Use of exogenous hTERT to immortalize primary human cells

A major obstacle to the immortalization of primary human cells and the establishment of human cell lines is telomere-controlled senescence. Telomere-controlled senescence is caused by the shortening of telomeres that occurs each time somatic human cells divide. The enzyme telomerase can prevent the erosion of telomeres and block the onset of telomere-controlled senescence, but its expression is restricted to the early stages of embryonic development, and in the adult, to rare cells of the blood, skin and digestive track. However, we and others have shown that the transfer of an exogenous hTERT cDNA, encoding the catalytic subunit of human telomerase, can be used to prevent telomere shortening, overcome telomere-controlled senescence, and immortalize primary human cells. Most importantly, hTERT alone can immortalize cells without causing cancer-associated changes or altering phenotypic properties. Primary human cells that have so far been established by the forced expression of hTERT alone include fibroblasts, retinal pigmented epithelial cells, endothelial cells, oesophageal squamous cells, mammary epithelial cells, keratinocytes, osteoblasts, and Nestin-positive cells of the pancreas. In this article, we discuss the use of hTERT to immortalize of human cells, the properties of hTERT-immortalized cells, and their applications to cancer research and tissue engineering.     

Selection of Specific Endophytic Bacterial Genotypes by Plants in Response to Soil Contamination

Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants. At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (ntdAa) and nitrotoluene monooxygenase (ntnM), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of ndoB-positive endophytes in Scirpus pungens, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants. Similarly, the numbers of alkB- or ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not with the numbers of alkB- or ndoB-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.     

Do men with more masculine voices have better immunocompetence?

The human voice is often considered to be a secondary sexual characteristic that signals underlying information about the immunocompetence of the speaker (i.e. the immunocompetence handicap hypothesis; ICHH). However, no studies have yet shown a relationship between vocal characteristics and biomarkers of immune function or self-reported health. In a sample of 108 men, we examined correlations between masculine vocal characteristics [i.e. relatively low fundamental frequency (F₀), low F₀ variability (F₀-SD), low formant position (P_f), and high vocal tract length (VTL)] in relation to salivary immunoglobulin-A (sIgA; a marker of mucosal immunity), testosterone (T), and well-validated measures of self-reported health status. Results showed that sIgA correlated with masculinized F₀, P_f, and VTL. Self-report health correlated with masculinized P_f and VTL. Anticipated future health correlated negatively with F₀-SD and sick role propensity (less interference of illness in daily life) correlated positively with VTL. Perceived susceptibility to infection correlated with more feminized F₀ and F₀-SD. Our results demonstrated a small relationship between men's vocal characteristics and one putative indicator of mucosal immunity along with self-identified health status. We suggest that more research is warranted to determine whether the masculinity of men's voices may serve as an indicator of their phenotypic quality.     

Decreased glutamate transport in acivicin resistant Leishmania tarentolae

Studies of drug resistance in the protozoan parasites of the genus Leishmania have been helpful in revealing biochemical pathways as potential drug targets. The chlorinated glutamine analogue acivicin has shown good activity against Leishmania cells and was shown to target several enzymes containing amidotransferase domains. We selected a Leishmania tarentolae clone for acivicin resistance. The genome of this resistant strain was sequenced and the gene coding for the amidotransferase domain-containing GMP synthase was found to be amplified. Episomal expression of this gene in wild-type L. tarentolae revealed a modest role in acivicin resistance. The most prominent defect observed in the resistant mutant was reduced uptake of glutamate, and through competition experiments we determined that glutamate and acivicin, but not glutamine, share the same transporter. Several amino acid transporters (AATs) were either deleted or mutated in the resistant cells. Some contributed to the acivicin resistance phenotype although none corresponded to the main glutamate transporter. Through sequence analysis one AAT on chromosome 22 corresponded to the main glutamate transporter. Episomal expression of the gene coding for this transporter in the resistant mutant restored glutamate transport and acivicin susceptibility. Its genetic knockout led to reduced glutamate transport and acivicin resistance. We propose that acivicin binds covalently to this transporter and as such leads to decreased transport of glutamate and acivicin thus leading to acivicin resistance.     

