miR-497 and miR-302b regulate ethanol-induced neuronal cell death through BCL2 protein and cyclin D2

In chronic alcoholism, brain shrinkage and cognitive defects because of neuronal death are well established, although the sequence of molecular events has not been fully explored yet. We explored the role of microRNAs (miRNAs) in ethanol-induced apoptosis of neuronal cells. Ethanol-sensitive miRNAs in SH-SY5Y, a human neuroblastoma cell line, were identified using real-time PCR-based TaqMan low-density arrays. Long-term exposure to ethanol (0.5% v/v for 72 h) produced a maximum increase in expression of miR-497 (474-fold) and miR-302b (322-fold). Similar to SH-SY5Y, long-term exposure to ethanol induced miR-497 and miR-302b in IMR-32, another human neuroblastoma cell line. Using in silico approaches, BCL2 and cyclin D2 (CCND2) were identified as probable target genes of these miRNAs. Cotransfection studies with 3'-UTR of these genes and miRNA mimics have demonstrated that BCL2 is a direct target of miR-497 and that CCND2 is regulated negatively by either miR-302b or miR-497. Overexpression of either miR-497 or miR-302b reduced expression of their identified target genes and increased caspase 3-mediated apoptosis of SH-SY5Y cells. However, overexpression of only miR-497 increased reactive oxygen species formation, disrupted mitochondrial membrane potential, and induced cytochrome c release (mitochondria-related events of apoptosis). Moreover, ethanol induced changes in miRNAs, and their target genes were substantially prevented by pre-exposure to GSK-3B inhibitors. In conclusion, our studies have shown that ethanol-induced neuronal apoptosis follows both the mitochondria-mediated (miR-497- and BCL2-mediated) and non-mitochondria-mediated (miR-302b- and CCND2-mediated) pathway.     

Impact of exogenous silicon addition on chromium uptake, growth, mineral elements, oxidative stress, antioxidant capacity, and leaf and root structures in rice seedlings exposed to hexavalent chromium

Hydroponic experiments were conducted to investigate the role of exogenous silicon (Si) addition in increasing hexavalent chromium (Cr VI) tolerance in rice seedlings. Rice seedlings were grown under 100 μM Cr(VI) stress without or with 10 μM Si. Chromium treatment decreased growth, photosynthetic pigments and protein, which was accompanied by a significant increase in Cr accumulation and lipid peroxidation (as malondialdehyde; MDA). However, Si addition alleviated Cr toxicity and promoted growth of rice by decreasing Cr accumulation, root-to-shoot Cr transport and MDA level. Contents of macro (Mg, Ca and K) as well as micronutrients (Zn and Fe) were decreased by Cr except Mn while Si addition prevented decrease in these nutrients induced by Cr. Antioxidant capacity and total phenolic contents were decreased by Cr while these indices improved by Si addition. Treatment of Cr decreased the length of leaf epidermal cells and stomatal frequency, and adversely affected chloroplasts containing mesophyll cells and integrity of xylem and phloem, and Si addition minimized these abnormalities. However, frequency of root hairs was increased by Cr treatment. Results showed that exogenous Si addition enhanced Cr(VI) tolerance in rice seedlings by decreasing Cr accumulation, root-to-shoot Cr transport and MDA level, and by increasing content of some mineral elements (K, Fe and Zn) and antioxidant capacity compared to the Cr treatment alone.     

Neuroinflammatory reactions in sickness behavior induced by bacterial infection: Protective effect of minocycline

The neurological changes elicited by bacterial infection are called sickness behavior. Minocycline (MIN) is neuroprotective with a remarkable brain tissue penetration. MIN was orally administered at a dose 90 mg/kg for 3 days, whereas Escherichia coli was given as a single intraperitoneal injection (0.2 mL of 24 h growth) on the third day. After 24 h of bacterial infection, behavioral tests namely open field and forced swimming were carried out, then animals were decapitated. Rats infected with E. coli displayed reduced struggling time in forced swimming test, as well as, exploration and locomotion in open field test with reduction in neurotransmitters (norepinephrine, dopamine, and serotonin) versus elevation in the inflammatory (tumor necrosis factor‐alpha, interferon‐gamma) and oxidative stress (thiobarbituric acid reactive substance, reduced glutathione) biomarkers. Inflammatory infiltrates of nuclear cells were observed in brains of infected rats. MIN administration prevented the deleterious effects of E. coli infection, thus protects against sickness behavior possibly via defending from neuroinflammation.     

Evaluation of adaptability to different seasons in goat breeds of semi-arid region in India through differential expression pattern of heat shock protein genes

The present study was conducted to examine differential expression pattern of HSP genes and adaptability in Indian goat breeds of semi-arid region. The study was conducted in five animals from each breed viz. Barbari, Sirohi, and Jhakrana during winter, thermo-neutral and summer seasons. The respiratory rate (RR) and rectal temperature (RT) of the goats were recorded at 09:00 h during the study period. The blood samples were collected for RNA isolation, cDNA synthesis, and quantitative analysis of HSP genes expression by quantitative RT-PCR. The RR increased significantly (p < 0.01) during summer as compared to winter and thermo-neutral season however, RT did not change (p > 0.05) during different seasons. The expression of HSP genes was significantly (p < 0.01) increased during summer (high THI) as compared to thermo-neutral season in all the goat breeds. Among HSPs, only HSP90 was upregulated (p < 0.01) in Jhakrana goats during winter as compared to thermo-neutral season. The deviation in expression of HSP genes during summer and winter with respect to thermo-neutral season was minimum in Barbari goats. Therefore, it can be concluded that Barbari goats possessed better adaptability during summer and winter as compared to Sirohi and Jhakrana goats in semi-arid climatic conditions of India.     

