Effects of Gibberellic Acid on Primary Terpenoids and Δ9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA₃) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA₃ caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Δ⁹-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA₃ showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA₃ can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

Polymorphism in the packing of aquaporin-1 tetramers in 2-D crystals

Hitherto, the packing arrangement of the aquaporin-1 (AQP1) tetramer in 2-dimensional (2-D) crystals (two-sided plane group p42(1)2) was observed to be largely similar (canonical crystal form) despite the difference in the source of the protein, the glycosylation state of the protein, the type of lipids, and the ratio of lipid to protein in the crystallization mixture. We report here our observation that the packing of AQP1 tetramers shows polymorphism in 2-D crystals generated in dioleoyl phosphatidylcholine bilayers. Apart from the canonical form, three additional allomorphs were identified. One was observed when small (0.25) lipid to protein ratio was used in the crystallization mixture while the other two were observed when the divalent cation content in the canonical crystals was modified. The various allomorphs were distinguished by different relative orientations of the AQP1 tetramer viewed in projection. The same, two-sided plane group p42(1)2 and similar unit cell dimensions were maintained in the different allomorphs as established by analysis of images of frozen-hydrated, nominally untilted crystals. Our results indicate that the interaction between the AQP1 monomers at the interface of the tetramers is flexible and is also strongly influenced by Mg(2+) ions with the cation effect materializing because of the intrinsic fluidity of the membrane.     

Tissue-enhanced plasma proteomic analysis for disease stratification in amyotrophic lateral sclerosis

Background

It is unclear to what extent pre-clinical studies in genetically homogeneous animal models of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder, can be informative of human pathology. The disease modifying effects in animal models of most therapeutic compounds have not been reproduced in patients. To advance therapeutics in ALS, we need easily accessible disease biomarkers which can discriminate across the phenotypic variants observed in ALS patients and can bridge animal and human pathology. Peripheral blood mononuclear cells alterations reflect the rate of progression of the disease representing an ideal biological substrate for biomarkers discovery.

Methods

We have applied TMTcalibrator?, a novel tissue-enhanced bio fluid mass spectrometry technique, to study the plasma proteome in ALS, using peripheral blood mononuclear cells as tissue calibrator. We have tested slow and fast progressing SOD1G93A mouse models of ALS at a pre-symptomatic and symptomatic stage in parallel with fast and slow progressing ALS patients at an early and late stage of the disease. Immunoassays were used to retest the expression of relevant protein candidates.

Results

The biological features differentiating fast from slow progressing mouse model plasma proteomes were different from those identified in human pathology, with only processes encompassing membrane trafficking with translocation of GLUT4, innate immunity, acute phase response and cytoskeleton organization showing enrichment in both species. Biological processes associated with senescence, RNA processing, cell stress and metabolism, major histocompatibility complex-II linked immune-reactivity and apoptosis (early stage) were enriched specifically in fast progressing ALS patients. Immunodetection confirmed regulation of the immunosenescence markers Galectin-3, Integrin beta 3 and Transforming growth factor beta-1 in plasma from pre-symptomatic and symptomatic transgenic animals while Apolipoprotein E differential plasma expression provided a good separation between fast and slow progressing ALS patients.

Conclusions

These findings implicate immunosenescence and metabolism as novel targets for biomarkers and therapeutic discovery and suggest immunomodulation as an early intervention. The variance observed in the plasma proteomes may depend on different biological patterns of disease progression in human and animal model.

     

Phytoestrogenic effects of black tea extract (Camellia sinensis) in an oophorectomized rat (Rattus norvegicus) model of osteoporosis

The adverse side effects of currently available anti-osteoporotic agents warrant the search for compounds with less toxic effects. In this study, we assessed the phytoestrogenic potentiality of whole aqueous extract of black tea (BTE) in a bilaterally oophorectomized rat model (2.5%, 1 ml/100 g body weight/day for 28 days). Although the supplementation was given for 28 days but, sign of revival of copulation period (estrous stage) from non-receptive diestrous stage was first noticed after 21 days of BTE supplementation in bilaterally oophorectomized rats. This was accompanied by a significant increase in serum estradiol level. To test whether this increase in serum estradiol level could have an influence upon the oophorectomy-induced damage of bone, we assessed marker parameters of bone resorption and osteoclastic activity (tartrate-resistant acid phosphatase), collagen degradation (urinary hydroxyproline), bone loss (bone ash mineral content) and bone breaking strength (bone density). Results indicated that increase in serum estradiol level after BTE supplementation could significantly diminish oophorectomy-induced decaying changes in bone. This study proposes that aqueous BTE may be assessed as a phytoestrogenic compound for prevention against estrogen deficiency-related osteoporotic damages.     

Mitochondrial complex I activity is impaired during HIV-1-induced T-cell apoptosis

Studies carried out till date to elucidate the pathways involved in HIV-1-induced T-cell depletion has revealed that apoptosis underlie the etiology, however, a clear molecular understanding of HIV-1-induced apoptosis has remained elusive. Although evidences pointing towards the importance of mitochondrial energy generating system in apoptosis exist but it's exact role remains to be clearly understood. Here, we describe for the first time specific downregulation of a complex I subunit NDUFA6 with simultaneous impairment of mitochondrial complex I activity in HIV infection. We also show that NDUFA6 gene silencing induces apoptosis and its overexpression reduces apoptosis in HIV-infected cells. Finally, sensitivity to complex I inhibitor Rotenone is reduced in HIV-1-infected T cells indicating an important role for it in the death process. Our data provide a novel molecular basis as to how the virus might interfere with host cell energy generating system during apoptotic cell death.     

