Purification and immunological characterization of a 12-kDa allergen from Epicoccum purpurascens

Exposure to Epicoccum purpurascens is implicated in respiratory allergies and asthma. Several allergens of clinical importance were identified in Epicoccum extract (EE), but only one allergen has been isolated and characterized. In the present study, a 12-kDa allergen was isolated from an Epicoccum spore-mycelial extract by concanavalin-A sepharose, reverse-phase hydrophobic and gel filtration chromatography. The purified protein was recognized as a single 12-kDa allergen on immunoblot with a serum pool of Epicoccum- sensitive patients. Of the 94 respiratory allergy patients tested intradermally, 17 showed marked positive skin reactions to EE and 12 of them reacted with the 12-kDa protein, indicating a diagnostic sensitivity of 70%. More than 80% patients' sera showed immunoglobulin E (IgE) reactivity to the purified protein in enzyme-linked immunosorbent assay and immunoblot, identifying it as a major allergen. Preincubation of pooled serum with the protein led to inhibition of IgE binding to solid-phase-bound EE (effective concentration 50%=180 ng). Twelve of the 17 serum samples showed significant basophil histamine release upon stimulation with purified protein. The protein induced significant proliferation of peripheral blood mononuclear cells of 13 patients. A high level of interleukin-4 in the culture supernatant of these cells indicated induction of a T-helper type 2 response. The purified 12-kDa protein is a clinically relevant allergen and has potential for the diagnosis and therapy of Epicoccum allergies.     

Mitochondrial epigenetics in bone remodeling during hyperhomocysteinemia

The studies into the pathophysiology of viral miRNAs are still in infancy; the interspecies regulation at the miRNA level fuels the spark of the investigation into the repertoire of virus–host interactions. Reports pertaining to the viral miRNAs role in modulating/evading the host immune response are surging up; we initiated this in silico study to speculate the role of human cytomegalovirus (HCMV)-encoded miRNAs on human antiviral mechanisms such as apoptosis and autophagy. The results indicate that both the above mechanisms were targeted by the HCMV miRNAs, located in the unique long region of the HCMV genome. The proapoptotic genes MOAP1, PHAP, and ERN1 are identified to be the potential targets for the miR-UL70-3p and UL148D, respectively. The ERN1 gene plays a role in the initiation of Endoplasmic reticulum stress-induced apoptosis as well as autophagosome formation. This study shows that HCMV employs its miRNA repertoire for countering the cellular apoptosis and autophagy, particularly the mitochondrial-dependent intrinsic pathway of apoptosis. In addition, the homology studies reveal no HCMV miRNA bears sequence homology with human miRNAs.     

Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein

Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.     

The X protein of hepatitis B virus binds to the F box protein Skp2 and inhibits the ubiquitination and proteasomal degradation of c-Myc

The HBx protein of hepatitis B virus is involved in deregulation of cell cycle and development of hepatocellular carcinoma. Since c-Myc also plays an important role in cell proliferation and tumor development, we studied its regulation by HBx in a human hepatoma cell line. Co-expression of HBx and c-Myc resulted in increased stability of intracellular c-Myc. HBx blocked the ubiquitination of Myc through a direct interaction with the F box region of Skp2 and destabilization of the SCF(Skp2) complex. We suggest that sustained presence of c-Myc combined with mitogenic activity inherent to HBx may be associated with cell cycle deregulation and transformation.     

Homocysteine-mediated activation and mitochondrial translocation of calpain regulates MMP-9 in MVEC

Hyperhomocysteinemia (HHcy) is associated with atherosclerosis, stroke, and dementia. Hcy causes extracellular matrix remodeling by the activation of matrix metalloproteinase-9 (MMP-9), in part, by inducing redox signaling and modulating the intracellular calcium dynamics. Calpains are the calcium-dependent cysteine proteases that are implicated in mitochondrial damage via oxidative burst. Mitochondrial abnormalities have been identified in HHcy. The mechanism of Hcy-induced extracellular matrix remodeling by MMP-9 activation via mitochondrial pathway is largely unknown. We report a novel role of calpains in mitochondrial-mediated MMP-9 activation by Hcy in cultured rat heart microvascular endothelial cells. Our observations suggested that calpain regulates Hcy-induced MMP-9 expression and activity. We showed that Hcy activates calpain-1, but not calpain-2, in a calcium-dependent manner. Interestingly, the enhanced calpain activity was not mirrored by the decreased levels of its endogenous inhibitor calpastatin. We presented evidence that Hcy induces the translocation of active calpain from cytosol to mitochondria, leading to MMP-9 activation, in part, by causing intramitochondrial oxidative burst. Furthermore, studies with pharmacological inhibitors of calpain (calpeptin and calpain-1 inhibitor), ERK (PD-98059) and the mitochondrial uncoupler FCCP suggested that calpain and ERK-1/2 are the major events within the Hcy/MMP-9 signal axis and that intramitochondrial oxidative stress regulates MMP-9 via ERK-1/2 signal cascade. Taken together, these findings determine the novel role of mitochondrial translocation of calpain-1 in MMP-9 activation during HHcy, in part, by increasing mitochondrial oxidative stress.     