Structure based molecular design, synthesis and biological evaluation of α-pyrone analogs as anti-HSV agent

Several options for treating Herpes Simplex Virus type 1 and type 2 are available. However, non-specific inhibition and drug resistance warrants the discovery of new anti-herpetic compounds with better therapeutic profile or different mode of action. The non-nucleoside inhibitors of HSV DNA polymerase target the site that is less important for the binding of a natural nucleoside or nucleoside inhibitors. In the present study, we have explored the possibility to find a new lead molecule based on α-pyrone analogs as non-nucleoside inhibitors using structure based modeling approach. The designed molecules were synthesized and evaluated for anti-HSV activity using MTT assay. The compound 5h with EC(50) 7.4μg/ml and CC(50) 52.5μg/ml was moderately active against HSV when compared to acyclovir. A plaque reduction assay was also carried out and results reveal that 5h is more effective against HSV-1 with better selective index of 12.8 than against HSV-2 (SI=3.6). The synthesized compounds were also evaluated for anti-HIV activity, but none were active.     

Expression of a heterologous S-adenosylmethionine decarboxylase cDNA in plants demonstrates that changes in S-adenosyl-L-methionine decarboxylase activity determine levels of the higher polyamines spermidine and spermine

We posed the question of whether steady-state levels of the higher polyamines spermidine and spermine in plants can be influenced by overexpression of a heterologous cDNA involved in the later steps of the pathway, in the absence of any further manipulation of the two synthases that are also involved in their biosynthesis. Transgenic rice (Oryza sativa) plants engineered with the heterologous Datura stramonium S-adenosylmethionine decarboxylase (samdc) cDNA exhibited accumulation of the transgene steady-state mRNA. Transgene expression did not affect expression of the orthologous samdc gene. Significant increases in SAMDC activity translated to a direct increase in the level of spermidine, but not spermine, in leaves. Seeds recovered from a number of plants exhibited significant increases in spermidine and spermine levels. We demonstrate that overexpression of the D. stramonium samdc cDNA in transgenic rice is sufficient for accumulation of spermidine in leaves and spermidine and spermine in seeds. These findings suggest that increases in enzyme activity in one of the two components of the later parts of the pathway leading to the higher polyamines is sufficient to alter their levels mostly in seeds and, to some extent, in vegetative tissue such as leaves. Implications of our results on the design of rational approaches for the modulation of the polyamine pathway in plants are discussed in the general framework of metabolic pathway engineering.     

Identification of G-proteins in rat parotid gland plasma membranes and granule membranes: presence of distinct components in granule membranes

We have identified by immunoblotting and ADP-ribosylation by cholera toxin and pertussis toxin the presence of Mr 43 and 46 KDa G_s, and 39 and 41 KDa G_i;.. subunits in rat parotid gland plasma membranes but not in granule membranes. A Mr 28 KDa polypeptide that served as substrate for ADP-ribosylation by both cholera toxin and pertussis toxin was present exclusively in granule membranes. Photoaffinity crosslinking of [-³²P]GTP showed the presence of high molecular weight GTP-binding proteins (Mr 160,100 KDa) in granule membranes. Six low molecular weight GTP-binding proteins (Mr 21–28 KDa) were differentially distributed in both plasma membranes and granule membranes. The present study identifies various GTP-binding proteins in rat parotid gland plasma membranes and granule membranes, and demonstrates the presence of distinct molecular weight GTP-binding proteins in granule membranes. These granule-associated GTP-binding proteins may be involved in secretory processes.     

Evidence that insulin-like growth factor-1 requires protein kinase C-epsilon, PI3-kinase and mitogen-activated protein kinase pathways to protect human vascular smooth muscle cells from apoptosis

Insulin-like growth factor (IGF)-1 has been implicated in the development of occlusive vascular lesions. Although its role in vascular smooth muscle cell (VSMC) growth and migration are fairly well characterized, anti-apoptotic signals of IGF-1 in human VSMC remain largely unknown. In this study, we examined IGF-1 signals that protect human and rat VSMC from staurosporine (STAU)- and c-myc- induced apoptosis, respectively. Treatment with STAU resulted in apoptotic DNA fragmentation, phosphatidylserine externalization and cell shrinkage, but only occasional VSMC 'blebbing'. STAU-induced death and IGF-1-mediated survival were concentration dependent, while time-lapse video microscopy showed that IGF-1 inhibited c-myc-induced apoptosis by 90%. Pretreatment with mitogen-activated protein kinase/extracellular signal regulated kinase kinase (MEK) inhibitors UO126 and PD098059, or with the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, reversed IGF-1-mediated human VSMC survival by 25-27% and 66%, respectively. Translocation studies showed that IGF-1 activated protein kinase C (PKC)-epsilon, but not PKC-alpha or PKC-delta, even in the presence of STAU, while pharmacological PKC inhibition (Ro-318220 or Go6976) implicated PKC-zeta or a novel PKC isozyme in IGF-1-mediated survival. Transient expression of activated PKC-epsilon but not activated PKC-zeta decreased myc-induced apoptosis in rat VSMC. In human VSMC, antisense oligodeoxynucleotides to PKC-epsilon partially reversed IGF-1-induced survival. In addition, IGF-1 elicited a mild but sustained activation of extracellular signal regulated kinase (ERK)1/2 in human VSMC that was abolished after 1 h in the presence of STAU. PKC downregulation reversed both IGF-1- and PMA-induced ERK activity, but platelet-derived growth factor (PDGF)-induced activity was unchanged. These results indicate for the first time that IGF-1 can protect human VSMC via multiple signals, including PKC-epsilon, PI3-K and mitogen-activated protein kinase pathways.