Design of antimicrobial peptide arenicin analogs with improved therapeutic indices

β‐Hairpin antimicrobial peptides are among the most potent peptide antibiotics of animal origin. Arenicins, isolated earlier from marine polychaeta lugworm Arenicola marina, belong to a family of β‐hairpin antimicrobial peptides and display a broad spectrum of biological activities. However, despite being potent antimicrobials, arenicins are partially unapplicable as therapeutics as a result of their relatively high cytotoxicity against mammalian cells. In this study, a template‐based approach was used to create therapeutically valuable analogs of arenicin‐1 and identify amino acid residues important for antibacterial and cytotoxic activities of the peptide. The plasmids encoding recombinant analogs were constructed by mutagenesis technique based on inverse PCR amplification of the whole arenicin‐1 expression plasmid. The analogs were produced as a part of the fusion proteins in Escherichia coli. It was shown that an obvious reduction in hemolytic activity without lose of antimicrobial activity can be achieved by a single amino acid substitution in the non‐polar face of the molecule with hydrophilic residues such as serine and arginine. As the result, the selective analog with 50‐fold improved therapeutic index was developed. The circular dichroism spectra demonstrated that the secondary structure of the analog was similar to the natural arenicin‐1 in water solution and sodium dodecyl sulfate micelles but significantly differed in the presence of dodecylphosphocholine micelles mimicking mammalian membranes. Similarly to arenicin‐1, the designed analog killed bacteria via induction of the membrane damage, assessed using the fluorescent dye SYTOX Green uptake. Our results afford molecular insight into mechanism of antimicrobial action of the designed arenicin analogs and their possible clinical application. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Mutations in PNKP Cause Recessive Ataxia with Oculomotor Apraxia Type 4

Hereditary autosomal-recessive cerebellar ataxias are a genetically and clinically heterogeneous group of disorders. We used homozygosity mapping and exome sequencing to study a cohort of nine Portuguese families who were identified during a nationwide, population-based, systematic survey as displaying a consistent phenotype of recessive ataxia with oculomotor apraxia (AOA). The integration of data from these analyses led to the identification of the same homozygous PNKP (polynucleotide kinase 3′-phosphatase) mutation, c.1123G>T (p.Gly375Trp), in three of the studied families. When analyzing this particular gene in the exome sequencing data from the remaining cohort, we identified homozygous or compound-heterozygous mutations in five other families. PNKP is a dual-function enzyme with a key role in different pathways of DNA-damage repair. Mutations in this gene have previously been associated with an autosomal-recessive syndrome characterized by microcephaly; early-onset, intractable seizures; and developmental delay (MCSZ). The finding of PNKP mutations associated with recessive AOA extends the phenotype associated with this gene and identifies a fourth locus that causes AOA. These data confirm that MCSZ and some forms of ataxia share etiological features, most likely reflecting the role of PNKP in DNA-repair mechanisms.     

Differentiation of Human Umbilical Cord Matrix Mesenchymal Stem Cells into Neural-Like Progenitor Cells and Maturation into an Oligodendroglial-Like Lineage

Mesenchymal stem cells (MSCs) are viewed as safe, readily available and promising adult stem cells, which are currently used in several clinical trials. Additionally, their soluble-factor secretion and multi-lineage differentiation capacities place MSCs in the forefront of stem cell types with expected near-future clinical applications. In the present work MSCs were isolated from the umbilical cord matrix (Wharton''s jelly) of human umbilical cord samples. The cells were thoroughly characterized and confirmed as bona-fide MSCs, presenting in vitro low generation time, high proliferative and colony-forming unit-fibroblast (CFU-F) capacity, typical MSC immunophenotype and osteogenic, chondrogenic and adipogenic differentiation capacity. The cells were additionally subjected to an oligodendroglial-oriented step-wise differentiation protocol in order to test their neural- and oligodendroglial-like differentiation capacity. The results confirmed the neural-like plasticity of MSCs, and suggested that the cells presented an oligodendroglial-like phenotype throughout the differentiation protocol, in several aspects sharing characteristics common to those of bona-fide oligodendrocyte precursor cells and differentiated oligodendrocytes.     

HIM1, a new yeast Saccharomyces cerevisiae gene playing a role in control of spontaneous and induced mutagenesis

We have identified a new Saccharomyces cerevisiae gene, HIM1, mapped on the right arm of the chromosome IV (ORF YDR317w), mutations in which led to an increase in spontaneous mutation rate and elevated the frequencies of mutations, induced by UV-light, nitrous acid, ethylmethane sulfonate and methylmethane sulfonate. At the same time, him1 mutation did not result in the increase of the sensitivity to the lethal action of these DNA-damaging agents. We tested the induced mutagenesis in double mutants carrying him1 mutation and mutations in other repair genes: apn1, blocking base excision repair; rad2, rev3, and rad54, blocking three principal DNA repair pathways; pms1, blocking mismatch repair; hsm2 and hsm3 mutations, which lead to a mutator effect. Epistatic analysis showed a synergistic interaction of him1 with pms1, apn1, and rad2 mutations, and epistasis with the rev3, the rad54, the hsm2, and the hsm3. To elucidate the role of the HIM1 in control of spontaneous mutagenesis, we checked the repair of DNA mispaired bases in the him1 mutant and discovered that it was not altered in comparison to the wild-type strain. In our opinion, our results suggest that HIM1 gene participates in the control of processing of mutational intermediates appearing during error-prone bypass of DNA damage.     

