Differential methylation of TSP50 and mTSP50 genes in different types of human tissues and mouse spermatic cells

Earlier studies identified human TSP50 as a testis-specific gene that encoded a threonine protease. Most importantly, TSP50 could be a cancer/testis antigen since there was a high frequency of reactivation in breast cancer biopsies. It was also found to be negatively regulated by the p53 gene. To further characterize this gene, we recently examined the DNA methylation patterns of the TSP50 gene promoter in normal human testis, as well as breast tissue and a testicular embryonic carcinoma cell line (HTECCL). Bisulfite genomic sequencing results demonstrated that the promoter exhibited mixed DNA methylation patterns in normal human testis, mainly non-methylation versus slight methylation, which could be attributed to the different stages spermatic cells go through during spermatogenesis. In contrast, it was methylated to a much greater extent in both breast tissue and HTECCL. To find out whether DNA methylation status was related to spermatogenesis stages, we analyzed DNA methylation patterns of the mTSP50 (the mouse ortholog of TSP50) promoter in spermatocytes and spermatozoa isolated from sexually mature mice. The results clearly demonstrated that each group of cells exhibited its preferential DNA methylation pattern that apparently was consistent with the gene expression status observed before. Taken together, our findings suggested that DNA methylation might regulate the TSP50 and mTSP50 gene expressions in different types of tissues and spermatic cells.     

Alteration of 23 S ribosomal RNA and erythromycin-induced resistance to lincomycin and spiramycin in Staphylococcus aureus

Functionally active “hybrid” 50 S ribosomal subunits can be reconstituted using 23 S RNA from Staphylococcus aureus (strain 1206) and 5 S RNA, as well as 50 S ribosomal proteins from Bacillus stearothermophilus. Using this system, resistance of S. aureus 50 S subunits to lincomycin and spiramycin was analyzed. When 23 S RNA from either phenotypically resistant (“induced resistance”) S. aureuscells or derived genetically resistant (“constitutive resistance”) S. aureus cells, were used, the reconstituted 50 S subunits showed the resistant phenotype similar to that seen in native 50 S subunits obtained from resistant cells; only very weak inhibition by the antibiotics was observed in poly (U) - directed polyphenylalanine synthesis involving these 50 S subunits. In contrast, the 50 S particles reconstituted using 23 S RNA from uninduced (sensitive) S. aureus were subject to greater inhibition by the antibiotics in cell-free poly-peptide synthesis. It is concluded that modification of 23 S RNA, presumably the previously observed methylation to form dimethyladenine, is responsible for the resistance to the antibiotics in this strain of S. aureus.     

Gene cloning,expression and characterisation of a new β-agarase,AgWH50C,producing neoagarobiose from Agarivorans gilvus WH0801

agWH50C, a novel β-agarase gene, was cloned from Agarivorans gilvus WH0801 by degenerate PCR and nested PCR. The gene agWH50C comprized a 2,223-bp, encoding a protein of 740 amino acids. Sequencing results demonstrated that AgWH50C shared 45 % sequence identity with a well characterized β-agarase, Aga50D, from Saccharophagus degradans 2–40. The mature agarase was expressed in Escherichia coli and purified by affinity chromatography. The optimum pH and temperature for AgWH50C activity were 6.0 and 30 °C. The K _m and V _max values for agarose were 12.55 mg/ml and 1.17 U/mg. Analysis of the hydrolysis products using linear ion trap mass spectrometry, Fourier transform-nuclear magnetic resonance spectrometry and thin-layer chromatography confirmed that the reaction product of AgWH50c was α-neoagarobiose alone. Therefore, our novel agarase has the potential for industrial applications to produce neoagarobiose as well as provides a key β-agarase for fermentation of agar biomass.     

Biocontrol Potential of Steinernema thermophilum and Its Symbiont Xenorhabdus indica Against Lepidopteran Pests: Virulence to Egg and Larval Stages

Under laboratory conditions, the biocontrol potential of Steinernema thermophilum was tested against eggs and larval stages of two important lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura (polyphagous pests), as well as Galleria mellonella (used as a model host). In terms of host susceptibility of lepidopteran larvae to S. thermophilum, based on the LC₅₀ 36 hr after treatment, G. mellonella (LC₅₀ = 16.28 IJ/larva) was found to be more susceptible than S. litura (LC₅₀ = 85 IJ/larva), whereas neither host was found to be significantly different from H. armigera (LC₅₀ = 54.68 IJ/larva). In addition to virulence to the larval stages, ovicidal activity up to 84% was observed at 200 IJ/50 and 100 eggs of H. armigera and S. litura, respectively. To our knowledge this is the first report of entomopathogenic nematode pathogenicity to lepidopteran eggs. Production of infective juvenile (IJ) nematodes/insect larva was also measured and found to be positively correlated with rate of IJ for H. armigera (r = 0.990), S. litura (r = 0.892), as well as G. mellonella (r = 0.834). Both Phase I and Phase II of symbiotic bacteria Xenorhabdus indica were tested separately against neonates of H. armigera and S. litura by feeding assays and found to be virulent to the target pests; phase variation did not affect the level of virulence. Thus S. thermophilum as well as the nematode’s symbiotic bacteria applied separately have the potential to be developed as biocontrol agents for key lepidopteran pests.     

