SNS/PN3 and SNS2/NaN sodium channel-like immunoreactivity in human adult and neonate injured sensory nerves

Two tetrodotoxin-resistant voltage-gated sodium channels, SNS/PN3 and SNS2/NaN, have been described recently in small-diameter sensory neurones of the rat, and play a key role in neuropathic pain. Using region-specific antibodies raised against different peptide sequences of their alpha subunits, we show by Western blot evidence for the presence of these channels in human nerves and sensory ganglia. The expected fully mature 260 kDa component of SNS/PN3 was noted in all injured nerve tissues obtained from adults; however, for SNS2/NaN, smaller bands were found, most likely arising from protein degradation. There was increased intensity of the SNS/PN3 260 kDa band in nerves proximal to the site of injury, whereas it was decreased distally, suggesting accumulation at sites of injury; all adult patients had a positive Tinel's sign at the site of nerve injury, indicating mechanical hypersensitivity. Injured nerves from human neonates showed similar results for both channels, but neonate neuromas lacked the SNS2/NaN 180 kDa molecular form, which was strongly present in adult neuromas. The distribution of SNS/PN3 and SNS2/NaN sodium channels in injured human nerves indicates that they represent targets for novel analgesics, and could account for some differences in the development of neuropathic pain in infants.     

A novel locus (RP24) for X-linked retinitis pigmentosa maps to Xq26-27.

Two genetic loci, RP2 and RP3, for X-linked retinitis pigmentosa (XLRP) have been localized to Xp11.3-11.23 and Xp21.1, respectively. RP3 appears to account for 70% of XLRP families; however, mutations in the RPGR gene (isolated from the RP3 region) are identified in only 20% of affected families. Close location of XLRP loci at Xp and a lack of unambiguous clinical criteria do not permit assignment of genetic subtype in a majority of XLRP families; nonetheless, in some pedigrees, both RP2 and RP3 could be excluded as the causative locus. We report the mapping of a novel locus, RP24, by haplotype and linkage analysis of a single XLRP pedigree. The RP24 locus was identified at Xq26-27 by genotyping 52 microsatellite markers spanning the entire X chromosome. A maximum LOD score of 4.21 was obtained with DXS8106. Haplotype analysis assigned RP24 within a 23-cM region between the DXS8094 (proximal) and DXS8043 (distal) markers. Other chromosomal regions and known XLRP loci were excluded by obligate recombination events between markers in those regions and the disease locus. Hemizygotes from the RP24 family have early onset of rod photoreceptor dysfunction; cone receptor function is normal at first, but there is progressive loss. Patients at advanced stages show little or no detectable rod or cone function and have clinical hallmarks of typical RP. Mapping of the RP24 locus expands our understanding of the genetic heterogeneity in XLRP and will assist in development of better tools for diagnosis.     

Protocol for in vitro propagation of Excoecaria agallocha L., a medicinally important mangrove species

An in vitro propagation protocol has been developed for Excoecaria agallocha L. (Euphorbiaceae), a mangrove species. Nodal segments were used for axillary shoot proliferation. One shoot from each node of binodal explants was observed 3 weeks after inoculation. The best axillary sprouting was seen on a newly formulated medium containing BA, Zeatin and IBA in concentrations of 13.3 μM, 4.65 μM and 1.23 μM, respectively. The new medium, first used in this study, has a specific composition of major nutrients, MS micronutrients and iron compounds. Nodal segments from rooted cuttings and seedlings responded better than those of mature tree explants. Multiple shoot induction was complemented with efficient shoot elongation, and repeated subculture of binodal segments from axillary shoots resulted in 10–12 shoots per explant in 3 months. Rooting was achieved by growing shoots in the new medium with 0.23 μM IBA. Regenerated plants were successfully acclimatized to the natural environment, and about 85% of plantlets survived under ex vitro conditions. This is the first report of micropropagation in the genus Excoecaria and also in mangrove tree species. Received: 11 August 1997 / Revision received: 10 February 1998 / Accepted: 2 March 1998     

Molecular Changes in Entamoeba histolytica in Response to Bacteria1

Entamoeba histolytica, the protozoan parasite, is the causative agent of amoebiasis. The degree of virulence, as inferred from invasiveness, of potentially pathogenic strains may be regulated by both host and parasite factors that determine the gut environment. One such factor that plays an important role is the bacterial flora in the gut. Previous studies have clearly shown that bacterial flora is an important determinant of virulence in E. histolytica. However, the exact nature of changes induced in E. histolytica in response to bacteria and their role in virulence is not clear. In this study the levels of a number of molecules potentially important in virulence mechanisms were determined in E. histolytica cells grown with and without normal human bacterial flora, using enzyme-linked immunosorbent assay. Significant changes were observed only after the E. histolytica cells had been adapted to grow with bacterial flora for a number of generations, and not in short term culture.     

