DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet

Using a bisubstituted caspase-3 target sequence: aspartate-glutamate-valine-aspartate, (z-DEVD)2 peptide derivative of the fluorophore, cresyl violet, we have obtained a cell permeant, fluorogenic, caspase substrate capable of detecting the site-specific presence of functionally active, caspase-3 and caspase-7 up-regulation within intact apoptotic cells. Addition of this substrate to induced and noninduced cell culture populations allows for the rapid site-specific detection of caspase up-regulation without the requirement for a wash step. We demonstrate here the use of (z-DEVD)2-cresyl violet substrate for the detection of apoptosis induction in Jurkat, THP-1, and MCF-7 cells using fluorescence microscopy and 96-well fluorescence plate reader analysis. Intracellular up-regulated DEVDase activity, which was clearly visible by fluorescence microscopy and 96-well fluorescence plate reader measurements, showed greater than 6-fold increases in fluorescence output in induced versus noninduced Jurkat cell samples. A simple fluorogenic substrate conversion method is demonstrated here for detecting apoptosis induction within intact living cells.     

Nuclear zinc in liver of cadmium and zinc treated rats

X-ray microanalysis was performed to detect quantitatively, the variation of the nuclear zinc in the liver cells of rats. The nuclear zinc concentration showed statistical decrease and increase in response to cadmium and zinc treatments, respectively. The results suggest that the liver responds differently to cadmium and zinc treatments. The difference in response to either treatment may reflect different mechanisms of zinc transport and metabolism in the liver. The difference in binding affinity of metallothionein (MT) may suggest the involvement of Mt in the metabolism and transport of zinc, an effect, which may be modified by treatment.     

A small stress protein acts synergistically with trehalose to confer desiccation tolerance on mammalian cells

The ability to desiccate mammalian cells while maintaining a high degree of viability would be very important in many areas of biological science, including tissue engineering, cell transplantation, and biosensor technologies. Certain proteins and sugars found in animals capable of surviving desiccation might aid this process. We report here that human embryonic kidney (293H) cells transfected with the gene for the stress protein p26 from Artemia and loaded with trehalose showed a sharp increase in survival during air-drying. Further, we find vacuum-drying greatly improved the ability of the cells to survive, and that the physical shape and structure of the cellular sample had a large influence on recovery following rehydration. Cells suspended in a rounded droplet survived desiccation markedly better than those spread as a thin film. Finally, we used alamarBlue to monitor cellular metabolism and Hema 3 to assess colony formation after vacuum-drying. AlamarBlue fluorescence indicated that the transfected 293H cells expressing p26 (E11'L) grew much better than the control 293H cells. In fact, immediate survival and colony formation in E11'L cells increased as much as 34-fold compared with control cells when the samples were dried to a water content of 0.2 g H2O/g dry weight, as measured by gravimetric analysis. These results indicate that p26 improves cell survival following drying and rehydration, and suggest that dry storage of mammalian cells is a likely possibility in the future.     

Probing a hydrogen bond pair and the FAD redox properties in the proline dehydrogenase domain of Escherichia coli PutA

The PutA flavoprotein from Escherichia coli combines DNA-binding, proline dehydrogenase (PRODH), and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities onto a single polypeptide. Recently, an X-ray crystal structure of PutA residues 87-612 was solved which identified a D370-Y540 hydrogen bond pair in the PRODH active site that appears to have an important role in shaping proline binding and the FAD redox environment. To examine the role of D370-Y540 in the PRODH active site, mutants D370A, Y540F, and D370A/Y540F were characterized in a form of PutA containing only residues 86-601 (PutA86-601) designed to mimic the known structural region of PutA (87-612). Disruption of the D370-Y540 pair only slightly diminished k(cat), while more noticeable affects were observed in K(m). The mutant D370A/Y540F showed the most significant changes in the pH dependence of k(cat)/K(m) and K(m) relative to wild-type PutA86-601 with an apparent pK(a) value of about 8.2 for the pH-dependent decrease in K(m). From the pH profile of D370A/Y540F inhibition by l-tetrahydro-2-furoic acid (l-THFA), the pH dependency of K(m) in D370A/Y540F is interpreted as resulting from the deprotonation of the proline amine in the E-S complex. Replacement of D370 and Y540 produces divergent effects on the E(m) for bound FAD. At pH 7.0, E(m) values of -0.026, -0.089 and -0.042 V were determined for the two-electron reduction of bound FAD in D370A, Y540F and D370A/Y540F, respectively. The 40-mV positive shift in E(m) determined for D370A relative to wild-type PutA86-601 (E(m)=-0.066 V, pH 7.0) indicates D370 has a key role in modulating the FAD redox environment.     

Prolonged expression of c-Fos protein in the lateral habenular nucleus of the Japanese monkey (Macaca fuscata) after eye enucleation

To elucidate the effect of traumatic stress on the lateral habenular nucleus, we investigated the time course of the expression of c-Fos protein in this nucleus of the Japanese monkey (Macaca fuscata) after enucleation of one eye using c-Fos protein immunocytochemistry. c-Fos protein-like immunoreactive neurons were significantly increased; the increase started 1 h after the enucleation and remained high for 3-9 h in the lateral habenular nucleus on both sides. These results suggest that the prolonged expression of c-Fos protein occurred in the lateral habenular nucleus after traumatic stress through multiple transsynaptic activations.     

