Quantitative Comparison of HTLV-1 and HIV-1 Cell-to-Cell Infection with New Replication Dependent Vectors

We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.     

A Local Role for the Small Ribosomal Subunit Primary Binder rpS5 in Final 18S rRNA Processing in Yeast

In vivo depletion of the yeast small ribosomal subunit (SSU) protein S5 (rpS5) leads to nuclear degradation of nascent SSUs and to a perturbed global assembly state of the SSU head domain. Here, we report that rpS5 plays an additional local role at the head/platform interface in efficient SSU maturation. We find that yeast small ribosomal subunits which incorporated an rpS5 variant lacking the seven C-terminal amino acids have a largely assembled head domain and are exported to the cytoplasm. On the other hand, 3′ processing of 18S rRNA precursors is inhibited in these ribosomal particles, although they associate with the putative endonuclease Nob1p and other late acting 40S biogenesis factors. We suggest that the SSU head component rpS5 and platform components as rpS14 are crucial constituents of a highly defined spatial arrangement in the head – platform interface of nascent SSUs, which is required for efficient processing of the therein predicted SSU rRNA 3′ end. Positioning of rpS5 in nascent SSUs, including its relative orientation towards platform components in the head-platform cleft, will depend on the general assembly and folding state of the head domain. Therefore, the suggested model can explain 18S precursor rRNA 3′ processing phenotypes observed in many eukaryotic SSU head assembly mutants.     

Heterologous ferredoxin reductase and flavodoxin protect Cos-7 cells from oxidative stress

Background

Ferredoxin-NADP(H) reductase (FNR) from Pisum sativum and Flavodoxin (Fld) from Anabaena PCC 7119 have been reported to protect a variety of cells and organisms from oxidative insults. In this work, these two proteins were expressed in mitochondria of Cos-7 cells and tested for their efficacy to protect these cells from oxidative stress in vitro.

Principal Findings

Cos-7/pFNR and Cos-7/pFld cell lines expressing FNR and Fld, respectively, showed a significantly higher resistance to 24 h exposure to 300–600 µM hydrogen peroxide measured by LDH retention, MTT reduction, malondialdehyde (MDA) levels and lipid peroxide (LPO; FOX assay) levels. However, FNR and Fld did not exhibit any protection at shorter incubation times (2 h and 4 h) to 4 mM hydrogen peroxide or to a 48 h exposure to 300 µM methyl viologen. We found enhanced methyl viologen damage exerted by FNR that may be due to depletion of NADPH pools through NADPH-MV diaphorase activity as previously observed for other overexpressed enzymes.

Significance

The results presented are a first report of antioxidant function of these heterologous enzymes of vegetal and cyanobacterial origin in mammalian cells.     

Immunohistochemical evidences showing the presence of thymulin containing cells located in involuted thymus and in peripheral lymphoid organs

Thymulin is a well-characterized thymic hormone that exists as a nonapeptide coupled to equimolar amounts of Zn2+. Thymulin is known to have multiple biological roles, including T cell differentiation, immune regulation, and analgesic functions. It has been shown that thymulin is produced by the reticulo-epithelial cells of the thymus, and it circulates in the blood from the moment of birth, maintain its serum level until puberty diminishing thereafter in life. To study the localization of this hormone, we prepared polyclonal and monoclonal antibodies against the commercial peptide and utilized immunocytochemical techniques for visualization. The results indicate that thymulin stains the thymic reticular cells, the outer layers of Hassall's corpuscles and a large round cellular type, which is keratin-negative and does not show affinity for the common leukocyte antigen (CD-45). In mice, this thymulin-positive cell remains in the thymus throughout life and even appears in relatively increased numbers in old involuted thymi. It also appears in thymus-dependent areas of the spleen and lymph nodes, demonstrating that at least one of the thymus cells containing this peptide can be found in peripheral lymphoid tissue.     