Transformation of Nicotiana tabacum with a native cry1Ia5 gene confers complete protection against Heliothis armigera

A cry1Ia5 insecticidal toxin coding gene has been cloned from an Indian isolate of Bacillus thuringiensis. Sequence analyses of the cry1Ia5 gene revealed the absence of potential polyadenylation signal sequences thus making it a suitable candidate for expression in plants without extensive modification. This possibility was examined by subcloning the cry1Ia5 gene into a plant expression vector and then transferring it to Nicotiana tabacum through Agrobacterium-mediated transformation. Our results demonstrate that N. tabacum with a stably integrated native cry1Ia5 gene afforded complete protection against predation by Heliothis armigera. Forty three percent of the transgenic plants displayed a high level of protection against insect predation. The protection obtained in transgenic plants with the cry1Ia5 gene was comparable to that obtained with the synthetically modified cry1A(b) or cry1A(c) genes. The results demonstrate that novel insecticidal genes already exist in nature that do not require extensive modifications for efficient expression in plants.     

A comparison of worker exposure to inhalable and total dust, inorganic arsenic, and borates using two types of particulate sampling assemblies in a borate mining and processing facility

This study describes a comparison of worker exposure to total and inhalable dust, inorganic As, and borates using two types of particulate sampling assemblies as part of a comprehensive industrial hygiene evaluation in a borate mining and processing facility. Employees were segmented into similar exposure groups (SEG) based on work location within the facility, job classification, and type of chemical agent. Approximately 10% of the employees from each SEG wore two personal sampling devices simultaneously for the purpose of collecting total and inhalable particulate fractions using a closed face, 37-mm mixed cellulose ester matched-weight filters (MMW), and Institute of Occupational Medicine (IOM) sampling assembly. Sample results indicated that the IOM concentrations were consistently higher than the corresponding MMW concentrations for all three agents. An analysis was performed to investigate a relationship between MMW and IOM. The data revealed correlation coefficient values of 0.72, 0.82, and 0.84 for total dust (n = 197), inorganic As (n = 137), and borates (n = 194), respectively. These positive correlation coefficients indicate that the IOM and MMW measurements are consistent with each other, and can be used for predicting exposure levels. The total dust and borate large mean ratios should be considered in developing inhalable fraction-based regulatory standards.     

LSD1 Overexpression Is Associated with Poor Prognosis in Basal-Like Breast Cancer,and Sensitivity to PARP Inhibition

LSD1, a lysine-specific histone demethylase, is overexpressed in several types of cancers and linked to poor outcomes. In breast cancer, the significance of LSD1 overexpression is not clear. We have performed an in silico analysis to assess the relationship of LSD1 expression to clinical outcome. We demonstrate that LSD1 overexpression is a poor prognostic factor in breast cancer, especially in basal-like breast cancer, a subtype of breast cancer with aggressive clinical features. This link is also observed in samples of triple negative breast cancer. Interestingly, we note that overexpression of LSD1 correlates with down-regulation of BRCA1 in triple negative breast cancer. This phenomenon is also observed in in vitro models of basal-like breast cancer, and is associated with an increased sensitivity to PARP inhibitors. We propose therefore that high expression levels of the demethylase LSD1 is a potential prognostic factor of poor outcome in basal-like breast cancer, and that PARP inhibition may be a therapeutic strategy of interest in this poor prognostic subtype with overexpression of LSD1.     