A comparison of desiccation tolerance among 12 species of chironomid larvae

Tolerance to desiccation was compared among 12 Japanese species of chironomid larvae under the condition of 60% in relative humidity at 25.5?°C. Three parameters were assessed: time to 50% survival (T ₅₀), water loss at 50% survival (WL₅₀) and water loss rate (WLR). T ₅₀, WL₅₀ and WLR were determined as measures of desiccation tolerance, dehydration tolerance, and dehydration resistance, respectively. T ₅₀ was 64.4–142 min for most species, except Propsilocerus akamusi (Tokunaga) which took 872 min. WL₅₀ was 60.6–82.4% for all species. WLR was only 0.0664% per minute for Pr. akamusi, while it was 0.629–1.50% for the other species. These results showed that Pr. akamusi had a high desiccation tolerance due to a high preventive ability of evaporation from body surface. T ₅₀ showed no significant relationships to WL₅₀ or WLR among the 12 species, while there was a significant positive relationship between WL₅₀ and WLR. These results suggest that chironomid species have a trade-off tendency that a species has a high tolerance – low resistance or a high resistance – low tolerance for dehydration.     

The Rad50 genes of diploid and polyploid wheat species. Analysis of homologue and homoeologue expression and interactions with Mre11

The MRN complex plays a central role in the DNA repair pathways of eukaryotic cells and takes part in many other processes, including cell cycle checkpoint signalling, meiosis, DNA replication and telomere maintenance. This complex is formed by the interaction of the products of the Mre11, Rad50 and Nbs1 genes. This paper reports the molecular characterization, expression and interactions of the Rad50 gene in several wheat species with different levels of ploidy. The homoeologous Rad50 wheat genes were found to show a high level of conservation. Most of the RAD50 domains and motifs previously described in other species were also present in wheat RAD50; these proteins are therefore likely to have similar functions. Interactions between the RAD50 wheat proteins and their MRE11 counterparts in the MRN complex were observed. The level of expression of Rad50 in each of the species examined was determined and compared with those previously reported for the Mre11 genes. In some cases similar levels of expression were seen, as expected. The expression of the RAD50 homoeologous genes was assessed in two polyploid wheat species using quantitative PCR. In both cases, an overexpression of the Rad50B gene was detected. Although the results indicate the maintenance of function of these species?? three homoeologous Rad50 genes, the biased expression of Rad50B might indicate ongoing silencing of one or both other homoeologues in polyploid wheat. To assess the consequences of such silencing on the formation of the MRN complex, the interactions between individual homoeologues of Rad50 and their genomic counterpart Mre11 genes were examined. The results indicate the inexistence of genomic specificity in the interactions between these genes. This would guarantee the formation of an MRN complex in wheat.     

Physiological effects and bioconcentration of triclosan on amphibian larvae

We examined the acute effects of triclosan (TCS) exposure, a common antimicrobial found as a contaminant in the field, on survival and physiology of amphibian larvae. LC50 values were determined after 96 h for North American larval species: Acris crepitans blanchardii, Bufo woodhousii woodhousii, Rana sphenocephala, and for a developmental model: Xenopus laevis. Amphibian larvae were most sensitive to TCS exposure during early development based upon 96-h LC50 values. Heart rates for X. laevis and North American larvae exposed to TCS were variable throughout development. Metabolic rates of X. laevis and R. sphenocephala larvae exposed to TCS were significantly affected in larvae exposed to [50% LC50] and [LC50]. Tissue uptake and tissue bioconcentration factor (BCF) of TCS were investigated in X. laevis, B. woodhousii woodhousii, and R. sphenocephala. In general, a significant increase was observed as exposure concentration increased. Tissue BCF values were dependent upon stage and species. While TCS concentrations used here are higher than environmental concentrations, exposure to TCS was dependent upon species and developmental stage, with early developmental stages being most sensitive to TCS exposure.     