3-Deazaadenosine mitigates arterial remodeling and hypertension in hyperhomocysteinemic mice

Chronic hyperhomocysteinemia (HHcy) is an important factor in development of arterial hypertension. HHcy is associated with activation of matrix metalloproteinases (MMPs); however, it is unclear whether HHcy-dependent extracellular matrix (ECM) accumulation plays a role in arterial hypertrophy and hypertension. We tested the hypothesis that in HHcy the mechanism of arterial hypertension involves arterial dysfunction in response to ECM accumulation between endothelial and arterial smooth muscle cells and subsequent endothelium-myocyte (E-M) uncoupling. To decrease plasma Hcy, dietary supplementation with 3-deazaadenosine (DZA), the S-adenosylhomocysteine hydrolase inhibitor, was administered to cystathionine beta-synthase (CBS) knockout (KO) mice. Mice were grouped as follows: wild type (WT; control), WT+DZA, CBSKO, and CBSKO+DZA (n = 4/group). Mean aortic blood pressure and heart rate were monitored in real time with a telemetric system before, during, and after DZA treatment (6 wk total). In vivo aorta function and morphology were analyzed by M-mode and Doppler echocardiography in anesthetized mice. Aorta MMP activity in unfixed cryostat sections was measured with DQ gelatin. Aorta MMP-2, MMP-9, and connexin 43 expression were measured by RT-PCR and Western blot analyses, respectively. HHcy caused increased aortic blood pressure and resistance, tachycardia, and increased wall thickness and ECM accumulation in aortic wall vs. control groups. There was a linear correlation between aortic wall thickness and plasma Hcy levels. MMP-2, MMP-9, and connexin 43 expression were increased in HHcy. In the CBSKO+DZA group, aortic blood pressure and levels of MMP and connexin 43 were close to those found in control groups. However, removal of DZA reversed the aortic lumen-to-wall thickness ratio in CBSKO mice, suggesting, in part, a role of vascular remodeling in the increase in blood pressure in HHcy. The results show that arterial hypertension in HHcy mice is, in part, associated with arterial remodeling and E-M uncoupling in response to MMP activation.     

The Water Channel Aquaporin 1 Is a Novel Molecular Target of Polychlorinated Biphenyls for in Utero Anomalies

