In vitro inhibition of human erythrocyte acetylcholinesterase (EC3.1.1.7) by an antineoplastic drug methotrexate

This work addresses the kinetic analysis of the interaction of methotrexate (MTX) with human erythrocyte membrane-bound acetylcholinesterase (AChE, EC 3.1. 1.7). It was found that the MTX effect was independent of time of incubation with AChE before the addition of substrate which proves its reversible action. The IC₅₀ was determined, by three methods, to be 0.73 mM. The Michaelis-Menten constant (K_s) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.13 mM in the control system, a value decreased by 30–61% in the MTX treated systems. The V_max was 1.27tmole/min/mg protein for the control system while it was decreased by 44–77% in the MTX treated systems. The Linexveaver-Buck plot, Dixon plot, and their secondary replots indicated that the nature of the inhibition was of the linear mixed type, i.e. uncompetitive and noncompetitive. The values of K_i(slope) and K_I(tntecept) were estimated as 1.67 and 0.34 mM, respectively.Abbreviations AChE acetylcholinesterase - ASCh acetylthiocholine - K_s Michaelis-Menten constant - V_max the limiting maximal velocity - K_i inhibition constant - MTX methotrexate     

Excision of pyrimidine dimers by several UV-sensitive mutants of S. pombe

Summary Nine radiation-sensitive mutants of S. pombe showing a variety of phenotypic characteristics were analysed for their ability to excise pyrimidine dimers after ultraviolet irradiation. From earlier studies using indirect parameters, it was expected that some would be excision-deficient. Data reported here show that all the mutants tested, like wild type cells, were able to remove a high percentage of pyrimidine dimers during post-irradiation incubation in several different holding media, but not in saline or phosphate buffer. These mutants included strains showing increased, as well as others which showed decreased, levels of UV-induced mutation frequency relative to that of the wild type at the same total dose.     

Involvement of ras in sexual differentiation but not in growth control in fission yeast

The function of the ras⁺ gene of Schizosaccharomyces pombe has been studied by constructing null and activated alleles of this gene. An activated allele (^rasVal ₁₂) inhibits conjugation but has no effect on cell growth, entry into stationary phase or sporulation. The phenotype of ^rasVal ₁₂ is distinct from that caused by elevating the intracellular level of cAMP. This supports the hypothesis that ras of fission yeast does not modulate adenylate cyclase in a manner analogous to S. cerevisiae RAS. Introduction of a human ras sequence into fission yeast cells containing a non-functional null allele of ras restored the sexual differentiation process thus indicating that the human sequence can complement S. pombe ras. Our data suggest that although ras genes are highly conserved across a considerable evolutionary divide, the cellular function of the ras gene product varies in different organisms.     

Thermosensitive Export of Amylases in the Yeast Schwanniomyces alluvius

Cultures of Schwanniomyces alluvius were grown aerobically at30?C in a phosphate-buffered defined medium containing solublestarch as sole carbon source. Extracellular amylolytic activity,minimal when cells were most actively dividing, increased dramaticallyat the end of exponential growth and became maximal during stationaryphase. When cultures were grown at 37?G, no increase in extracellularamylolytic activity was detectable. This lack was only partlyascribable to thermal inactivation of the amylases. When culturesgrowing at 30?C were shifted to 37?C, excretion was arrested.Cultures growing at the restrictive temperature could be madeto excrete full amylolytic activity at the permissive temperature,only if the shift was made before the end of exponential growth. ¹NRCC contribution No. 23556. (Received December 10, 1983; Accepted April 20, 1984)     

In vitro antibiofilm and anti‐adhesion effects of magnesium oxide nanoparticles against antibiotic resistant bacteria