Evolution and functional characterization of the RH50 gene from the ammonia-oxidizing bacterium Nitrosomonas europaea

The family of ammonia and ammonium channel proteins comprises the Amt proteins, which are present in all three domains of life with the notable exception of vertebrates, and the homologous Rh proteins (Rh50 and Rh30) that have been described thus far only in eukaryotes. The existence of an RH50 gene in bacteria was first revealed by the genome sequencing of the ammonia-oxidizing bacterium Nitrosomonas europaea. Here we have used a phylogenetic approach to study the evolution of the N. europaea RH50 gene, and we show that this gene, probably as a component of an integron cassette, has been transferred to the N. europaea genome by horizontal gene transfer. In addition, by functionally characterizing the Rh50_Ne protein and the corresponding knockout mutant, we determined that NeRh50 can mediate ammonium uptake. The RH50_Ne gene may thus have replaced functionally the AMT gene, which is missing in the genome of N. europaea and may be regarded as a case of nonorthologous gene displacement.     

The role of amino terminus of mouse Cx50 in determining transjunctional voltage-dependent gating and unitary conductance

Amino-terminus and carboxyl-terminus of connexins have been proposed to be responsible for the transjunctional voltage-dependent gating (V_j-gating) and the unitary gap junction channel conductance (γ_j). To better understand the molecular structure(s) determining the V_j-gating properties and the γ_j of Cx50, we have replaced part of the amino-terminus of mCx50 by the corresponding domain of mCx36 to engineer a chimera Cx50-Cx36N, and attached GFP at the carboxyl-terminus of mCx50 to construct Cx50-GFP. The dual whole-cell patch-clamp technique was used to test the resulting gap junction channel properties in N2A cells. The Cx50-Cx36N gap junction channel lowered the sensitivity of steady-state junctional conductance to V_j (G_j/V_j relationship), slowed V_j-gating kinetics, and reduced γ_j as compared to Cx50 channel. Cx50-GFP gap junction channel showed similar V_j-gating properties and γ_j to Cx50 channel. We further characterized a mutation, Cx50N9R, where the Asn (N) at the ninth position of Cx50 was replaced by the corresponding Arg (R) at Cx36. The G_j/V_j relationship of Cx50N9R channel was significantly changed; most strikingly, the macroscopic residual conductance (G_min) was near zero. Moreover, the single Cx50N9R channel only displayed one open state (γ_j = 132 ± 4 pS), and no substate could be detected. Our data suggest that the NT of Cx50 is critical for both the V_j-gating and the γ_j, and the introduction of a positively charged Arg at the ninth position reduced the G_min with a correlated disappearance of the substate at the single channel level.     

Fumigant Toxicity of Plant Essential Oils to Plutella xylostella (Lepidoptera: Yponomeutidae) and Cotesia glomerata (Hymenoptera: Braconidae)

The fumigant toxicity of 66 plant essential oils to Plutella xylostella (L.) larvae and Cotesia glomerata (L.) adults was examined using a vapor-phase toxicity bioassay and compared with that of dichlorvos. Responses varied according to oil and insect species used. Based on 24 h LD₅₀ values, pennyroyal oil [10.77 mg/filter paper (4.25 cm diameter)] was the most toxic fumigant, followed by rosemary and sage (Dalmatin) oils (15.15 mg/paper). Potent fumigant toxicity was also produced from armoise, buchu leaf, cedarleaf, coriander, eucalyptus, howood, lavender, myrtle, niaouli, peppermint, and rosewood oils (LD₅₀, 21.29–27.31 mg/paper). All essential oils were less effective than dichlorvos (LD₅₀, 0.52 mg/paper). Against adult C. glomerata, dichlorvos (LD₅₀, 0.03 mg/paper) was the most toxic fumigant, whereas the LD₅₀ values of the 14 essential oils ranged from 1.59 to 8.51 mg/paper. Based on selective toxicity ratio (STR, P. xylostella LD₅₀/C. glomerata LD₅₀), the 14 essential oils (STR, 2.5–14.5) are more selective than dichlorvos (STR, 17.3). The essential oils tested merit further study as potential fumigants for the control of P. xylostella in greenhouses because of their selective toxicity to adult C. glomerata and their much greater activity as a fumigant.     

Influence of auxins on somatic embryogenesis and alkaloid accumulation in Leucojum aestivum callus

In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer’s disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM).     