Incidence of Wolbachia in aquatic insects

Wolbachia is a genus of intracellular bacteria typically found within the reproductive systems of insects that manipulates those systems of their hosts. While current estimates of Wolbachia incidence suggest that it infects approximately half of all arthropod species, these estimates are based almost entirely on terrestrial insects. No systematic survey of Wolbachia in aquatic insects has been performed. To estimate Wolbachia incidence among aquatic insect species, we combined field‐collected samples from the Missouri River (251 samples from 58 species) with a global database from previously published surveys. The final database contained 5,598 samples of 2,687 total species (228 aquatic and 2,459 terrestrial). We estimate that 52% (95% CrIs: 44%–60%) of aquatic insect species carry Wolbachia, compared to 60% (58%–63%) of terrestrial insects. Among aquatic insects, infected orders included Odonata, Coleoptera, Trichoptera, Ephemeroptera, Diptera, Hemiptera, and Plecoptera. Incidence was highest within aquatic Diptera and Hemiptera (69%), Odonata (50%), and Coleoptera (53%), and was lowest within Ephemeroptera (13%). These results indicate that Wolbachia is common among aquatic insects, but incidence varies widely across orders and is especially uncertain in those orders with low sample sizes such as Ephemeroptera, Plecoptera, and Trichoptera.     

Pyrophosphate-bridged complexes with picomolar toxicity

Recently, we have observed the emergence of a new series of pyrophosphate-bridged coordination complexes. Such complexes have been prepared by overcoming the ready hydrolysis of the pyrophosphate moiety. To date, no exploration has been conducted on the cytotoxicity of such complexes. Three pyrophosphate-bridged complexes, namely {[Ni(phen)₂]₂(μ-P₂O₇)}·27H₂O, {[Cu(phen)(H₂O)]₂(μ-P₂O₇)}·8H₂O and {[Co(phen)₂]₂(μ-P₂O₇)}·6MeOH, (where phen is 1,10′-phenanthroline) were chosen for their comparative structural similarities and suitable aqueous solubility. Cytotoxicity studies in the adriamycin-resistant ovarian cancer cell line A2780/AD demonstrated highly significant efficacy, with values as low as 160 pM for the cobalt complex at 72 h. The underlying mechanism for such exceptional toxicity is investigated focusing on DNA interactions, topoisomerase I enzyme inhibition and oxidative stress (followed by intracellular glutathione levels). The role of hydrolysis in uptake and toxicity is also explored (followed by electronic absorption spectroscopy, ³¹P NMR, and confocal microscopy) and the complexes are compared to cisplatin controls. Overall a clear picture of the extraordinary toxicity emerged. The results demonstrate a new class of prodrugs with significant potential for future development for the treatment of drug-resistant cancer cell lines.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.     

Nelfinavir,an HIV-1 Protease Inhibitor,Induces Oxidative Stress–Mediated,Caspase-Independent Apoptosis in Leishmania Amastigotes

Background

Visceral leishmaniasis has now emerged as an important opportunistic disease in patients coinfected with human immunodeficiency virus type-1 (HIV-1). Although the effectiveness of HIV-1 protease inhibitors, such as nelfinavir, in antiretroviral therapies is well documented, little is known of the impact of these drugs on Leishmania in coinfected individuals.

Methodology and Principal Findings

Here, we show that nelfinavir generates oxidative stress in the parasite, leading to altered physiological parameters such as an increase in the sub-G1 DNA content, nuclear DNA fragmentation and loss of mitochondrial potential, which are all characteristics of apoptosis. Pretreatment of axenic amastigotes with the caspase inhibitor z-VAD-fmk did not inhibit the increase in sub-G1 DNA content in nelfinavir-treated parasites, suggesting therefore that this antiviral agent does not kill Leishmania amastigotes in a caspase-dependent manner. Furthermore, we observed that the mitochondrial resident protein endonuclease G is involved. We also demonstrate that parasites overexpressing GSH1 (the rate limiting enzyme of glutathione biosynthesis) were more resistant to nelfinavir when compared to untransfected controls.

Conclusions and Significance

These data suggest that nelfinavir induces oxidative stress in Leishmania amastigotes, culminating in caspase-independent apoptosis, in which DNA is degraded by endonuclease G. This study provides a rationale for future, long-term design of new therapeutic strategies to test nelfinavir as a potential antileishmanial agent as well as for possible future use in Leishmania/HIV-1 coinfections.