Phytomass change in natural phytocenosis as an indicator of technogenic pollution of soils with heavy metals

This study considered the possibility of using plant community phytomass for the assessment of soil pollution with heavy metals (HM) from industrial wastes. The three-year-long field experiment was run under the regional natural meadow vegetation; the polymetallic galvanic slime was used as an industrial waste contaminant. It is shown that soil contamination primarily causes decrease of phytomass in the growing phytocenosis. The vegetation experiments determined nonlinear dependence of cultivated and wild plant biomass on the level of soil contamination; it is described by the equations of logistic and Gaussian regression. In the absence of permanent contaminants, the soil is self-cleaned over time. It reproduces phytomass mainly due to the productivity increase of the most pollution-tolerant species in the remaining phytocenosis. This phenomenon is defined as environmental hysteresis. Soil pollution by industrial waste leads to the loss of plant biodiversity. The research shows that the study of the HM impact on ecosystems is expedient given the consideration of the “soil–phytocenosis–pollutant” complex in the “dose–response” aspect. The reaction of phytocenosis on HM showing decline in phytomass leads to serious limitations in the choice of accumulating plants, because the adsorbed HM are rejected through phytomass.     

The human spindle assembly checkpoint protein Bub3 is required for the establishment of efficient kinetochore-microtubule attachments

The spindle assembly checkpoint monitors the status of kinetochore-microtubule (K-MT) attachments and delays anaphase onset until full metaphase alignment is achieved. Recently, the role of spindle assembly checkpoint proteins was expanded with the discovery that BubR1 and Bub1 are implicated in the regulation of K-MT attachments. One unsolved question is whether Bub3, known to form cell cycle constitutive complexes with both BubR1 and Bub1, is also required for proper chromosome-to-spindle attachments. Using RNA interference and high-resolution microscopy, we analyzed K-MT interactions in Bub3-depleted cells and compared them to those in Bub1- or BubR1-depleted cells. We found that Bub3 is essential for the establishment of correct K-MT attachments. In contrast to BubR1 depletion, which severely compromises chromosome attachment and alignment, we found Bub3 and Bub1 depletions to produce defective K-MT attachments that, however, still account for significant chromosome congression. After Aurora B inhibition, alignment defects become severer in Bub3- and Bub1-depleted cells, while partially rescued in BubR1-depleted cells, suggesting that Bub3 and Bub1 depletions perturb K-MT attachments distinctly from BubR1. Interestingly, misaligned chromosomes in Bub3- and Bub1-depleted cells were found to be predominantly bound in a side-on configuration. We propose that Bub3 promotes the formation of stable end-on bipolar attachments.     

Recent decrease in chinstrap penguin (<Emphasis Type="Italic">Pygoscelis antarctica</Emphasis>) populations at two of Admiralty Bay’s islets on King George Island,South Shetland Islands,Antarctica

We examined the breeding populations of chinstrap penguins (Pygoscelis antarctica) on Chabrier Rock and Shag Island within Admiralty Bay, King George Island, South Shetland Islands, Antarctica from 2002 to 2004. When comparing our results to historic data from 1979, we found an overall decline of 57% in the last 25 years, mirroring the population trend of this species in other regions of the Antarctic Peninsula. Our results are discussed in relation to factors hypothesized to be driving the declines found at other sites, as well as the importance of consistent annual censuses to accurately determine population trends.     

Molecular identification of mammalian phosphopentomutase and glucose-1,6-bisphosphate synthase, two members of the alpha-D-phosphohexomutase family

The molecular identity of mammalian phosphopentomutase has not yet been established unequivocally. That of glucose-1,6-bisphosphate synthase, the enzyme that synthesizes a cofactor for phosphomutases and putative regulator of glycolysis, is completely unknown. In the present work, we have purified phosphopentomutase from human erythrocytes and found it to copurify with a 68-kDa polypeptide that was identified by mass spectrometry as phosphoglucomutase 2 (PGM2), a protein of the alpha-d-phosphohexomutase family and sharing about 20% identity with mammalian phosphoglucomutase 1. Data base searches indicated that vertebrate genomes contained, in addition to PGM2, a homologue (PGM2L1, for PGM2-like 1) sharing about 60% sequence identity with this protein. Both PGM2 and PGM2L1 were overexpressed in Escherichia coli, purified, and their properties were studied. Using catalytic efficiency as a criterion, PGM2 acted more than 10-fold better as a phosphopentomutase (both on deoxyribose 1-phosphate and on ribose 1-phosphate) than as a phosphoglucomutase. PGM2L1 showed only low (<5%) phosphopentomutase and phosphoglucomutase activities compared with PGM2, but was about 5-20-fold better than the latter enzyme in catalyzing the 1,3-bisphosphoglycerate-dependent synthesis of glucose 1,6-bisphosphate and other aldose-bisphosphates. Furthermore, quantitative real-time PCR analysis indicated that PGM2L1 was mainly expressed in brain where glucose-1,6-bisphosphate synthase activity was previously shown to be particularly high. We conclude that mammalian phosphopentomutase and glucose-1,6-bisphosphate synthase correspond to two closely related proteins, PGM2 and PGM2L1, encoded by two genes that separated early in vertebrate evolution.