Analysis of Rev1p and Pol zeta in mitochondrial mutagenesis suggests an alternative pathway of damage tolerance

Ultraviolet light is a potent DNA damaging agent that induces bulky lesions in DNA which block the replicative polymerases. In order to ensure continued DNA replication and cell viability, specialized translesion polymerases bypass these lesions at the expense of introducing mutations in the nascent DNA strand. A recent study has shown that the N-terminal sequences of the nuclear translesion polymerases Rev1p and Pol zeta can direct GFP to the mitochondrial compartment of Saccharomyces cerevisiae. We have investigated the role of these polymerases in mitochondrial mutagenesis. Our analysis of mitochondrial DNA point mutations, microsatellite instability, and the spectra of mitochondrial mutations indicate that these translesion polymerases function in a less mutagenic pathway in the mitochondrial compartment than they do in the nucleus. Mitochondrial phenotypes resulting from the loss of Rev1p and Pol zeta suggest that although these polymerases are responsible for the majority of mitochondrial frameshift mutations, they do not greatly contribute to mitochondrial DNA point mutations. Analysis of spontaneous mitochondrial DNA point mutations suggests that Pol zeta may play a role in general mitochondrial DNA maintenance. In addition, we observe a 20-fold increase in UV-induced mitochondrial DNA point mutations in rev deficient strains. Our data provides evidence for an alternative damage tolerance pathway that is specific to the mitochondrial compartment.     

Recent advancement in the discovery and development of COX-2 inhibitors: Insight into biological activities and SAR studies (2008–2019)

Cyclooxygenase-2 is a very important physiological enzyme playing key roles in various biological functions especially in the mechanism of pain and inflammation, among other roles, making it a molecule of high interest to the pharmaceutical community as a target. COX 2 enzyme is induced only during inflammatory processes or cancer and reflects no role in the guarding stomach lining. Thus, selective COX-2 inhibition can significantly reduce the adverse effects including GI tract damage and hepatotoxic effects of traditional NSAIDs like aspirin, ibuprofen, etc. Recent developments on COX-2 inhibitors is primarily focused on improving the selectivity index of the drug towards COX-2 along with enhancing the potency of the drug by modifying the scaffolds of Coxibs currently in the market like Celecoxib, Indomethacin, Oxaprozin, etc. We have reported the progress on new COX-2 inhibitors in the last decade (2008–2019) focussing on five heterocyclic rings- Pyrazole, Indole, Oxazole, Pyridine and Pyrrole. The addition of various moieties to these core rings and their structure-activity relationship along with their molecular modelling data have been explored in the article. This review aims to aid medicinal chemists in the design and discovery of better COX-2 inhibitors constructed on these five heterocyclic pharmacophores.     

Ultrastructure of the antennal sensilla of Alabama argillacea (Hübner, 1823) (Lepidoptera: Erebidae)

Insects have several types of sensilla, the characterization of which has been fundamental to understanding the mechanisms of sensory perception in different species. This study aimed to describe the ultrastructure of the sensilla present on the antennae of Alabama argillacea (Hübner, 1823) (Lepidoptera: Erebidae), an important pest of cotton (Gossypium hirsutum L.) crops, as well as their possible variation between sexes. To do this, the antennae of males and females of A. argillacea were analyzed using scanning electron microscopy. Sensilla morphometry was assessed using photomicrographs, from which the lengths and basal and apical diameters of sensilla were measured using the ImageJ program. Seven types of sensilla were identified on the antennae of A. argillacea: sensilla trichodea, sensilla chaetica, sensilla auricillica, sensilla coeloconica, sensilla styloconica, sensilla basiconica, and sensilla Böhm bristles. Differences between the sensilla of males and females were found in their lengths and basal diameters in the distal and proximal regions. This suggests that sensilla functionality may not only vary from one species to another, but also between sexes within the same species. Thus, further transmission electron microscopy and single sensillum recording studies may provide more detailed information on the sensilla of A. argillacea and their functions.     

紫彩血蛤鳃的组织化学和扫描电镜研究

紫彩血蛤（Ｎｕｔｔａｌｌｉａｏｌｉｖａｃｅａ）的鳃上皮细胞核多为椭圆形,核内染色体呈丝状;结缔组织细胞的核多为圆形,部分核内有块状Ｆｅｕｌｇｅｎ阳性颗粒,ＲＮＡ在鳃丝上皮细胞最丰富,有的形成直径０．５２μｍ的颗粒,结缔组织和鳃丝上皮细胞内均有直径０．６３μｍ左右的ＰＡＳ阳性颗粒,吞噬细胞呈极强的ＡＮＡＥ阳性,鳃丝上皮细胞呈较弱的阳性。ＡＣＰ和ＡＫＰ在吞噬细胞和外缘上皮细胞中的活性较强,其它部位较弱     