Using Bioconductor Package BiGGR for Metabolic Flux Estimation Based on Gene Expression Changes in Brain

Predicting the distribution of metabolic fluxes in biochemical networks is of major interest in systems biology. Several databases provide metabolic reconstructions for different organisms. Software to analyze flux distributions exists, among others for the proprietary MATLAB environment. Given the large user community for the R computing environment, a simple implementation of flux analysis in R appears desirable and will facilitate easy interaction with computational tools to handle gene expression data. We extended the R software package BiGGR, an implementation of metabolic flux analysis in R. BiGGR makes use of public metabolic reconstruction databases, and contains the BiGG database and the reconstruction of human metabolism Recon2 as Systems Biology Markup Language (SBML) objects. Models can be assembled by querying the databases for pathways, genes or reactions of interest. Fluxes can then be estimated by maximization or minimization of an objective function using linear inverse modeling algorithms. Furthermore, BiGGR provides functionality to quantify the uncertainty in flux estimates by sampling the constrained multidimensional flux space. As a result, ensembles of possible flux configurations are constructed that agree with measured data within precision limits. BiGGR also features automatic visualization of selected parts of metabolic networks using hypergraphs, with hyperedge widths proportional to estimated flux values. BiGGR supports import and export of models encoded in SBML and is therefore interoperable with different modeling and analysis tools. As an application example, we calculated the flux distribution in healthy human brain using a model of central carbon metabolism. We introduce a new algorithm termed Least-squares with equalities and inequalities Flux Balance Analysis (Lsei-FBA) to predict flux changes from gene expression changes, for instance during disease. Our estimates of brain metabolic flux pattern with Lsei-FBA for Alzheimer’s disease agree with independent measurements of cerebral metabolism in patients. This second version of BiGGR is available from Bioconductor.     

In Vivo Assessment of Cold Tolerance through Chlorophyll-a Fluorescence in Transgenic Zoysiagrass Expressing Mutant Phytochrome A

Chlorophyll-a fluorescence analysis provides relevant information about the physiology of plants growing under abiotic stress. In this study, we evaluated the influence of cold stress on the photosynthetic machinery of transgenic turfgrass, Zoysia japonica, expressing oat phytochrome A (PhyA) or a hyperactive mutant phytochrome A (S599A) with post-translational phosphorylation blocked. Biochemical analysis of zoysiagrass subjected to cold stress revealed reduced levels of hydrogen peroxide, increased proline accumulation, and enhanced specific activities of antioxidant enzymes compared to those of control plants. Detailed analyses of the chlorophyll-a fluorescence data through the so-called OJIP test exhibited a marked difference in the physiological status among transgenic and control plants. Overall, these findings suggest an enhanced level of cold tolerance in S599A zoysiagrass cultivars as reflected in the biochemical and physiological analyses. Further, we propose that chlorophyll-a fluorescence analysis using OJIP test is an efficient tool in determining the physiological status of plants under cold stress conditions.     

Host Erythrocyte Environment Influences the Localization of Exported Protein 2, an Essential Component of the Plasmodium Translocon

Malaria parasites replicating inside red blood cells (RBCs) export a large subset of proteins into the erythrocyte cytoplasm to facilitate parasite growth and survival. PTEX, the parasite-encoded translocon, mediates protein transport across the parasitophorous vacuolar membrane (PVM) in Plasmodium falciparum-infected erythrocytes. Proteins exported into the erythrocyte cytoplasm have been localized to membranous structures, such as Maurer''s clefts, small vesicles, and a tubovesicular network. Comparable studies of protein trafficking in Plasmodium vivax-infected reticulocytes are limited. With Plasmodium yoelii-infected reticulocytes, we identified exported protein 2 (Exp2) in a proteomic screen of proteins putatively transported across the PVM. Immunofluorescence studies showed that P. yoelii Exp2 (PyExp2) was primarily localized to the PVM. Unexpectedly, PyExp2 was also associated with distinct, membrane-bound vesicles in the reticulocyte cytoplasm. This is in contrast to P. falciparum in mature RBCs, where P. falciparum Exp2 (PfExp2) is exclusively localized to the PVM. Two P. yoelii-exported proteins, PY04481 (encoded by a pyst-a gene) and PY06203 (PypAg-1), partially colocalized with these PyExp2-positive vesicles. Further analysis revealed that with P. yoelii, Plasmodium berghei, and P. falciparum, cytoplasmic Exp2-positive vesicles were primarily observed in CD71⁺ reticulocytes versus mature RBCs. In transgenic P. yoelii 17X parasites, the association of hemagglutinin-tagged PyExp2 with the PVM and cytoplasmic vesicles was retained, but the pyexp2 gene was refractory to deletion. These data suggest that the localization of Exp2 in mouse and human RBCs can be influenced by the host cell environment. Exp2 may function at multiple points in the pathway by which parasites traffic proteins into and through the reticulocyte cytoplasm.