RNA,proteins and polyamines during gametophytic and androgenetic development of pollen in Nicotiana tabacum and Datura innoxia

Variations in polyamines, proteins and RNA during in vivo gametogenesis and in vitro androgenesis in Datura innoxia and in Nicotiana tabacum were studied. Spermidine was the major polyamine during gametogenesis in both species. Marked differences in proteins, RNA and polyamines were evident during meiosis and at the first haploid mitosis. In Nicotiana an unknown amine (X₆₀) appears at the beginning of the first haploid mitosis. At the same time a rapid increase in the concentrations of RNA and proteins is observed. In Datura, at the time of the first haploid mitosis there is large increase in amine and RNA levels followed by an arginine peak. During androgenesis, putrescine and spermidine were the major polyamines in both species. In Nicotiana during androgenesis an unknown amine (X₈₁) was observed together with putrescine and spermidine. This unknown compound peaks during the developmental stages of embryogenesis. In Datura androgenic induction was marked by an arginine peak followed by an increase in the putrescine and spermidine levels associated with maximum RNA. These biochemical events are tentatively correlated with structural changes during pollen development. The significance of these results is discussed in relation to the role of polyamines during gametogenesis and androgenesis.     

Self-association of the Lentivirus protein, Nef

Background

The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization.

Results

By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV_MAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface.

Conclusions

We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.     

Selective reactivity of 2-mercaptoethanol with 5beta,6beta-epoxide in steroids from Withania somnifera

2-Mercaptoethanol reacts selectively with the 5beta,6beta-epoxy steroids isolated from Withania somnifera substituting the epoxide by a six-membered oxyethylene-2'-thio ring whereas it failed to show such reactivity on 6alpha,7alpha-epoxy withasteroids. The structure of the product has been elucidated by spectroscopic methods, especially applying extensive 2D NMR methods. The anticancer activity of withaferin A was lost in the reaction product indicating that its activity is also linked to the free 5beta,6beta-epoxide functional group.     

Regulation of the Met receptor-tyrosine kinase by the protein-tyrosine phosphatase 1B and T-cell phosphatase

The non-receptor protein-tyrosine phosphatases (PTPs) 1B and T-cell phosphatase (TCPTP) have been implicated as negative regulators of multiple signaling pathways including receptor-tyrosine kinases. We have identified PTP1B and TCPTP as negative regulators of the hepatocyte growth factor receptor, the Met receptor-tyrosine kinase. In vivo, loss of PTP1B or TCPTP enhances hepatocyte growth factor-mediated phosphorylation of Met. Using substrate trapping mutants of PTP1B or TCPTP, we have demonstrated that both phosphatases interact with Met and that these interactions require phosphorylation of twin tyrosines (Tyr-1234/1235) in the activation loop of the Met kinase domain. Using confocal microscopy, we show that trapping mutants of both PTP1B and the endoplasmic reticulum-targeted TCPTP isoform, TC48, colocalize with Met and that activation of Met enables the nuclear-localized isoform of TCPTP, TC45, to exit the nucleus. Using small interfering RNA against PTP1B and TCPTP, we demonstrate that phosphorylation of Tyr-1234/1235 in the activation loop of the Met receptor is elevated in the absence of either PTP1B or TCPTP and further elevated upon loss of both phosphatases. This enhanced phosphorylation of Met corresponds to enhanced biological activity and cellular invasion. Our data demonstrate that PTP1B and TCPTP play distinct and non-redundant roles in the regulation of the Met receptor-tyrosine kinase.     

Minnelide: A Novel Therapeutic That Promotes Apoptosis in Non-Small Cell Lung Carcinoma In Vivo

Background

Minnelide, a pro-drug of triptolide, has recently emerged as a potent anticancer agent. The precise mechanisms of its cytotoxic effects remain unclear.

Methods

Cell viability was studied using CCK8 assay. Cell proliferation was measured real-time on cultured cells using Electric Cell Substrate Impedence Sensing (ECIS). Apoptosis was assayed by Caspase activity on cultured lung cancer cells and TUNEL staining on tissue sections. Expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA, APAF-1) was estimated by qRTPCR. Effect of Minnelide on proliferative cells in the tissue was estimated by Ki-67 staining of animal tissue sections.

Results

In this study, we investigated in vitro and in vivo antitumor effects of triptolide/Minnelide in non-small cell lung carcinoma (NSCLC). Triptolide/Minnelide exhibited anti-proliferative effects and induced apoptosis in NSCLC cell lines and NSCLC mouse models. Triptolide/Minnelide significantly down-regulated the expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA) and up-regulated pro-apoptotic APAF-1 gene, in part, via attenuating the NF-κB signaling activity.

Conclusion

In conclusion, our results provide supporting mechanistic evidence for Minnelide as a potential in NSCLC.     