Abundance of type I toxin–antitoxin systems in bacteria: searches for new candidates and discovery of novel families

Small, hydrophobic proteins whose synthesis is repressed by small RNAs (sRNAs), denoted type I toxin–antitoxin modules, were first discovered on plasmids where they regulate plasmid stability, but were subsequently found on a few bacterial chromosomes. We used exhaustive PSI-BLAST and TBLASTN searches across 774 bacterial genomes to identify homologs of known type I toxins. These searches substantially expanded the collection of predicted type I toxins, revealed homology of the Ldr and Fst toxins, and suggested that type I toxin–antitoxin loci are not spread by horizontal gene transfer. To discover novel type I toxin–antitoxin systems, we developed a set of search parameters based on characteristics of known loci including the presence of tandem repeats and clusters of charged and bulky amino acids at the C-termini of short proteins containing predicted transmembrane regions. We detected sRNAs for three predicted toxins from enterohemorrhagic Escherichia coli and Bacillus subtilis, and showed that two of the respective proteins indeed are toxic when overexpressed. We also demonstrated that the local free-energy minima of RNA folding can be used to detect the positions of the sRNA genes. Our results suggest that type I toxin–antitoxin modules are much more widely distributed among bacteria than previously appreciated.     

Kalirin: a novel genetic risk factor for ischemic stroke

Cerebrovascular and cardiovascular diseases are the leading causes of death and disability worldwide. They are complex disorders resulting from the interplay of genetic and environmental factors, and may share several susceptibility genes. Several recent studies have implicated variants of the Kalirin (KALRN) gene with susceptibility to cardiovascular and metabolic phenotypes, but no studies have yet been performed in stroke patients. KALRN is involved, among others, in the inhibition of inducible nitric oxide synthase, in the regulation of ischemic signal transduction, and in neuronal morphogenesis, plasticity, and stability. The goal of the present study was to determine whether SNPs in the KALRN region on 3q13, which includes the Ropporin gene (ROPN1), predispose to ischemic stroke (IS) in a cohort of Portuguese patients and controls. We genotyped 34 tagging SNPs in the KALRN and ROPN1 chromosomal region on 565 IS patients and 517 unrelated controls, and performed genotype imputation for 405 markers on chromosome 3. We tested the single-marker association of these SNPs with IS. One SNP (rs4499545) in the ROPN1–KALRN intergenic region and two SNPs in KALRN (rs17286604 and rs11712619) showed significant (P < 0.05) allelic and genotypic (unadjusted and adjusted for hypertension, diabetes, and ever smoking) association with IS risk. Thirty-two imputed SNPs also showed an association at P < 0.05, and actual genotyping of three of these polymorphisms (rs7620580, rs6438833, and rs11712039) validated their association. Furthermore, rs11712039 was associated with IS (0.001 < P < 0.01) in a recent well-powered genomewide association study (Ikram et al. 2009). These studies suggest that variants in the KALRN gene region constitute risk factors for stroke and that KALRN may represent a common risk factor for vascular diseases.     

A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker

Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools. Therefore, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows rapid curing of an Escherichia coli-A. pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A. pleuropneumoniae chromosome. A cassette containing the Tn903 kanamycin resistance determinant (km(r)) and the sacB gene expressed from the A. pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A. pleuropneumoniae. The resultant stable plasmid cointegrates were kanamycin-resistant, sucrose-sensitive, and urease-positive. A simple counterselection on sucrose-containing agar plates without an additional transconjugation step allowed the efficient isolation of urease-negative A. pleuropneumoniae mutants that had lost the km(r)-sacB cassette.     