Molecular polymorphisms in Palestinian Figs (Ficus carica L.) as revealed by Random Amplified Polymorphic DNA (RAPD)

The genetic diversity of 13 local Palestinian fig genotypes was investigated using RAPD markers. Among the 30 tested primers, 28 revealed various banding patterns and 2 generated no polymorphic bands. In addition, 13 primers (46.4%) produced good amplification products with high intensity and pattern stability. A total of 94 DNA fragments (loci), separated by electrophoresis on agarose gel were detected, ranging in size from 190 to 1300 bp. Of these fragments, 72 (76.6%) were polymorphic and 22 (23.4%) were monomorphic. A minimum of three and a maximum of eight DNA fragments were obtained using (OPH-02 and OPT-10) as well as (OPA-13, OPA-18 and OPY-07) primers respectively. The maximum percentage of polymorphic markers was 100.0 (Z-5, Z-12, and OPT-10) and the minimum was 60.0 (OPH-02). Primers OPY-07 and OPA-13 revealed high collective resolving power (Rp) values with 4.640 and 4.760 respectively and therefore, they were the most useful RAPD primers to assess the genetic diversity in the Palestinian figs. Genetic distance matrix showed an average distance range from 0.186 to 0.559 with a mean of 0.373. Thus, the cultivars tested in this study were characterized by large divergence at the DNA level. To our knowledge, this is the first report using RAPD marker to assess genetic diversity of Palestinian figs.     

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.     

Enhancement in the germination, growth and yield of okra (Abelmoschus esculentus) using pre-sowing magnetic treatment of seeds

The effect of pre-sowing magnetic treatments was investigated on germination, growth and yield of okra (Abelmoschus esculentus cv. Sapz pari). The dry okra seeds were exposed to sinusoidal magnetic field induced by an electromagnet. The average magnetic field exposure was 99 mT for 3 and 11 min and seeds with no magnetic field treatment were considered as control. Both treated and non-treated seeds were sown in experimental plots (120 m2) under similar conditions. Samples were collected at regular intervals for statistical analysis. A significant increase (P < 0.05) was observed in germination percentage, number of flowers per plant, leaf area (cm2), plant height (cm) at maturity, number of fruits per plant, pod mass per plant and number of seeds per plant. The 99 mT for 11 min exposure showed better results as compared to control.     

Female spontaneously hypertensive rats have greater renal anti-inflammatory T lymphocyte infiltration than males

T cells contribute to hypertension in male experimental models; data in females is lacking even though women are more likely to develop immune disorders. The goal of this study was to determine whether immune cells contribute to hypertension in female spontaneously hypertensive rats (SHR) and define the T cell profile in whole blood and kidneys of male and female SHR. We hypothesized that inflammatory cells contribute to hypertension in female SHR; however, male SHR have a higher blood pressure so we hypothesize they will have a heightened inflammatory profile. The lymphocyte inhibitor mycophenolate mofetil (MMF) was administered in a dose-dependent manner to SHR. At the highest dose (50 mg·kg(-1)·day(-1)), blood pressure was significantly decreased in both sexes, yet the percent decrease in blood pressure was greater in females (female: 12 ± 1%; males: 7 ± 1%, P = 0.01). Circulating and renal T cell profiles were defined using analytical flow cytometry. Female SHR had more circulating CD3(+), CD4(+), and pro-inflammatory CD3(+)CD4(+)RORγ(+) Th17 cells, whereas males had more immune-suppressive CD3(+)CD4(+)Foxp3(+) T regulatory cells. In the kidney, females had greater numbers of CD8(+) and T regulatory cells than males, whereas males had greater CD4(+) and Th17 cell infiltration. MMF decreased circulating and renal T cells in both sexes (P < 0.0001), although the effect of MMF on T cell subtypes was sex specific with females having greater sensitivity to MMF-induced decreases in lymphocytes. In conclusion, there is a lymphocyte contribution to the maintenance of hypertension in the female SHR and sex of the animal impacts the T cell profile.     

Increase of CD4+CD25highFoxP3+ cells impairs in vitro human microbicidal activity against Mycobacterium tuberculosis during latent and acute pulmonary tuberculosis

BackgroundRegulatory T cells (Tregs) play a critical role during Mycobacterium tuberculosis (Mtb) infection, modulating host responses while neutralizing excessive inflammation. However, their impact on regulating host protective immunity is not completely understood. Here, we demonstrate that Treg cells abrogate the in vitro microbicidal activity against Mtb.MethodsWe evaluated the in vitro microbicidal activity of peripheral blood mononuclear cells (PBMCs) from patients with active tuberculosis (TB), individuals with latent tuberculosis infection (LTBI, TST+/IGRA+) and healthy control (HC, TST-/IGRA-) volunteers. PBMCs, depleted or not of CD4⁺CD25⁺ T-cells, were analyzed to determine frequency and influence on microbicidal activity during in vitro Mtb infection with four clinical isolates (S1, S5, R3, and R6) and one reference strain (H37Rv).ResultsThe frequency of CD4⁺CD25^highFoxP3⁺ cells were significantly higher in Mtb infected whole blood cultures from both TB patients and LTBI individuals when compared to HC. Data from CD4⁺CD25⁺ T-cells depletion demonstrate that increase of CD4⁺CD25^highFoxP3⁺ is associated with an impairment of Th-1 responses and a diminished in vitro microbicidal activity of LTBI and TB groups.ConclusionsTregs restrict host anti-mycobacterial immunity during active disease and latent infection and thereby may contribute to both disease progression and pathogen persistence.