Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30?kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

OPTIMIZATION OF AFFINITY DIGESTION FOR THE ISOLATION OF CELLULOSOMES FROM Clostridium thermocellum

The affinity digestion process for cellulase purification consisting of binding to amorphous cellulose, and amorphous cellulose hydrolysis in the presence of dialysis (Morag et al., 1991), was optimized to obtain high activity recoveries and consistent protein recoveries in the isolation of Clostridium thermocellum cellulase. Experiments were conducted using crude supernatant prepared from C. thermocellum grown on either Avicel or cellobiose. While no difference was observed between Avicel-grown or cellobiose-grown cellulase in the adsorption step, differences were observed during the hydrolysis step. The optimal amorphous cellulose loading was found to be 3 mg amorphous cellulose per milligram supernatant protein. At this loading, 90–100% of activity in the crude supernatant was adsorbed. Twenty-four-hour incubation with the amorphous cellulose during the adsorption stage was found to result in maximal and stable adsorption of activity to the substrate. By fitting the adsorption data to the Langmuir model, an adsorption constant of 410 L/g and a binding capacity of 0.249 g cellulase/g cellulose were obtained. The optimal length of time for hydrolysis was found to be 3 hr for cellulase purified from Avicel cultures and 4 hr for cellulase purified from cellobiose cultures. These loadings and incubation times allowed for more than 85% activity recovery.     

Genital microsporidiosis in women with AIDS: A post-mortem study

BackgroundMicrosporidiosis is a life threatening opportunistic infection of AIDS patients. The infection is usually restricted to specific anatomical areas, but could become systemic depending on the involved species. Genital microsporidiosis in female patients is rare.ObjectiveTo report genital microsporidiosis in female AIDS patients.MethodsTissues samples from the genital tract (ovary, fallopian tubes and uterus) of eight deceased women who died of wasting syndrome associated to AIDS and disseminated microsporidiosis at the Institute of Tropical Medicine Pedro Kourí were collected between 1997 and 2005. Using an indirect immunohistochemistry assay the microsporidia species involved in those cases were identified.ResultsWe report several cases of microsporidial infection of the female genital tract. Six out of eight women with the disseminated form of the disease showed the presence of microsporidia in the genital tract. Encephalitozoon cuniculi and Encephalitozoon hellem were identified in the internal lining epithelium of the fallopian tubes and endometrium.ConclusionsMicrosporidia species could disseminate to other organs and become systemic in severe immunocompromised cases. To our knowledge this is the greatest number of female genital tract microsporidiosis cases so far reported in humans.     

Evidence for progressive phosphorus limitation over long-term ecosystem development: Examination of a biogeochemical paradigm

Aims

To test predictions of ecosystem theory for changes in P cycling over primary succession, we determined soil phosphorus (P) in labile, primary mineral, organic, and occluded forms along a chronosequence of five wave cut terraces known as the “Ecological Staircase”. The Ecological Staircase terraces (T1-T5) transition naturally from fertile native coastal forests in California, USA, to diminutive pygmy vegetation over the span of?>?500,000 years of pedogenesis.

Methods

Soil P fractions were quantified to a depth of 40 cm on T1-T5 using a modified Hedley P fractionation procedure.

Results

Overall results confirmed the Walker and Syers Model of Phosphorus Transformations During Pedogenesis: total P declined from youngest (194 mg/kg P) to oldest (127 mg/kg P) sites; primary-mineral P decreased sharply from T1 to older sites; and occluded P dominated P pools at the oldest pygmy sites (T3-T5). In addition, foliar P concentrations declined markedly in the pygmy forest, and N/P of vegetation (T1: 6.03, T5: 14.4) and N/P_organic of mineral soils (T1: 6.10, T5: 25.3) increased significantly over time.

Conclusions

Results point to P as the primary limiting nutrient in the pygmy forest, exemplifying the terminal steady-state of ecosystem retrogression that underlies the persistence of this unique ecosystem.