The characterization of complex continuous norms of reaction

Recent focus on the array of phenotypes expressed under differing environmental conditions, or phenotypic plasticity, has led to increased understanding of its genetic basis as well as its adaptive significance. However, the quantification of plasticity has proven difficult, hampered by both the limited number of environments over which plasticity may typically be assessed and by the need to assume, a priori, the general form of reaction norms under study. Our understanding of the shapes of continuous norms of reaction and, consequently, the subtle differences that may exist in shapes among genotypes or populations is rudimentary. Here, we propose the use of the loess smoothing function to analyze complex norms of reaction and to quantify total plasticity over many environments. A thermogradient incubator offers an ideal means to provide many environments for a demonstration of the use of the loess method. We test seed germination in three populations of two monocarpic plant species for population differentiation in plasticity to temperature. First, we test for differentiation in norms of reaction to 30 temperature environments among three populations of the monocarpic perennial, Lobelia inflata . The second demonstration assesses plasticity to eight temperature environments of three populations of the arctic-alpine annual, Koenigia islandica . Our demonstration shows that the loess technique can detect significant genetic differentiation among populations in complex norms of reaction for both species studied, and suggests that the use of this procedure should be considered where the form of norms of reaction might be complex. The general applicability of the approach is discussed.     

Delusions of grandeur: Seed count is not a good fitness proxy under individual variation in phenology

The concept of fitness is central to evolutionary biology, yet it is difficult to define and to measure. In plant biology, fitness is often measured as seed count. However, under an array of circumstances, seed count may be a biased proxy of fitness, for example when individuals vary in allocation to sexual versus asexual reproduction. A more subtle example, but also likely to be important in natural populations, is when interindividual variation in conditions during development results in variation in offspring quality among seed parents. In monocarpic (semelparous) plants, this is expected to result from variation in effective season length experienced among individuals that reach reproductive maturity at different times. Here, we manipulate growing season length to ask whether seed count is an accurate representation of parental fitness in the monocarpic herb Lobelia inflata. Simple seed count suggests a paradoxical fitness advantage under constrained‐season length. However, we find that the apparent fitness advantage of a constrained‐season length is overridden by low relative per‐seed fitness. Furthermore, the fitness deficit in the constrained environment is associated primarily with an accelerating decrease in viability and seedling survival in seeds derived from fruits produced progressively later in the season. In this study, the overall fitness value of a seed under a constrained season is 0.774 of that observed under a long season.     

Na + /H + exchanger isoform 1 activity in AQP2-expressing cells can be either proliferative or anti-proliferative depending on extracellular pH

We have previously shown in renal cells that expression of the water channel Aquaporin-2 increases cell proliferation by a regulatory volume mechanism involving Na+/H+ exchanger isoform 2. Here, we investigated if Aquaporin-2 (AQP2) also modulates Na+/H+ exchanger isoform 1-dependent cell proliferation. We use two AQP2-expressing cortical collecting duct models: one constitutive (WT or AQP2-transfected RCCD1 cell line) and one inducible (control or vasopressin-induced mpkCCDc14 cell line). We found that Aquaporin-2 modifies Na+/H+ exchanger isoform 1 (NHE1) contribution to cell proliferation. In Aquaporin-2-expressing cells, Na+/H+ exchanger isoform 1 is anti-proliferative at physiological pH. In acid media, Na+/H+ exchanger isoform 1 contribution turned from anti-proliferative to proliferative only in AQP2-expressing cells. We also found that, in AQP2-expressing cells, NHE1-dependent proliferation changes parallel changes in stress fiber levels: at pH 7.4, Na+/H+ exchanger isoform 1 would favor stress fiber disassembly and, under acidosis, NHE1 would favor stress fiber assembly. Moreover, we found that Na+/H+ exchanger-dependent effects on proliferation linked to Aquaporin-2 relied on Transient Receptor Potential Subfamily V calcium channel activity. In conclusion, our data show that, in collecting duct cells, the water channel Aquaporin-2 modulates NHE1-dependent cell proliferation. In AQP2-expressing cells, at physiological pH, the Na+/H+ exchanger isoform 1 function is anti-proliferative and, at acidic pH, Na+/H+ exchanger isoform 1 function is proliferative. We propose that Na+/H+ exchanger isoform 1 modulates proliferation through an interplay with stress fiber formation.