Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl β-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.     

Radiosensitivity of mammalian cell lines engineered to overexpress cytosolic glutathione peroxidase

Reactive oxygen species are believed to be involved in radiation lethality. Glutathione peroxidase is an intracellular enzyme with antioxidant functions. To determine whether increasing the cellular antioxidant capacity can confer radiation resistance, the effect of overexpression of glutathione peroxidase on radiosensitivity was determined in two different cell types. An expression construct including the bovine cytosolic glutathione peroxidase cDNA was used to overexpress this enzyme in cells of the human lymphoblast cell line Sup-T1 as well as the Chinese hamster ovary cell line AA8. Supplementation of the culture media with 30 nM sodium selenite was included to obtain optimal glutathione peroxidase activity. Northern blot analysis confirmed the presence of the construct mRNA, and a standard coupled spectrophotometric assay demonstrated significantly increased glutathione peroxidase activity in the transfected cell lines. An approximately 8-fold increase was found in the Sup-T1 cells, and an approximately 30-fold increase was obtained in the Chinese hamster ovary AA8 cells. Clonogenic survival was assayed in the overexpressing cells and compared to that in control cells transfected with vector alone. Despite significantly increased glutathione peroxidase activity, no observable radioprotection was conferred in either of the two cell lines studied, indicating that increased glutathione peroxidase activity is insufficient to confer radioresistance in the two cell types examined. These data are discussed in the context of using antioxidants as adjuncts to clinical radiotherapy.     

A critical review of the use of Clara cell secretory protein (CC16) as a biomarker of acute or chronic pulmonary effects

Biomarkers associated with asthma aetiology and exacerbation have been sought to shed light on this multifactorial disease. One candidate is the serum concentration of the Clara cell secretory protein (CC16, sometimes referred to as CC10 or uteroglobin). In this review, we examine serum CC16's relation to asthma aetiology and exacerbation. There is evidence that acute exposures to certain pulmonary irritants can cause a transient increase in serum CC16 levels, and limited evidence also suggests that a transient increase in serum CC16 levels can be caused by a localized pulmonary inflammation. Research also indicates that a transient increase in serum CC16 is not associated with measurable pulmonary damage or impairment of pulmonary function. The biological interpretation of chronic changes in serum CC16 is less clear. Changes in serum CC16 concentrations (either transient or chronic) are not specific to any one agent, disease state, or aetiology. This lack of specificity limits the use of serum CC16 as a biomarker of specific exposures. To date, many of the critical issues that must be understood before serum CC16 levels can have an application as a biomarker of effect or exposure have not been adequately addressed.     

Testing a molecular protocol to monitor the presence of golden mussel larvae (Limnoperna fortunei) in plankton samples

The golden mussel (Limnoperna fortunei, Mollusca: Mytilidae)is an emerging invasive species in freshwater environments inSouth America, causing extensive environmental and economicimpacts. A molecular method to detect larvae of the golden musselin plankton samples has been recently developed and holds promisefor becoming an important way to monitor the expansion of goldenmussel populations. In the present study, we conduct, for thefirst time, field tests of this method by comparing its performancewith alternative sampling efforts (microscopy and manual searchfor adults). In addition, we test different modifications ofthe molecular method to deal with PCR inhibition in environmentalsamples. The results indicate that the molecular method is veryefficient, being faster and more sensitive that microscopy methods.Therefore, the molecular method tested in the present studycan represent an invaluable tool in large-scale monitoring effortsof the golden mussel throughout its introduced range.     

Impact of the erineum strain of <Emphasis Type="Italic">Colomerus vitis</Emphasis> (Acari: Eriophyidae) on the development of plants of grapevine cultivars of Iran

The present experiment was aimed at determining the influence of the grape erineum strain of Colomerus vitis (GEM) (Acari: Eriophyidae) on responses of local grapevine cultivars. GEM was applied at five density levels to each of five cultivars, i.e. Shahani, Sahebi Uroomie, Khalili Bovanat, Rishbaba and Sezdang Ghalat (listed from early to late grape ripening). The experiment was performed in a full factorial design (12 replicates each) and effects of the mite on the relative content of leaf chlorophyll, internode and cane length, leaf area and weight, number and size of the erinea, and percentage of leaves with erinea were investigated. Also mite density on leaves and in buds was assessed. Data were analyzed with a two-way ANOVA followed by Tukey’s test to separate means among treatment levels and cultivars. The relative content of chlorophyll (expressed in Spad units) in infested leaves was reduced along with an increase in mite density and it was shown to be highly significant at the two higher mite density levels for Khalili Bovanat, Rishbaba and Sezdang Ghalat; Shahani and Sahebi Uroomie leaves appeared to be less affected by mite infestation. The highest mite density treatment displayed a strong correlation with weight (positive correlation) and size (negative correlation) of the leaves of four cultivars; leaves of Sahebi Uroomie appeared to be less affected. The reduced internode length was weak in infested plants. Most infested plants produced shorter canes and their lengths appeared to have a strong negative correlation with the highest mite density in four cultivars; canes of Sahebi Uroomie did not appear affected. At the highest mite density, canes of Khalili Bovanat and Sahebi Uroomie displayed the most and the least shortening effects, respectively. The percentage of leaves with erinea, as well as the number of erinea per leaves and the diameter of erinea increased along with the mite population density. The mite densities in buds (April 2014) and on leaves with erinea (in November 2013) were higher at the highest treatment level in the medium-late (Rishbaba) and late ripening (Sezdang Ghalat) cultivars, than in the early and early-medium ripening ones. Almost all data collected in the current experiment allowed the conclusion that Sahebi Uroomie and Shahani were less affected than the other cultivars (Khalili Bovanat, Rishbaba and Sezdang Ghalat).     

