Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.     

Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus)

Summary Results from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities. These results are consistent with the hypothesis that peptides having significant sequence homology with human neuropeptide Y and porcine neuropeptide Y are present in the nerve fibers that permeate the islet.     

Use of somatic cell nuclear transfer to study meiosis in female cattle carrying a sex-dependent fertility-impairing X-chromosome abnormality

Animal models have played an important part in establishing our knowledge base on reproduction, development, and the occurrence and impact of chromosome abnormalities. Translocations involving the X chromosome and an autosome are unique in that they elicit sex-dependent infertility, with male carriers rendered sterile by synaptic anomalies during meiosis, whereas female carriers conceive but repeatedly abort. Until now the limited access to relevant fetal oocytes has precluded direct study of meiotic events in female carriers. Because somatic cell nuclear transfer (SCNT) circumvents meiotic problems associated with fertility disturbances in translocation carriers, we used SCNT to generate embryos, fetuses, and calves from a cell line derived from a deceased subfertile X-autosome translocation carrier cow to study the meiotic configurations in carrier oocytes. Data from 33 replicates involving 2470 oocyte-donor-cell complexes were assessed for blastocyst development and of these, 42 blastocysts were transferred to 21 recipients. Fourteen pregnancies were detected on day 35 of gestation. One of these was sacrificed for ovary retrieval on day 94 and three went to term. Features of oocytes from the fetal ovary and from the newborn ovaries were examined. Of the pachytene spreads analyzed, 16%, 82%, and 1.5% exhibited quadrivalent, trivalent/univalent, and bivalent/univalent/univalent structures, respectively, whereas among the diakinesis/metaphase I spreads, 16% ring, 75% chain, and 8.3% bivalent/bivalent configurations were noted, suggesting that the low fertility among female carriers may be related to synaptic errors in a predominant proportion of oocytes. Our results indicate that fibroblasts carrying the X-autosome translocation can be used for SCNT to produce embryos, fetuses, and newborn clones to study such basic aspects of development as meiosis and to generate carriers that cannot easily be reproduced by conventional breeding.     

Biomarker discovery for arsenic exposure using functional data. Analysis and feature learning of mass spectrometry proteomic data

Plasma biomarkers of exposure to environmental contaminants play an important role in early detection of disease. The emerging field of proteomics presents an attractive opportunity for candidate biomarker discovery, as it simultaneously measures and analyzes a large number of proteins. This article presents a case study for measuring arsenic concentrations in a population residing in an As-endemic region of Bangladesh using plasma protein expressions measured by SELDI-TOF mass spectrometry. We analyze the data using a unified statistical method based on functional learning to preprocess mass spectra and extract mass spectrometry (MS) features and to associate the selected MS features with arsenic exposure measurements. The task is challenging due to several factors, the high dimensionality of mass spectrometry data, complicated error structures, and a multiple comparison problem. We use nonparametric functional regression techniques for MS modeling, peak detection based on the significant zero-downcrossing method, and peak alignment using a warping algorithm. Our results show significant associations of arsenic exposure to either under- or overexpressions of 20 proteins.     

Condensing and fluidizing effects of ganglioside GM1 on phospholipid films

Mixed monolayers of the ganglioside G_M1 and the lipid dipalmitoylphosphatidlycholine (DPPC) at air-water and solid-air interfaces were investigated using various biophysical techniques to ascertain the location and phase behavior of the ganglioside molecules in a mixed membrane. The effects induced by G_M1 on the mean molecular area of the binary mixtures and the phase behavior of DPPC were followed for G_M1 concentrations ranging from 5 to 70 mol %. Surface pressure isotherms and fluorescence microscopy imaging of domain formation indicate that at low concentrations of G_M1 (<25 mol %), the monolayer becomes continually more condensed than DPPC upon further addition of ganglioside. At higher G_M1 concentrations (>25 mol %), the mixed monolayer becomes more expanded or fluid-like. After deposition onto a solid substrate, atomic force microscopy imaging of these lipid monolayers showed that G_M1 and DPPC pack cooperatively in the condensed phase domain to form geometrically packed complexes that are more ordered than either individual component as evidenced by a more extended total height of the complex arising from a well-packed hydrocarbon tail region. Grazing incidence x-ray diffraction on the DPPC/G_M1 binary mixture provides evidence that ordering can emerge when two otherwise fluid components are mixed together. The addition of G_M1 to DPPC gives rise to a unit cell that differs from that of a pure DPPC monolayer. To determine the region of the G_M1 molecule that interacts with the DPPC molecule and causes condensation and subsequent expansion of the monolayer, surface pressure isotherms were obtained with molecules modeling the backbone or headgroup portions of the G_M1 molecule. The observed concentration-dependent condensing and fluidizing effects are specific to the rigid, sugar headgroup portion of the G_M1 molecule.     

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

Introduction

The possible role of UCP2 in modulating mitochondrial Ca²⁺-uptake (mCa²⁺-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.

Methods

Thus, we analyzed mCa²⁺-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca²⁺-transients from mitochondrial ((Ca²⁺)m) and intracellular compartment ((Ca²⁺)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2^-/- mice.

Results

Isolated mitochondria showed a Ru360 sensitive mCa²⁺-uptake, which was significantly decreased in UCP2^-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca²⁺-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2^-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2^-/- cardiomyocytes (Ca²⁺)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca²⁺)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca²⁺-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa²⁺-uptake in WT mitoplasts and (Ca²⁺)m of cardiomyocytes leading to an increase of (Ca²⁺)c while no ATP dependent effect was observed in UCP2^-/-.

