Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae

DNA polymerases (Pol) α, δ and ε are necessary for replication of nuclear DNA. Po1δ interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Po18. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase δ gene (POL3). We have attempted to clone two recessive extragenic suppressors of such is mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdpl and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, D1S2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.     

Carbonic anhydrase,carbondioxide levels and growth of Nitrosomonas

The ammonia oxidizing bacterium Nitrosomonas europaea was grown either (a) with added bicarbonate in the absence of added CO₂ (bubbled through the culture), (b) with added bicarbonate plus low added CO₂ (0.03% v/v), or (c) without added bicarbonate with high added CO₂ (1% v/v). Cell doubling times of 12 h were observed in 1% cultures; doubling times of 2 to 3-fold longer wre found with 0.03% CO₂ and/or bicarbonate grown cultures. The specific activity of carbonic anhydrase was 40–80% lower in cultures grown on 1% CO₂. These results are compared with those in heterotrophic and photosynthetic microorganisms.Scientific Contribution Number 1241 from the New Hampshire Agricultural Experiment Station     

Structure-activity relationships of pyrazole-4-carbodithioates as antibacterials against methicillin–resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious hospital-acquired infections and is responsible for significant morbidity and mortality in residential care facilities. New agents against MRSA are needed to combat rising resistance to current antibiotics. We recently reported 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) as a new bacteriostatic agent against MRSA that appears to act via a novel mechanism. Here, twenty nine analogs of HMPC were synthesized, their anti-MRSA structure-activity relationships evaluated and selectivity versus human HKC-8 cells determined. Minimum inhibitory concentrations (MIC) ranged from 0.5 to 64?μg/mL and up to 16-fold selectivity was achieved. The 4-carbodithioate function was found to be essential for activity but non-specific reactivity was ruled out as a contributor to antibacterial action. The study supports further work aimed at elucidating the molecular targets of this interesting new class of anti-MRSA agents.     

BSD2 is a Rubisco‐specific assembly chaperone,forms intermediary hetero‐oligomeric complexes,and is nonlimiting to growth in tobacco

The folding and assembly of Rubisco large and small subunits into L₈S₈ holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco‐BSD2 assembly complexes in the model C₃ plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.m²) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L₈S₈ pool. RNAi‐silencing BSD2 production in transplastomic tobacco producing bacterial L₂ Rubisco had no effect on leaf photosynthesis, cell ultrastructure, or plant growth. Genetic crossing the same RNAi‐bsd2 alleles into wild‐type tobacco however impaired L₈S₈ Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis, or maize BSD2 reinstated Rubisco biogenesis in BSD2‐silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate, or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly, and is produced in amounts nonlimiting to tobacco growth.     

FURIN and placental syncytialisation: a cautionary tale

FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.Subject terms: Proteases, Differentiation     

TMEM41B and VMP1 are scramblases and regulate the distribution of cholesterol and phosphatidylserine

TMEM41B and VMP1 are integral membrane proteins of the endoplasmic reticulum (ER) and regulate the formation of autophagosomes, lipid droplets (LDs), and lipoproteins. Recently, TMEM41B was identified as a crucial host factor for infection by all coronaviruses and flaviviruses. The molecular function of TMEM41B and VMP1, which belong to a large evolutionarily conserved family, remains elusive. Here, we show that TMEM41B and VMP1 are phospholipid scramblases whose deficiency impairs the normal cellular distribution of cholesterol and phosphatidylserine. Their mechanism of action on LD formation is likely to be different from that of seipin. Their role in maintaining cellular phosphatidylserine and cholesterol homeostasis may partially explain their requirement for viral infection. Our results suggest that the proper sorting and distribution of cellular lipids are essential for organelle biogenesis and viral infection.     

Evaluation of Antioxidant,Antibacterial and Enhancement of Antibiotic Action by Punica granatum Leaves Crude Extract and Enriched Fraction against Multidrug-Resistant Bacteria

The aim of this study was to evaluate the phytochemical composition, antioxidant, and antimicrobial potential of crude extract and fractions of Punica granatum leaves. The extract was produced by turbo extraction, after which hexanic, ethyl acetate, and aqueous fractions were obtained by partitioning. The chemical analyses were performed by thin layer chromatography and high-performance liquid chromatography, and the antioxidant activities were assayed by DPPH^. and ABTS^.+. Minimal inhibitory and bactericidal concentrations (MIC/MBC) were applied to twenty-two bacteria. Most strains susceptible to extract/fractions and resistant to antibiotics were selected, and ampicillin, azithromycin, ciprofloxacin, and gentamicin were associated with the ethyl acetate fraction (EAF) against multidrug-resistant strains in modulatory and checkboard models. The data from chromatographic analyses showed flavonoids and tannins in the extract, as well as the enrichment of EAF in phenols, mainly flavonoids. The flavonoids were connected to the electron transfer activity demonstrated in the DPPH^. and ABTS^.+ assays. Gram-positive strains are more susceptible to EAF. The subinhibitory concentrations of P. granatum enhanced the antimicrobial activity of the agents and reduced the EAF individual MIC, and the combination of EAF and antibiotics demonstrated a synergistic effect. These results present a promising approach for developing a therapy in which antioxidant extracts and fractions can be used in combination with antibiotics.