In vitro plantlet regeneration of “dwarf” Indian olive (Elaeocarpus robustus Roxb.): a fruit plant of Bangladesh

A plantlet regeneration protocol was developed on pot-grown mature plants of Elaeocarpus robustus Roxb. cv. Dwarf from nodal and leaf explants. The best yield of adventitious shoots was achieved from the leaf-derived calli in a modified MS (MMS₁, half strength of major salts, full strength of minor salts, and vitamins) medium containing 4.0 μM BA + 4.0 μM Kn + 0.5 μM NAA + 15% coconut water (CW). The shoot multiplication rate was amplified about twofold per culture after the addition of 15% CW to the medium. The rate of shoot multiplication reached maximum at the 5th subculture, and it maintained this rate throughout the 3 subsequent subcultures. The best rooting in vitro was investigated by subculturing the microcuttings in an MMS₂ (half strength of both major salts and minor salts and full strength of vitamins) medium containing 1.0 μM IBA in the dark for one initial week at 30°C, followed by subculturing them in a plant-growth regulator (PGR)-free medium in the light. The plantlets raised in vitro were successfully established under ex vitro conditions.     

Ribotyping of rhizobia nodulating Acacia mangium and Paraserianthes falcataria from different geographical areas in Indonesia using PCR-RFLP-SSCP (PRS) and sequencing

Acacia mangium and Paraserianthes falcataria are leguminous tree species widely grown for timber in Indonesia and other tropical countries, yet little is known about the identity of their rhizobial symbionts. Polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the 16S rRNA gene was used along with sequencing to assess the diversity of 57 rhizobia isolated from nodules of A. mangium and P. falctaria in Indonesia. In total, 26 rhizobia isolated from A. mangium were analysed by PRS and sequencing. The PRS patterns indicated that 12 (46%) clustered with Bradyrhizobium elkanii , 13 (50%) with B. lianoningense / japonicum and one (4%) with Mesorhizobium loti . Thirty-one isolates were analysed from P. falcataria : five (16%) clustered with B. elkanii and 26 (84%) with B. lianoningense / japonicum. These results were confirmed by phylogenetic analysis of sequences. Intraspecific diversity of the 16S rRNA genes from rhizobia nodulating A. mangium and P. falcataria revealed by PRS was low, only one genotype was found within the isolates that clustered with B. elkanii and two within the B. liaoningense / japonicum group. These Bradyrhizobium species are apparently ubiquitous throughout the Indonesian archipelago and it is clear why the two tree species are able to successfully establish outside their native range without the need for inoculation with indigenous rhizobia.     

Assessment of Genetic Stability Among In Vitro Plants of Arachis retusa Using RAPD and AFLP Markers for Germplasm Preservation

Arachis retusa Krapov. et W. C. Gregory et Valls is endemic in the West-central region of Brazil, occurring In areas endangered by human actions. The establishment of in vitro preservation methods for wild species of Arachis is an alternative to seed banks for germplasm storage, multiplication and distribution. The risk of genetic changes Induced by tissue culture and the monitoring of the genetic stability of the biological material before, during and after storage must be considered In the context of conservation. Random amplified polymorphlc ONA （RAPO） and amplified fragment length polymorphism （AFLP） fingerprinting were used to evaluate the genetic stability of in vitro plants originated from cotyledons and embryo axes of A. retusa. Cotyledons originated shoots through direct organogenesls and embryo axes displayed muItishoot formation Induced by 110 mmol/L and 8.8 mmol/L BAP, respectively. Ninety genomlc regions （loci） generated from RAPO and 372 from AFLP analyses were evaluated. All amplified fragments detected by both techniques in plants derived from the two explant types were monomorphic. The results Indicate that the recovered shoots are genetically stable at the assessed genomic regions.