On approaches to the functional restoration of salivary glands damaged by radiation therapy for head and neck cancer,with a review of related aspects of salivary gland morphology and development

Radiation therapy for cancer of the head and neck can devastate the salivary glands and partially devitalize the mandible and maxilla. As a result, saliva production is drastically reduced and its quality adversely altered. Without diligent home and professional care, the teeth are subject to rapid destruction by caries, necessitating extractions with attendant high risk of necrosis of the supporting bone. Innovative techniques in delivery of radiation therapy and administration of drugs that selectively protect normal tissues can reduce significantly the radiation effects on salivary glands. Nonetheless, many patients still suffer severe oral dryness. I review here the functional morphology and development of salivary glands as these relate to approaches to preventing and restoring radiation-induced loss of salivary function. The acinar cells are responsible for most of the fluid and organic material in saliva, while the larger ducts influence the inorganic content. A central theme of this review is the extent to which the several types of epithelial cells in salivary glands may be pluripotential and the circumstances that may influence their ability to replace cells that have been lost or functionally inactivated due to the effects of radiation. The evidence suggests that the highly differentiated cells of the acini and large ducts of mature glands can replace themselves except when the respective pools of available cells are greatly diminished via apoptosis or necrosis owing to severely stressful events. Under the latter circumstances, relatively undifferentiated cells in the intercalated ducts proliferate and redifferentiate as may be required to replenish the depleted pools. It is likely that some, if not many, acinar cells may de-differentiate into intercalated duct-like cells and thus add to the pool of progenitor cells in such situations. If the stress is heavy doses of radiation, however, the result is not only the death of acinar cells, but also a marked decline in functional differentiation and proliferative capacity of all of the surviving cells, including those with progenitor capability. Restoration of gland function, therefore, seems to require increasing the secretory capacity of the surviving cells, or replacing the acinar cells and their progenitors either in the existing gland remnants or with artificial glands.     

Changes in the bacterial community and lin genes diversity during biostimulation of indigenous bacterial community of hexachlorocyclohexane (HCH) dumpsite soil

In this study hexachlorocyclohexane (HCH) contaminated soil (with HCH level 84 g/kg of soil) from HCH dumpsite (Ummari village, Lucknow, India) was used to demonstrate biostimulation approach for HCH bioremediation. Different nutrients (molasses and ammonium phosphate) were used in different pits having contaminated soil to stimulate the indigenous microbial community. There was a substantial reduction in the total HCH content of the soil in 12 months long experiment. Maximum reduction was seen in the pit that received a combination of molasses and ammonium phosphate. A change in the microbial community concomitant with degradation of HCH was observed. Sphingomonads, which are known degraders of HCH, were found to dominate the experimental pits. Moreover changes in linA and linB gene (primary genes involved in HCH degradation) diversity and number were also seen as revealed by T-RFLP and RT-PCR respectively. The study suggests the prospects of biostimulation in decontaminating soils heavily contaminated with HCH.     

Qualitative and quantitative variations in withanolides and expression of some pathway genes during different stages of morphogenesis in Withania somnifera Dunal

Withania somnifera Dunal is an important and extensively studied medicinal plant; however, there is no report available that relates withanolide content and its profile in relation to the expression of pathway genes during different morphogenic stages. In this study, withanolide A, withaferin A, and withanone, the major withanolides of W. somnifera, were measured in different in vitro stages during organogenesis, viz., shoot to root (direct rhizogenesis)/root to shoot (indirect via callus phase) transition vis-à-vis expression levels of key pathway genes involved in withanolide biosynthetic pathways. The morphogenic transitions were found to be tightly linked to the pattern of accumulation of withanolides. The high expression levels of most of the pathway genes in in vitro shoots in comparison to in vitro root and callus tissues exhibited a direct co-relation with the maximum withanolide content (>2.7 mg/gDW). The biogenesis of withaferin A, a major constituent of the leaves, was however found to be tightly linked to shoots/green tissue. In addition, we were also able to establish an efficient regeneration system from roots for their further utilization in biotechnological applications.     

Trehalose determination in linseed subjected to osmotic stress. HPAEC‐PAD analysis: an inappropriate method

Trehalose is a non‐reducing disaccharide involved in stress tolerance in plants. To understand better the role of trehalose in the osmotic stress response in linseed (Linum usitatissimum), trehalose content in leaves was studied. First, the method commonly used for sugar determination, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD), gave unsatisfactory results and the separation efficiency could not be improved by varying the elution conditions. The same problem was also found in the model plant: Arabidopsis thaliana. After clearly highlighting a co‐elution of trehalose in these two species by a trehalase assay and liquid chromatography‐high resolution mass spectrometry analysis, gas chromatography–mass spectrometry (GC‐MS) was used as the analytical method instead. These results confirmed that trehalose content is currently overestimated by HPAEC‐PAD analysis, approximately 7 and 13 times for A. thaliana and linseed respectively. Thus GC‐MS gave more satisfactory results for trehalose quantification in plants. With this method, trehalose accumulation was observed in linseed during an osmotic stress (?0.30 MPa), the quantity (31.49 nmol g^–1 dry weight after 48 h) appears too low to assign an osmoprotector or osmoregulator role to trehalose in stressed linseed.