Conclusion

Our results indicate regulatory effects of UCP2 on mCa²⁺-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.     

Life cycle thinking in small and medium enterprises: the results of research on the implementation of life cycle tools in Polish SMEs—Part 3: LCC-related aspects

Purpose

This article is the third of a series of articles presenting the results of research on the implementation of life cycle management tools in small- and medium-sized companies in Poland. The purpose of the three-part series of articles is to present the results of research on the implementation of life cycle tools in Polish small and medium enterprises (SMEs). This work is part of a project financed by the Polish Agency for Enterprise Development (PAED) which began in February 2011. It was carried out by the Wielkopolska Quality Institute—a business environment institution associated with the Polish Centre for LCA (PCLCA). The main practical objective of the project was to support SMEs in their business development, e.g. by expanding their horizons beyond the sphere of their operation and identifying new areas for the improvement and promotion of the products and services on offer. The specific objective of the analysis involving the assessment of life-cycle costs of products and services was an attempt to answer the question to determine whether the assessment carried out in accordance with the life-cycle cost (LCC) methodology is a good tool for cost management in this type of business. Part 3 describes the results of studies on the assessment of the implementation of LCC in SMEs conducted in 50 companies involved in the project.

Methods

In order to assess the effectiveness of the project and the effectiveness of the implementation of LCA and LCC, a survey was conducted of small- and medium-sized businesses where the implementation works had been fully completed. In total, 50 organisations agreed to participate in the LCC survey (while 46 in the LCA—part 2 paper), which was 71 % of all the companies where the LCA and LCC studies had been carried out within the project. The survey was conducted using individual in-depth interviews. Questions to the representatives of the companies referred both to aspects of their operating in the market (characteristics of a company, its market share, management systems, environmental policy, suppliers, clients) and the implementation of their environmental service (assessment of its effectiveness, motivation, difficulties in its implementation), as well as opinions on the potential applications of LCA in their current operations.

Results and discussion

The experience and observations of LCC experts resulting from their cooperation with the analysed organisations are largely supported by the results of the survey. The overall impression gained from the project is that the small- and medium-sized enterprises considered have a problem with accepting and understanding the life-cycle perspective, and show limited interest in taking liability for environmental and cost aspects beyond the mandatory legal standards and boundaries of their business operations. Nevertheless, the LCC analyses aroused much bigger interest among the companies than the environmental due to the fact that the cost aspects in companies undergoing normal development are seen as an important source of information about the structure of the costs generated with respect to the products or services provided. It is important to note that a very important factor encouraging businesses to join the studies was the fact that they were cost-free. Moreover, the planned introduction of a new product onto the market was the argument that often influenced the decision to implement the LCC. The survey has shown that companies rarely perform cost analyses including all stages of the life cycle of a product or service. Although the awareness of the importance of conducting economic researches for the entire life cycle of a product or service is great, it turned out to be problematic to unambiguously define the practical use of such an analysis, at least at the present stage of development of the companies surveyed.

Conclusions

The results obtained in the survey indicate that in the case of simple products, with a short life cycle, complex cost analyses may seem less useful. For more complex products or services, with long periods of use, high reliability required, and high operating costs, the analyses presented are useful tools that increase the economic efficiency of the projects implemented. It appears that from the point of view of polish SMEs, the usefulness of an LCA is seen mainly from the angle of opportunities for cost reduction (preferably in business) and increased sales (marketing). A good solution would be to conduct relatively simple, but integrated LCA/LCC analyses in SMEs so that the companies would clearly see the economic effects of the proposed environmental improvements.     

RNase HI Is Essential for Survival of Mycobacterium smegmatis

RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleotides are present. Here we investigated the importance of RNase H type I encoding genes for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis genome without affecting the growth rate of the mutant. Further, we demonstrate that deletion of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Finally, we question the possibility of existence of RNase HI related alternative mode of initiation of DNA replication in M. smegmatis, the process initially discovered in Escherichia coli. We suspect that synthetic lethality of double mutant lacking RNases H type I is caused by formation of R-loops leading to collapse of replication forks. We report Mycobacterium smegmatis as the first bacterial species, where function of RNase H type I has been found essential.     

Coiled‐coil length: Size does matter

Protein evolution is governed by processes that alter primary sequence but also the length of proteins. Protein length may change in different ways, but insertions, deletions and duplications are the most common. An optimal protein size is a trade‐off between sequence extension, which may change protein stability or lead to acquisition of a new function, and shrinkage that decreases metabolic cost of protein synthesis. Despite the general tendency for length conservation across orthologous proteins, the propensity to accept insertions and deletions is heterogeneous along the sequence. For example, protein regions rich in repetitive peptide motifs are well known to extensively vary their length across species. Here, we analyze length conservation of coiled‐coils, domains formed by an ubiquitous, repetitive peptide motif present in all domains of life, that frequently plays a structural role in the cell. We observed that, despite the repetitive nature, the length of coiled‐coil domains is generally highly conserved throughout the tree of life, even when the remaining parts of the protein change, including globular domains. Length conservation is independent of primary amino acid sequence variation, and represents a conservation of domain physical size. This suggests that the conservation of domain size is due to functional constraints. Proteins 2015; 83:2162–2169. © 2015 Wiley Periodicals, Inc.