Effect of reactive oxygen species on the metabolism of tryptophan in rat brain: Influence of age

In an earlier study, oxidation of tryptophan hydroxylase was implicated as its affinity was decreased with aging in rat brain. To establish any potential link between its oxidative damage and aging, we have determined the activities of antioxidant enzymes in midbrain, pons and medulla of 2, 12 and 24 month old Fisher 344 BNF1 rats. The results obtained suggest that the activities of antioxidant enzymes varied considerably with age and brain regions studied. Activities of Cu/Zn superoxide dismutase and glutathione peroxidase were found to increase from 2 to 12 months and then decrease in 24 month old rats. However catalase activity decreased consistently with the age. A parallel increase in the carbonyl content was observed in these brain regions indicating the oxidation of proteins. Reactive oxygen species when included in the incubation mixture decreased the activity of tryptophan hydroxylase in a concentration dependent manner. The loss of tryptophan hydroxylase activity induced by hydrogen peroxide and superoxide anion was prevented by catalase. However superoxide dismutase did not provide such protection. Sulfhydryl agents, cysteine, glutathione and dithiothreitol partially prevented the loss of activity. These studies suggest an involvement of reactive oxygen species for sulfhydryl oxidation of tryptophan hydroxylase in aging.     

Protein kinase B/Akt regulates coxsackievirus B3 replication through a mechanism which is not caspase dependent

The role of signaling pathways including the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) during viral infection has gained much recent attention. Our laboratory reported on an important regulatory role for extracellular signal-regulated kinases (ERK1/2), subfamily members of the MAPKs, during coxsackievirus B3 (CVB3) infection. However, the role of the PI3K pathway in CVB3 infection has not been well characterized. CVB3 is the most common known viral infectant of heart muscle that directly injures and kills infected cardiac myocytes during the myocarditic process. In the present study, we investigated the role of protein kinase B (PKB) (also known as Akt), a general downstream mediator of survival signals through the PI3K cascade, in regulating CVB3 replication and virus-induced apoptosis in a well-established HeLa cell model. We have demonstrated that CVB3 infection leads to phosphorylation of PKB/Akt on both Ser-473 and Thr-308 residues through a PI3K-dependent mechanism. Transfection of HeLa cells with a dominant negative mutant of Akt1 or pretreatment of wild-type HeLa cells with the specific PI3K inhibitor LY294002 significantly suppresses viral RNA expression, as reflected in diminished viral capsid protein expression and viral release. Dominant negative Akt1 and LY294002 also increase apoptosis in infected cells, which can be reversed by addition of the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk). Interestingly, blocking of apoptosis by zVAD.fmk does not reverse the viral RNA translation blockade, indicating that the inhibitory effect of dominant negative Akt1 on viral protein expression is not caspase dependent. In addition, we showed that the attachment of virus to its receptor-coreceptor complex is not sufficient for PKB/Akt activation and that postentry viral replication is required for Akt phosphorylation. Taken together, these data illustrate a new and imperative role for Akt in CVB3 infection in HeLa cells and show that the PI3K/Akt signaling is beneficial to CVB3 replication.     

Similar pattern in cardiac differentiation of human embryonic stem cell lines, BG01V and ReliCellhES1, under low serum concentration supplemented with bone morphogenetic protein-2

Human embryonic stem cells (hESCs) can differentiate into cardiomyocytes, but the efficiency of this process is highly variable. So, developing generic differentiation protocols and their empirical testing on a range of independently derived hESC lines pose a daunting challenge due to considerable diversity in culture methods practiced between lines. Maintenance of BG01V and ReliCellhES1 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual passaging. We assessed cardiac differentiation from both the cell lines via embryoid body (EB) formation. Subsequent culture in low fetal bovine serum (5%)-containing medium produced spontaneously contracting EBs, in the presence of bone morphogenetic protein-2 (BMP-2; 25 ng/ml). Derived cardiomyocytes expressed cardiac genes and proteins and responded to functional assays. Further, the activation of the Smad signaling machinery evoked by BMP-2 has been confirmed through inhibitor studies. Therefore, in our hands, the same differentiation conditions functioned in two independently derived hESC lines. Similar studies in other lines may facilitate development of universal protocols. The present data may also provide valuable insights for testing of other factors that might promote cardiomyocyte differentiation in low-serum formulations.     

DNA transfer into human lung cells is improved with Tat-RGD peptide by caveoli-mediated endocytosis

Cell lines and primary cells exhibit varying degrees of resistance to DNA transfection strategies. In this study, we employed the synthetic peptide Tat-RGD (TR), composed of the HIV-1 derived translocation peptide Tat fused to the integrin binding RGD motif, as a tool for improving DNA transfer into pulmonary cells. Binding experiments between DNA and TR and cytotoxicity measurements of TR treated cells were undertaken to optimize DNA and TR concentrations for transfection. Addition of a complex of TR and DNA (TRD) to A549 cells yielded significant transgene expression. When TRD was combined with Lipofectamine (TRDL), the expression was increased by 5-fold over Lipofectamine (DL) and by approximately 30-fold over TRD-mediated transfections. Also, in primary smooth muscle cells (SMC) and fibroblasts (FB) derived from pulmonary arteries, an increase in TRDL-mediated transfection efficiency was observed by a factor of approximately 2 and approximately 3 over that of DL. Laser scanning confocal microscopy for visualizing TR-dependent DNA uptake demonstrated that the internalization of TRDL complexes is linked to caveoli in the plasma membrane. Interfering with caveoli formation by methyl-b-cyclo-dextrin drastically decreased the transfection efficiency by TR. In conclusion, the Tat-RGD peptide mediates efficient gene delivery in human pulmonary cells, in particular when combined with a standard cationic lipid based transfection reagent. The enhancement of DNA uptake by Tat-RGD is suggested to be mediated by caveoli-dependent endocytosis.