Despite serious health risks in humans and wild life, the underlying mechanisms that explain the gene-environment effects of chemical toxicants are largely unknown. Polychlorinated biphenyls (PCBs) are one of the most ubiquitous environmental toxicants worldwide, with reported epidemiological evidence for reproductive and neurocognitive anomalies in humans. Here, we show that Aroclor 1254, a mixture of structurally distinct PCBs, causes preterm birth in interleukin (IL)-10^-/- mice at a dose that does not show any adverse effects in wild type mice, highlighting the significance of IL-10 as an anti-toxicant cytokine. Aroclor 1254-treated IL-10^-/- mice demonstrated increased amniotic fluid, intrauterine growth restriction, and reduced litter size with postnatal neuromotor defects. Further, our results identify aquaporin 1 (AQP1), a potent effector of fluid volume regulation and angiogenic activity, as a novel placental target of PCBs. In vivo or in vitro exposure to Aroclor 1254 coupled with IL-10 deficiency significantly reduced the protein content of AQP1. Reduced uterine AQP1 levels were associated with defective spiral artery transformation. Importantly, recombinant IL-10 reversed PCB-induced in vivo and in vitro effects. These data demonstrate for the first time that the IL-10-AQP1 axis is a novel regulator of PCB-induced in utero effects.The health consequences of environmental toxicants are likely to have critical effects during in utero fetal development because of the complex signaling cascades, high cellular proliferation rates, and differentiation events. Mammalian reproduction involves a complex but highly choreographed sequence of molecular processes. These processes include interactions between the hormonally stimulated uterus and the developing blastocyst, implantation, placental and fetal development, and parturition (1, 2). Although the hormonal milieu, metabolic changes, and placental microenvironment are programmed in a pregnancy compatible manner, pregnancy presents itself as an immunological and hormonal paradox (3, 4). The role of steroid hormones is well known in uterine receptivity, implantation, local immune modulation, and pregnancy success (5). If not temporally produced and regulated, their dysfunction lead to infertility or pregnancy loss. Man-made chemicals like polychlorinated biphenyls (PCBs)² act like hormones and interfere with their cognate receptor functions impacting normal biological processes (6, 7). Although the genotoxic effects of PCBs have been investigated intensively and epidemiological studies have highlighted their health risks (6, 7), the mechanisms responsible for reproductive and neurodevelopmental effects still remain enigmatic. The overarching goal of our studies is to identify unknown pathways and targets that impart adverse effects on pregnancy. In this study, we directed our efforts toward establishing an experimental system to evaluate the in utero gene-environment effects of PCBs using wild type mice and their counterparts deficient in pregnancy compatible anti-inflammatory cytokines such as interleukin 10 (IL-10).IL-10 is a potent anti-inflammatory cytokine that controls inflammatory insult in most organs, particularly at the maternal-fetal interface. IL-10 is produced by gestational tissue and maternal immune cells in the intrauterine microenvironment in humans (8, 9) and in mice (10). We and others have reported that IL-10^-/- mice experience preterm birth and resorptions in response to low doses of inflammatory triggers such as lipopolysaccharide (LPS) (11, 12) or poly(I-C) (13). Importantly, the pregnancy outcome in treated IL-10^-/- mice can be rescued by giving an exogenous dose of IL-10 (11, 14). We have also demonstrated poor IL-10 production in placental and decidual tissues from preterm labor deliveries and missed abortions (15, 16). These data suggest that an inflammatory environment coupled with genetic stress (IL-10 deficiency) may lead to adverse pregnancy outcomes. In consideration of these observations, we hypothesize that exposure to toxicants such as PCBs mimics the physiological counterpart of inflammation that predisposes to adverse pregnancy outcomes when combined with genetic deficiency in loci crucial for pregnancy success such as IL-10.PCBs are chlorinated aromatic hydrocarbon compounds consisting of a group of 209 structurally diverse congeners, identified based on the position of chlorine atoms (7). Since the start of their manufacture in the 1920s until their ban in late 1970s, PCBs were globally valued for their noninflammability and high heat and chemical stability and thus were used widely in a multitude of commercial and industrial applications (7, 17). Improper disposal and accidental release of these compounds led to their introduction into the environment, placing them in the list of widespread environmental contaminants. Subsequently, their lipophilicity facilitated their bioaccumulation in the food chain and bio-concentration at successively higher levels (6, 18-21). PCBs have now been detected globally, in different environmental matrices, wild life, food, and humans (6, 18, 20). Convincing evidence exist for their toxicity, both in humans as well as in laboratory animals (7). From epidemiological studies in humans it has been observed that exposure to PCBs causes various reproductive anomalies that include irregular and shorter menstrual cycles, delayed conception, miscarriage, reduced lactating time, low birth weight, preterm birth, small for gestational age infants, and higher incidence of still-births and mortality among children (22-27). PCB congeners may work in an aryl hydrocarbon receptor-dependent or -independent pathway (6, 7, 28). Despite the knowledge that PCBs affect either aryl hydrocarbon receptor or estrogen receptor signaling, there is a paucity of molecular mechanisms underlying the most sensitive developmental effects of PCBs, and thus new pathways and targets need to be identified.Aroclor 1254 is a mixture of more than one hundred different PCB congeners and may impart cumulative adverse effects on female reproductive health (29, 30). In this study, we show that Aroclor 1254 exposure induces preterm birth in IL-10^-/- mice with reduced litter size and birth weight, increased amniotic fluid, and postnatal neurocognitive defects. Importantly, we have identified aquaporin 1 (AQP1) as a novel target of PCB action at the maternal-fetal interface. Our findings for the first time provide direct experimental evidence for a protective role of IL-10 against PCB exposure. These findings may have implications for the understanding and management of environmental toxicant-induced female reproductive anomalies in humans.     

16S rRNA gene profiling of rhizospheric microbial community of Eichhornia crassipes

Molecular Biology Reports - The rhizosphere of a plant is an important interface for the plant-microbe interaction that plays a significant role in the uptake and removal of heavy metal from...