Delineation of the structural determinants of the N-methyl-D-aspartate receptor glycine binding site

In this study, we have further delineated the domains of the N-methyl-D-aspartate receptor NR1 subunit that contribute to the glycine co-agonist binding site. Taking an iterative approach, we have constructed truncation mutants of the NR1 subunit, transiently expressed them in HEK-293 cells, and determined the binding of the glycine site antagonist [3H]L-689,560. Amino acids 380-811 were sufficient to form a glycine binding site with affinities for [3H]L-689,560 and glycine that were not significantly different from wild-type NR1. More extensive deletions, from either the amino- or the carboxy-terminal end, resulted in loss of ligand binding. Additional constructs were made starting from amino acids 380-843 of NR1, replacing the transmembrane (TMI-TMIII) domain with intervening linker sequences while retaining the TMIV domain so as to anchor the polypeptide to the membrane. Although robust amounts of polypeptides were synthesised by transfected cells, only low levels of [3H]L-689,560 binding sites could be detected. This suggests that only a small proportion of the synthesised polypeptide folds in the appropriate manner so as to form a ligand binding site. These data indicate that although it is possible to reduce the glycine binding site to minimal so-called S1 and S2 domains, efficient folding of the polypeptide so as to form a ligand binding site may require sequences within the TMI-TMIII domain.     

Knockout of the alanine racemase gene in Lactobacillus plantarum results in septation defects and cell wall perforation

A stable mutant of Lactobacillus plantarum deficient in alanine racemase (Alr) was constructed by two successive homologous recombination steps. When the mutant was supplemented with D-alanine, growth and viability were unaffected. Surprisingly, deprivation of d-alanine during exponential growth did not result in a rapid and extensive lysis as observed in Alr-deficient strains of Escherichia coli or Bacillus subtilis. Rather, the starved mutant cells underwent a growth arrest and were gradually affected in viability with a decrease in colony forming units over 99% in less than 24 h. Additionally, fluorescent techniques demonstrated a loss of cell envelope integrity in the starved cells. Prolonged d-alanine starvation resulted in cells with an aberrant morphology. Scanning and transmission electron microscopy analyses revealed an increase in cell length, deficiencies in septum formation, thinning of the cell envelope and perforation of the cell wall in the septum region. We discuss the involvement of peptidoglycan hydrolases in these phenotypic defects in the context of the crucial role played by D-alanine in peptidoglycan biosynthesis and teichoic acids substitution.     

An examination of Stone Age/Bronze Age adzes and axes of red deer (<Emphasis Type="Italic">Cervus elaphus</Emphasis> L.) antler from the Leine Valley,near Hannover

Red deer antler was, together with stone, one of the most important raw materials for tool manufacture in prehistoric times. Both the physical characteristics of this material and the manufacturing technology of antler tools are examined. The use of experimental tests and analytical studies of the recovered antler finds allow us to propose, with a great degree of confidence, that the main use of antler axes during the Stone Age/Bronze Age was in woodworking.     

Influence of initial fixation protocol on the appearance of primary cilia on the rabbit corneal endothelial cell apical surface as assessed by scanning electron microscopy

The objective was to examine primary cilia at the apical surface of corneal endothelial cells after using different fixatives. Female albino rabbits (2 kg) were euthanised at 15:00 h and the corneas fixed immediately (usually with an isotonic 2% glutaraldehyde-cacodylate fixative) either after dissection, by application fixative at 4 degrees C, by immersion of the eyeball in fixative at room temperature (RT), or by application of an isotonic or a hypertonic (Karnovsky-type) fixative at RT. Images at 2000x were taken from the central corneal region, and number and length of primary cilia assessed. The length was the same regardless of method (overall average of 1.67+/-0.70 microm), but the incidence of primary cilia was hypertonic fixative (87% of cells) >cold drop fixation (71%), >whole globe immersion (68%) >dissect then fix methods (67%) >RT drop fixation (34%). The first four methods however yielded cells with unacceptable artefacts (especially distortion). More details should be provided of the primary fixation method used.     

Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins. Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme. Site-directed mutagenesis was used to examine the important roles of these residues. Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography. Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP+ but still retained substantial activity with NAD+. Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD+ and NADP+ with an increase in activity nearly 3-fold over that of the wild type. However, approximately 30-fold higher in K(m) for NADP+ than that of the wild type enzyme for NAD+ was found for mutants Q15K-W37R and Q15R-W37R. Similarly, the K(m) values for PGE(2) of mutants were also shown to increase over that of the wild type. Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP+ but not with NAD+. The triple mutants showed a decrease in K(m) for NADP+ but an increase in K(m) for PGE(2). Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K(m) values for both NADP+ and PGE(2) to levels comparable to those of the wild type. These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD+ to NADP+ by changing a few critical residues near the N-terminal end.