A bacterial model for studying effects of human mutations in vivo: Escherichia coli strains mimicking a common polymorphism in the human MTHFR gene

A simple bacterial model for studying effects of human mutations in vivo, when homologous genes exist in bacterial and human cells, is presented. We have constructed Escherichia coli strains bearing different alleles of the metF gene, an ortologue of human MTHFR gene, coding for 5,10-methylenetetrahydrofolate reductase. These strains bear a null mutation in the chromosomal metF gene and different metF alleles on plasmid(s), and thus there are merozygotes mimicking wild-type homozygotes, heterozygotes and recessive mutant homozygotes. The A177V mutantion in metF corresponds to one of the most common MTHFR polymorphism, A222V, which has been shown to be associated with increased levels of homocysteine in plasma that, in turn, causes many serious medical problems. Results of relatively simple and quick experiments with these strains are compatible with previously published reports on effects of the A222V substitution in the product of MTHFR gene. In addition, these results suggest either impairment of formation of heterodimers and/or heterotetramers by wild-type and A177V metF variants or dominance of the wild-type polypepides in such structures. Moreover, positive effects of folic acid and vitamins B2 and B12 on physiology of the mutant cells, suggested on the basis of clinical studies, is confirmed. Therefore, we conclude that the bacterial model described in this report may be a useful tool in studies on human mutations.     

Teriflunomide – The common drug with underestimated oxygen - Dependent anticancer potential

Leflunomide (LFN) is a well-known immunomodulatory and anti-inflammatory prodrug of teriflunomide (TFN). Due to pyrimidine synthesis inhibition TFN also exhibits potent anticancer effect. Because, there is the strict coupling between the pyrimidine synthesis and the mitochondrial respiratory chain, the oxygen level could modify the cytostatic TNF effect.The aim of the study was to evaluate the cytostatic effect of pharmacologically achievable teriflunomide (TFN) concentrations at physiological oxygen levels, i.e. 1% hypoxia and 10% tissue normoxia compared to 21%oxygen level occurred in routine cell culture environment.The TFN effect was evaluated using TB, MTT and FITC Annexin tests for human primary (SW480) and metastatic (SW620) colon cancer cell lines at various oxygen levels.We demonstrated significant differences between proliferation, survival and apoptosis at 1, 10 and 21% oxygen in primary and metastatic colon cancer cell lines (SW480, SW620) under TFN treatment. The cytostatic TFN effect was more pronounced at hypoxia compared to tissue and atmospheric normoxia in both cancer cell lines, however metastatic cells were more resistant to antiproliferative and proapoptotic TFN action. The early apoptosis was predominant in physiological oxygen tension while in atmospheric normoxia the late apoptosis was induced.Our findings showed that anticancer TFN effect is more strong in physiological oxygen compared to atmospheric normoxia. It suggests that results obtained from in vitro studies could be underestimated. Thus, it gives assumption for future comprehensive studies at real oxygen environment involving TNF use in combination with other antitumor agents affecting oxygen-dependent pyrimidine synthesis.     

Muscle‐derived exophers promote reproductive fitness

Caenorhabditis elegans body wall muscles release vesicles called exophers that transport muscle‐synthesized yolk proteins to nourish the next generation.     

Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2

Numerous p53 missense mutations possess gain-of-function activities. Studies in mouse models have demonstrated that the stabilization of p53 R172H (R175H in human) mutant protein, by currently unknown factors, is a prerequisite for its oncogenic gain-of-function phenotype such as tumour progression and metastasis. Here we show that MDM2-dependent ubiquitination and degradation of p53 R175H mutant protein in mouse embryonic fibroblasts is partially inhibited by increasing concentration of heat shock protein 70 (HSP70/HSPA1-A). These phenomena correlate well with the appearance of HSP70-dependent folding intermediates in the form of dynamic cytoplasmic spots containing aggregate-prone p53 R175H and several molecular chaperones. We propose that a transient but recurrent interaction with HSP70 may lead to an increase in mutant p53 protein half-life. In the presence of MDM2 these pseudoaggregates can form stable amyloid-like structures, which occasionally merge into an aggresome. Interestingly, formation of folding intermediates is not observed in the presence of HSC70/HSPA8, the dominant-negative K71S variant of HSP70 or HSP70 inhibitor. In cancer cells, where endogenous HSP70 levels are already elevated, mutant p53 protein forms nuclear aggregates without the addition of exogenous HSP70. Aggregates containing p53 are also visible under conditions where p53 is partially unfolded: 37°C for temperature-sensitive variant p53 V143A and 42°C for wild-type p53. Refolding kinetics of p53 indicate that HSP70 causes transient exposure of p53 aggregate-prone domain(s). We propose that formation of HSP70- and MDM2-dependent protein coaggregates in tumours with high levels of these two proteins could be one of the mechanisms by which mutant p53 is stabilized. Moreover, sequestration of p73 tumour suppressor protein by these nuclear aggregates may lead to gain-of-function phenotypes.     

