochemical data indicate that tyrosinase and phenoloxidase are distinctly different enzymes in spite of possessing both monophenol monooxygenase activity as well as o-diphenoloxidase activity. Similarly, insect dopachrome isomerase also differs significantly from its mammalian counterpart in several of its properties including the nature of the enzymatic reaction. In addition, there are considerable differences in the eumelanogenic pathways of these two animal groups that include the utility of substrates, use of dihydroxyindoles and the nature of eumelanin pigment. Thus, the biochemistry and molecular biology of melanogenesis in mammals and insects are significantly different. The advantages of generating different eumelanin pigments and intermediates by the insects are discussed.     

PDI reductase acts on Akita mutant proinsulin to initiate retrotranslocation along the Hrd1/Sel1L-p97 axis

In mutant INS gene–induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production.     

Localization of cyclooxygenase-2 in mice testis and assessment of its possible role through suppressing its expression using nimesulide: a preferential cyclooxygenase-2 inhibitor

Present generation non-steroidal anti-inflammatory drugs (NSAID) are potent inhibitors of the enzyme cyclooxygenase-2 (COX-2). Even though they exhibit reduced incidence of gastrotoxicity, severe nephrotoxicity and other side effects have been widely reported with respect to usage of these drugs. Since COX-2 levels are not only upregulated by inflammation but also by other stimuli such as cytokines, growth factors, mitogens and steroid hormones, we investigated the localization of COX-2 and activity of both COX-1 and COX-2 in mice testis. To correlate the localization of COX-2 with its function we suppressed COX-2 expression with the aid of nimesulide a preferential COX-2 inhibitor. We found COX-2 was constitutively expressed in the Leydig cells of mice testis suggesting a role on testosterone synthesis. Suppression of COX-2 resulted in increased concentration of most of polyunsaturated fatty acids especially arachidonic acid (AA). Prostaglandin (PG) levels which showed an initial decline during nimesulide treatment had a reversible effect during prolonged treatment. These findings state that cyclooxygenase is constitutively expressed in mice testis and continuous inhibition of COX-2 interferes in maturation of sperm.     

Identification and quantification of histidine-rich glycoprotein (HRG) in the blood plasma of six marine bivalves

Histidine-rich Glycoprotein (HRG) is a metal-binding protein described from the blood plasma of a pteriomorph bivalve, the marine mussel Mytilus edulis L. We demonstrate here, using Cd-Immobilized Metal Affinity Chromatography (IMAC), SDS-PAGE, Western Blotting, and ELISA, that HRG is present in three additional pteriomorphs and two heterodont bivalves. ELISA indicates that HRG is the predominant blood plasma protein in all six species (41 to 61% of total plasma proteins by weight). Thus, HRG appears to be a widespread metal-binding protein in the plasma of bivalves.