The aim of the current investigation was to determine the antibacterial and antibiofilm potential of MgO nanoparticles (NPs) against antibiotic‐resistant clinical strains of bacteria. MgO NPs were synthesized by a wet chemical method and further characterized by scanning electron microscopy and energy dispersive X‐ray. Antibacterial activity was determined by broth microdilution and agar diffusion methods. The Bradford method was used to assess cellular protein leakage as a result of loss of membrane integrity. Microtiter plate assay following crystal violet staining was employed to determine the effect of MgO NPs on biofilm formation and removal of established biofilms. MIC values ranged between 125 and 500 μg/mL. Moreover, treatment with MgO NPs accelerated rate of membrane disruption, measured as a function of leakage of cellular proteins. Leakage of cellular protein content was greater among gram‐negative bacteria. Cell adherence assay indicated 25.3–49.8% inhibition of bacterial attachment to plastic surfaces. According to a static biofilm method, MgO NPs reduced biofilm formation potential from 31% to 82.9% in a time‐dependent manner. Moreover, NPs also significantly reduced the biomass of 48, 72, 96 and 120 hr old biofilms (P < 0.05). Cytotoxicity experiments using a neutral red assay revealed that MgO NPs are non‐toxic to HeLa cells at concentrations of 15–120 μg/mL. These data provide in vitro scientific evidence that MgO NPs are effective and safe antibiofilm agents that inhibit adhesion, biofilm formation and removal of established biofilms of multidrug‐resistant bacteria.

     

Meta-analysis Reveals Genome-Wide Significance at 15q13 for Nonsyndromic Clefting of Both the Lip and the Palate,and Functional Analyses Implicate GREM1 As a Plausible Causative Gene

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13×10⁻¹⁴ for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32–1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94–1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47–9.61, P_diff<0.05). While we did not find lip or palate defects in Grem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions.     

Future growth in biotechnology in the developing countries

Conditions in developing countries are nowhere near what they should be to reap the benefits of modern biotechnology. Be that as it may, there are possibilities in which intelligent thinking and rational planning by the people concerned, combined with input from international agencies and national centres of excellence, can lead to potential economic gains. One possibility is the establishment of collaborative research on a regional basis to tackle common problems and to train each other's manpower in acquired skills. Similarity of circumstances would make these programmes more relevant and cost effective. To ensure the success of joint programmes in the local setting, foreign input in the form of finances, provision of critical laboratory materials, and expert advice in the selection of technically solvable problems becomes inevitable. Thus participation by the developed-country agencies and laboratories in the developing countries presents a purposeful pooling of efforts that will sow the seeds for sustainable development of the developing world. Given that, new technology would prove a boon and a blessing for humanity in general and for the developing world in particular.     

Genetic interactions reveal the antagonistic roles of FT/TSF and TFL1 in the determination of inflorescence meristem identity in Arabidopsis

During the transition to the reproductive phase, the shoot apical meristem switches from the developmental program that generates vegetative organs to instead produce flowers. In this study, we examined the genetic interactions of FLOWERING LOCUS T (FT)/TWIN SISTER OF FT (TSF) and TERMINAL FLOWER 1 (TFL1) in the determination of inflorescence meristem identity in Arabidopsis thaliana. The ft‐10 tsf‐1 mutants produced a compact inflorescence surrounded by serrated leaves (hyper‐vegetative shoot) at the early bolting stage, as did plants overexpressing TFL1. Plants overexpressing FT or TSF (or both FT and TFL1) generated a terminal flower, as did tfl1‐20 mutants. The terminal flower formed in tfl1‐20 mutants converted to a hyper‐vegetative shoot in ft‐10 tsf‐1 mutants. Grafting ft‐10 tsf‐1 or ft‐10 tsf‐1 tfl1‐20 mutant scions to 35S::FT rootstock plants produced a normal inflorescence and a terminal flower in the scion plants, respectively, although both scions showed similar early flowering. Misexpression of FT in the vasculature and in the shoot apex in wild‐type plants generated a normal inflorescence and a terminal flower, respectively. By contrast, in ft‐10 tsf‐1 mutants the vasculature‐specific misexpression of FT converted the hyper‐vegetative shoot to a normal inflorescence, and in the ft‐10 tsf‐1 tfl1‐20 mutants converted the shoot to a terminal flower. TFL1 levels did not affect the inflorescence morphology caused by FT/TSF overexpression at the early bolting stage. Taking these results together, we proposed that FT/TSF and TFL1 play antagonistic roles in the determination of inflorescence meristem identity, and that FT/TSF are more important than TFL1 in this process.