A Critical Examination of Escherichia coli Esterase Activity

The ability of Escherichia coli to grow on a series of acetylated and glycosylated compounds has been investigated. It is surmised that E. coli maintains low levels of nonspecific esterase activity. This observation may have ramifications for previous reports that relied on nonspecific esterases from E. coli to genetically encode nonnatural amino acids. It had been reported that nonspecific esterases from E. coli deacetylate tri-acetyl O-linked glycosylated serine and threonine in vivo. The glycosylated amino acids were reported to have been genetically encoded into proteins in response to the amber stop codon. However, it is our contention that such amino acids are not utilized in this manner within E. coli. The current results report in vitro analysis of the original enzyme and an in vivo analysis of a glycosylated amino acid. It is concluded that the amber suppression method with nonnatural amino acids may require a caveat for use in certain instances.The central question addressed in this paper is whether the glycosylated amino acids GlcNAc-Ser and GalNAc-Thr have been genetically encoded into proteins in vivo (1, 2). The reports for the incorporation of these two amino acids are unique from all other reports (3) that have incorporated unnatural amino acids using the recoded UAG codon and Methanococcus jannaschii orthogonal pairs in that these two amino acids required further processing by the host organism before incorporation (see Fig. 1). Here we posit that the primary barrier to their incorporation would appear to be the fact that the host organism used in the original reports, Escherichia coli, maintains very low levels of nonspecific esterase activity. In fact, the original reports used citations from mammalian biology to substantiate the nonspecific esterase mechanism (see below).Open in a separate windowFIGURE 1.Proposed product of an esterase with GlcNAc-Ser and other esterase substrates discussed in this study.E. coli is likely the most thoroughly studied microorganism. This is especially true in regard to carbohydrate and amino acid uptake and utilization (4). Therefore, it should not be surprising that it has long been known that esterified carbon sources are not metabolized by E. coli in standard assays used to probe for microorganism lipase and esterase activity (5). Such results and our current analysis underscore the limitations of the reports that triacetyl O-linked glycosylated amino acids (GlcNAc-Ser and GalNAc-Thr) were deacetylated in E. coli by endogenous “nonspecific” esterases. The deacetylated amino acids were then believed to have been genetically encoded into full-length proteins in vivo (1, 2).In these previous studies the glycosylated amino acids were provided to the growth media as their tetraacetate analogs, and it was construed from the mass spectra and lectin binding assays that the ester groups of the saccharide had all been hydrolyzed. The notion that E. coli rapidly hydrolyzes a simple ester is not easily reconciled with what is commonly observed when the ester functional group is introduced into cultures of E. coli. For example, we were prompted by reports that claimed to have harvested β-hydroxy esters from E. coli (6). There was nothing in such a report to indicate that the E. coli strain used had undergone a drastic genetic modification beyond the introduction of one enzyme derived from yeast. The enzyme from yeast was expressed in E. coli to asymmetrically reduce β-keto esters to the corresponding β-hydroxy esters. The reduction was accomplished in 87% yield and was performed in whole cells. It stands to reason that such a report having claimed to extract significant amounts of an esterified product would not be possible if E. coli maintained even moderate levels of nonspecific esterase activity. The fact that E. coli maintains low levels of endogenous esterases and lipases has been quite pivotal for a number of studies that have used this organism as the host to express esterase genes in vivo (see below).Nonspecific esterase activity is common in eukaryotic organisms, for example, our ability to hydrolyze triacylglycerides to access an important energy source, but this stands in stark contrast to E. coli where it is possible to directly extract O-acetylated oligosaccharides (7) and other simple esters (6) in high yields. These reports are consistent with the observation that UDP-2,3-diacylglucosamine accumulates in E. coli when genes from lipid biosynthesis are deleted (8). E. coli is also the preferred host for evaluating esterase and lipase activity when screening genes from cultured and uncultured organisms (9, 10). Screening for lipase activity from various microorganisms is often performed on tributyrin agar plates (11). The results are typically the same as for triacetin, and it is repeatedly observed that E. coli does not naturally grow on triesters of glycerol (12, 13). These and many other similar esterase screens (14) would not have been feasible if E. coli produced even moderate levels of a lipase or nonspecific esterase.In the present article we use a combination of our current findings and a thorough review of the relevant literature to conclude that E. coli may not maintain sufficient levels of nonspecific esterase activity to permit the in vivo incorporation of the glycosylated amino acids by the mechanism reported (Fig. 1). Our conclusion is further supported by isothermal calorimetry measurements of Zhang et al. (1) original enzyme showing it retains considerable wild-type activity. We also show that the amino acid GlcNAc-Ser appears to be metabolized in E. coli.     

Variation in pollen concentration of the mostallergenic taxa in Poznań (Poland), 1995–1996

The airborne pollen concentration of the four mostfrequent and most allergenic taxa in Poland; Alnus, Betula, Poaceae, and Artemisia atPozna in the years 1995–1996 has been analyzed,using a Hirst-type volumetric spore trap. Theappearance of the earliest pollen producing taxa wasobserved as early as January and February, which isrelevant information for people subject to allergiesin the Pozna region, where Spring usually beginsin March. The periods of high and very high pollenconcentration of individual taxa have been comparedfor the two years.     

Assessment between pollen seasons in areas with different urbanization level related to local vegetation sources and differences in allergen exposure

Pollen data recorded by a single sampler in any given city often fail to reflect particular events occurring in surrounding areas. This is frequently overlooked when interpreting aerobiological results, and therefore pollen data obtained in urban areas may not necessarily be representative of the situation in more rural areas of the same city. Our purpose is to assess differences between allergenic pollen concentrations recorded in an urban area (Eskulap) and a rural/suburban (Morasko) of the same city from 2005 to 2007. Anova-Manova Scheffe, Spearman correlation and Mixed-design ANOVA whitin-subjects effects tests were applied. The results obtained have showed longer pollen seasons, earlier starts of flowering and later conclusion in the urban area. The participation of arboreal pollen in the pollen fall of both sites had enough significative similarities to determine that is linked to regional conditions, while differences in the atmospheric pollen content of herbaceous plants between sites should be due to local conditions.