Proteomic Analysis of Gastrocnemius Muscle in Rats with Streptozotocin-Induced Diabetes and Chronically Exposed to Fluoride

Administration of high doses of fluoride (F) can alter glucose homeostasis and lead to insulin resistance (IR). This study determined the profile of protein expression in the gastrocnemius muscle of rats with streptozotocin-induced diabetes that were chronically exposed to F. Male Wistar rats (60 days old) were randomly distributed into two groups of 18 animals. In one group, diabetes was induced through the administration of streptozotocin. Each group (D-diabetic and ND-non-diabetic) was further divided into 3 subgroups each of which was exposed to a different F concentration via drinking water (0 ppm, 10 ppm or 50 ppm F, as NaF). After 22 days of treatment, the gastrocnemius muscle was collected and submitted to proteomic analysis (2D-PAGE followed by LC-MS/MS). Protein functions were classified by the GO biological process (ClueGO v2.0.7+Clupedia v1.0.8) and protein-protein interaction networks were constructed (PSICQUIC, Cytoscape). Quantitative intensity analysis of the proteomic data revealed differential expression of 75 spots for ND0 vs. D0, 76 for ND10 vs.D10, 58 spots for ND50 vs. D50, 52 spots for D0 vs. D10 and 38 spots for D0 vs. D50. The GO annotations with the most significant terms in the comparisons of ND0 vs. D0, ND10 vs. D10, ND50 vs. D50, D0 vs. D10 and D0 vs. D50, were muscle contraction, carbohydrate catabolic processes, generation of precursor metabolites and energy, NAD metabolic processes and gluconeogenesis, respectively. Analysis of subnetworks revealed that, in all comparisons, proteins with fold changes interacted with GLUT4. GLUT4 interacting proteins, such as MDH and the stress proteins HSPB8 and GRP78, exhibited decreased expression when D animals were exposed to F. The presence of the two stress proteins indicates an increase in IR, which might worsen diabetes. Future studies should evaluate whether diabetic animals treated with F have increased IR, as well as which molecular mechanisms are involved.     

Selection for resistance to a nucleopolyhedrosis virus in laboratory populations of the cotton bollworm, Heliothis zea

Resistance to a nucleopolyhedrosis virus (Baculovirus heliothis) did not develop in laboratory populations of the cotton bollworm, Heliothis zea. A selection pressure of LD₅₀ to 70 was maintained throughout 20 to 25 generations of selection. No significant changes in LD₅₀, slope, or intercept of dose-mortality lines were detected. Laboratory populations under selection were as susceptible to the virus as nonselected or wild populations of H. zea. The resistance ratio (LD₅₀ of selected generation/initial generation) ranged from 0.5 to 1.2.     

Ouabain-resistant Na,K-ATPases and cardenolide tolerance in the large milkweed bug,Oncopeltus fasciatus

Oncopeltus fasciatus tolerated 1954× and 7288×, respectively, the LD₅₀ ouabain dose of Schistocerca gregaria and Periplaneta americana when ouabain was injected into the haemocoel of these insects. The maximal ouabain dose that could be injected into O. fasciatus (200 nmol) resulted in no mortality; this dose is higher than the lethal ouabain doses recorded for vertebrates and invertebrates. The ouabain concentration resulting in 50% inhibition (I₅₀) of Na,K-ATPase activity was determined in lyophilates of nervous tissue of O. fasciatus and brain and recta of S. gregaria and were 2.0 × 10⁻⁴, 2.0 × 10⁻⁶ and 1.0 × 10⁻⁶ M, respectively. The I₅₀ value for ouabain inhibition of Na,K-ATPase activity in the nervous tissue of O. fasciatus is higher than the I₅₀ values for nervous tissue in most other insects as well as many other invertebrate and vertebrate tissues. Thus, the presence of ouabain-resistant Na,K-ATPases appears to be a factor in the tolerance and sequestration of plant cardenolides in O. fasciatus.     

Interaction of Population Levels of Fusarium oxysporum f. sp. vasinfectum and Meloidogyne incognita on Cotton

In autoclaved greenhouse soil without Fusarium oxysporum f. sp. vasinfectum, Meloidogyne incognita did not cause leaf or vascular discoloration of 59-day-old cotton plants. Plants had root galls with as few as 50 Meloidogyne larvae per plant. Root galling was directly proportional to the initial nematode population level. Fusarium wilt symptoms occurred without nematodes with 77,000 fungus propagules or more per gram of soil. As few as 50 Meloidogyne larvae accompanying 650 fungus propagules caused Fusarium wilt. With few exceptions, leaf symptoms appeared sooner as numbers of either or both organisms increased. In soils infested with both organisms, the extent of fungal invasion and colonization was well correlated with the extent of nematode galling and other indications of the Fusarium wilt syndrome.     

