Memory T(H)2 cells induce alternatively activated macrophages to mediate protection against nematode parasites

Although primary and memory responses against bacteria and viruses have been studied extensively, T helper type 2 (T(H)2) effector mechanisms leading to host protection against helminthic parasites remain elusive. Examination of the intestinal epithelial submucosa of mice after primary and secondary infections by a natural gastrointestinal parasite revealed a distinct immune-cell infiltrate after challenge, featuring interleukin-4-expressing memory CD4(+) T cells that induced IL-4 receptor(hi) (IL-4R(hi)) CD206(+) alternatively activated macrophages. In turn, these alternatively activated macrophages (AAMacs) functioned as important effector cells of the protective memory response contributing to parasite elimination, demonstrating a previously unknown mechanism for host protection against intestinal helminths.     

Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p)

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 μM) and thonzonium bromide (EC(50) = 69 μM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 μM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.     

Numerical Modeling of Bi-polar (AC) Pulse Electroporation of Single Cell in Microchannel to Create Nanopores on its Membrane

AC electroporation of a single cell in a microchannel was numerically studied. A \(15\,\upmu\) m diameter cell was considered in a microchannel \(25\,\upmu\) m in height and the influences of AC electric pulse on its membrane were numerically investigated. The cell was assumed to be suspended between two electroporative electrodes embedded on the walls of a microchannel. An amplitude and a time span of applied electric pulse were chosen to be 80 kV/m and \(10\,\upmu\) s, respectively. For different frequency values (50, 100, 200, and 500 kHz), simulations were performed to show how the cell membrane was electroporated and the creation of nanopores. Obtained numerical results show that the most and the largest nanopores are created around poles of cell (nearest points of cell membrane to the electrodes). The numerical simulations also demonstrate that increased frequency will slightly decrease electroporated area of the cell membrane; additionally, growth of the created nanopores will be stabilized. It has also been proven that size and number of the created nanopores will be decreased by moving from the poles to the equator of the cell. There is almost no nanopore created in the vicinity of the equator. Frequency affects the rate of generation of nanopores. In case of AC electroporation, creation of nanopores has two phases that periodically repeat over time. In each period, the pore density sharply increases and then becomes constant. Enhancement of the frequency will result in decrease in time span of the periods. In each period, size of the created nanopores sharply increases and then slightly decreases. However, until the AC electric pulse is present, overall trends of creation and development of nanopores will be ascending. Variation of the size and number of created nanopores can be explained by considering time variation of transmembrane potential (difference of electric potential on two sides of cell membrane) which is clear in the results presented in this study.     

Mycoplasma orale infection affects K+ and Cl− currents in the HSG salivary gland cell line

Summary The relations between K⁺ channel and Cl⁻ channel currents and mycoplasma infection status were studied longitudinally in HSG cells, a human submandibular gland cell line. The K⁺ channel currents were disrupted by the occurrence of mycoplasma infection: muscarinic activation of K⁺ channels and K⁺ channel expression as estimated by ionomycin- or hypotonically induced K⁺ current responses were all decreased. Similar decreases in ionomycin- and hypotonically induced responses were observed for Cl⁻ channels, but only the latter decrease was statistically significant. Also, Cl⁻ currents could be elicited more frequently than K⁺ currents (63% of cases versus 0%) in infected cells when tested by exposure to hypotonic media, indicating that mycoplasma infection affects K⁺ channels relatively more than Cl⁻ channels. These changes occurred in the originally infected cells, were ameliorated when the infection was cleared with sparfloxacin, and recurred when the cells were reinfected. Such changes would be expected to result in hyposecretion of salivary fluid if they occurredin vivo.     

Genomic Structure of the Human Gene for Spinocerebellar Ataxia Type 2 (SCA2) on Chromosome 12q24.1

Spinocerebellar ataxia type 2 (SCA2) is a member of a group of neurodegenerative diseases that are caused by instability of a DNA CAG repeat. We report the genomic structure of theSCA2gene. Its 25 exons, encompassing approximately 130 kb of genomic DNA, were mapped onto the physical map of the region. Exonic sizes varied from 37 to 890 bp, and intronic sizes ranged from 323 bp to more than 15 kb. The CAG repeat was contained in the 5′ coding region of the gene in exon 1. Determination of the splice junction sequences indicated the presence of only one deviation from the GT-AG rule at the donor splice site of intron 9, which contained a GC instead of a GT dinucleotide. Exon 10, immediately downstream from this rare splice donor site, was alternatively spliced. Alternative splicing does not affect the reading frame and is predicted to encode an isoform containing 70 amino acids less.     

Identification of mutations that decrease the stability of a fragment of Saccharomyces cerevisiae chromosome III lacking efficient replicators

Eukaryotic chromosomes are duplicated during S phase and transmitted to progeny during mitosis with high fidelity. Chromosome duplication is controlled at the level of replication initiation, which occurs at cis-acting replicator sequences that are spaced at intervals of approximately 40 kb along the chromosomes of the budding yeast Saccharomyces cerevisiae. Surprisingly, we found that derivatives of yeast chromosome III that lack known replicators were replicated and segregated properly in at least 96% of cell divisions. To gain insight into the mechanisms that maintain these "originless" chromosome fragments, we screened for mutants defective in the maintenance of an "originless" chromosome fragment, but proficient in the maintenance of the same fragment that carries its normal complement of replicators (originless fragment maintenance mutants, or ofm). We show that three of these Ofm mutations appear to disrupt different processes involved in chromosome transmission. The OFM1-1 mutant seems to disrupt an alternative initiation mechanism, and the ofm6 mutant appears to be defective in replication fork progression. ofm14 is an allele of RAD9, which is required for the activation of the DNA damage checkpoint, suggesting that this checkpoint plays a key role in the maintenance of the "originless" fragment.