Human RAD50 Deficiency in a Nijmegen Breakage Syndrome-like Disorder

The MRE11/RAD50/NBN (MRN) complex plays a key role in recognizing and signaling DNA double-strand breaks (DSBs). Hypomorphic mutations in NBN (previously known as NBS1) and MRE11A give rise to the autosomal-recessive diseases Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively. To date, no disease due to RAD50 deficiency has been described. Here, we report on a patient previously diagnosed as probably having NBS, with microcephaly, mental retardation, ‘bird-like’ face, and short stature. At variance with this diagnosis, she never had severe infections, had normal immunoglobulin levels, and did not develop lymphoid malignancy up to age 23 years. We found that she is compound heterozygous for mutations in the RAD50 gene that give rise to low levels of unstable RAD50 protein. Cells from the patient were characterized by chromosomal instability; radiosensitivity; failure to form DNA damage-induced MRN foci; and impaired radiation-induced activation of and downstream signaling through the ATM protein, which is defective in the human genetic disorder ataxia-telangiectasia. These cells were also impaired in G1/S cell-cycle-checkpoint activation and displayed radioresistant DNA synthesis and G2-phase accumulation. The defective cellular phenotype was rescued by wild-type RAD50. In conclusion, we have identified and characterized a patient with a RAD50 deficiency that results in a clinical phenotype that can be classified as an NBS-like disorder (NBSLD).     

Transformation of pWWO in Rhizobium leguminosarum DPT to Engineer Toluene Degrading Ability for Rhizoremediation

Rhizoremediation of organic xenobiotics is based on interactions between plants and their associated micro-organisms. The present work was designed to engineer a bacterial system having toluene degradation ability along with plant growth promoting characteristics for effective rhizoremediation. pWWO harboring the genes responsible for toluene breakdown was isolated from Pseudomonas putida MTCC 979 and successfully transformed in Rhizobium DPT. This resulted in a bacterial strain (DPT^T) which had the ability to degrade toluene as well as enhance growth of host plant. The frequency of transformation was recorded 5.7 × 10⁻⁶. DPT produced IAA, siderophore, chitinase, HCN, ACC deaminase, solubilized inorganic phosphate, fixed atmospheric nitrogen and inhibited the growth of Fusarium oxysporum and Macrophomina phaseolina in vitro. During pot assay, 50 ppm toluene in soil was found to inhibit the germination of Cajanus cajan seeds. However when the seeds bacterized with toluene degrading P. putida or R. leguminosarum DPT were sown in pots, again no germination was observed. Non-bacterized as well as bacterized seeds germinated successfully in toluene free soil as control. The results forced for an alternative mode of application of bacteria for rhizoremediation purpose. Hence bacterial suspension was mixed with soil having 50 ppm of toluene. Germination index in DPT treated soil was 100% while in P. putida it was 50%. Untreated soil with toluene restricted the seeds to germinate.     

Tetrahymena: An Alternative Model Host for Evaluating Virulence of Aeromonas Strains

An easier assessment model would be helpful for high-throughput screening of Aeromonas virulence. The previous study indicated the potential of Tetrahymena as a permissive model to examine virulence of Aeromonas hydrophila. Here our aim was to assess virulence of Aeromonas spp. using two model hosts, a zebrafish assay and Tetrahymena-Aeromonas co-culture, and to examine whether data from the Tetrahymena thermophila model reflects infections in the well-established animal model. First, virulence of 39 Aeromonas strains was assessed by determining the 50% lethal dose (LD₅₀) in zebrafish. LD₅₀ values ranging from 1.3×10² to 3.0×10⁷ indicated that these strains represent a high to moderate degree of virulence and could be useful to assess virulence in the Tetrahymena model. In Tetrahymena-Aeromonas co-culture, we evaluated the virulence of Aeromonas by detecting relative survival of Aeromonas and Tetrahymena. An Aeromonas isolate was considered virulent when its relative survival was greater than 60%, while the Aeromonas isolate was considered avirulent if its relative survival was below 40%. When relative survival of T. thermophila was lower than 40% after co-culture with an Aeromonas isolate, the bacterial strain was regarded as virulent. In contrast, the strain was classified as avirulent if relative survival of T. thermophila was greater than 50%. Encouragingly, data from the 39 Aeromonas strains showed good correlation in zebrafish and Tetrahymena-Aeromonas co-culture models. The results provide sufficient data to demonstrate that Tetrahymena can be a comparable alternative to zebrafish for determining the virulence of Aeromonas isolates.     

Microbiological Reduction of Quininone to Quinidine

A yeast identified as Hansenula anomala var. schneggii was found capable of reducing quininone to quinidine in 50% yields in 7 days of fermentation.