GD3 recruits reactive oxygen species to induce cell proliferation and apoptosis in human aortic smooth muscle cells

Sialic acid containing glycosphingolipids (gangliosides) are expressed on the surface of all mammalian cells and have been implicated in regulating various biological phenomena; however, the detailed signaling mechanisms involved in this process are not known. We report here a novel aspect of disialoganglioside, GD3-mediated regulation of cell proliferation and cell death via the recruitment of reactive oxygen species (ROS). A low concentration (2.5-10 microm) of GD3, incubated with human aortic smooth muscle cells for a short period of time (10-30 min), stimulates superoxide generation via the activation of both NADPH oxidase and NADH oxidase activity. This leads to downstream signaling leading to cell proliferation and apoptosis. However, [(3)H]GD3 incubated with the cells under such conditions was found in a trypsin-sensitive fraction that was separable from endogenous GD3. The exact mechanism causing ROS generation and downstream signaling remains to be elucidated. The uptake of GD3 was accompanied by a 2.5-fold stimulation in the activity of mitogen-activated protein (MAP) kinase and 5-fold stimulation in cell proliferation. Preincubation of cells with membrane-permeable antioxidants, pyrrolidine dithiocarbamate, and N-acetylcysteine abrogated the superoxide generation and cell proliferation. In contrast, at higher concentrations (50-200 microm) GD3 inhibited the generation of superoxides but markedly stimulated the generation of nitric oxide (NO) (10-fold compared with control). This in turn stimulated mitochondrial cytochrome c release and intrachromosomal DNA fragmentation, which lead to apoptosis. In sum, at a low concentration, GD3 recruits superoxides to activate p44 MAPK and stimulates cell proliferation. In contrast, at high concentrations GD3 recruits nitric oxide to scavenge superoxide radicals that triggered signaling events that led to apoptosis. These observations might have relevance in regard to the potential role of GD3 in aortic smooth muscle cell proliferation and apoptosis that may contribute to plaque rupture in atherosclerosis.     

Effect of low level exposure of lead and cadmium on hepatic estradiol metabolism in female rats

Toxic effect of metal cations on female reproduction and gonadal functions was studied. Adult synchronized female rats were treated intraperitoneally with lead acetate and cadmium acetate separately and in combination (0.025, 0.05 and 0.1 mg/kg body wt) for 15 days. The metabolizing enzymes (17beta-hydroxy steroid oxidoreductase and UDP glucoronyl transferase) activities decreased with increasing dose showing significant change compared to control. Also, significant decrease in cytochrome P450 (CYP450) content was found after the treatment. Displacement of zinc bound to metallothionein was more in cadmium treated rats compared to other groups. In all these parameters, treatment in combination of lead and cadmium showed intermediate results indicating some kind of competition between the two metals. But the histological studies showed that combined treatment caused more cytotoxic effect than cadmium and lead alone. These results indicated that metal cations tested did have a direct inhibitory effect on metabolizing enzyme activities.     

The Th1-specific costimulatory molecule,m150, is a posttranslational isoform of lysosome-associated membrane protein-1

In an earlier report, we had shown a 150-kDa protein termed as M150, isolated from the surface of activated macrophages, to possess costimulatory activity for CD4(+) T cells. Significantly, this protein was found to specifically elicit Th1 responses. In this study, we characterize M150, which belongs to a unique subset of the lysosome-associated membrane protein-1 glycoprotein. Interestingly, the costimulatory activity of M150 depends on its posttranslational modification, which has a distinct glycosylation pattern restricted to macrophages. Furthermore, it has been demonstrated that in addition to stimulating Th1-specific responses, M150 is also capable of driving differentiation of naive CD4(+) T cells into the Th1 subset. This altered posttranslational modification of housekeeping protein appears to represent a novel pathway by which APCs can additionally regulate T cell responses.     

Differential action of iodine on mitochondria from human tumoral- and extra-tumoral tissue in inducing the release of apoptogenic proteins

Iodide is actively concentrated in the thyroid gland for thyroid hormone biosynthesis. Excess iodine has been observed to induce apoptosis in thyrocytes and mammary cells. The mechanism of iodine induced apoptosis is poorly understood. Among various cell organelles, mitochondria is known to provide conducive environment for the organification of iodine, i.e. iodination of different proteins. Mitochondria also play a central role in execution of apoptosis. To study the role of mitochondria in iodine induced apoptosis, we investigated the direct interaction of iodine and human breast mitochondria vis-a-vis its role in the initiation of apoptosis in vitro. We observed that mitochondria isolated from the tumor (TT) and extra-tumoral tissue (ET) of human breast display significant uptake of iodine. Mitochondrial proteins were observed to be predominantly iodinated in ET but not in TT mitochondria. Treatment with iodine showed an increase in mitochondrial permeability transition of TT and decrease in ET. Iodine induced released factor(s) other than cytochrome c from tumor mitochondria initiate(s) apoptosis in vitro, while those from ET mitochondria were non-apoptogenic in nature. To our knowledge, this is first report demonstrating that iodine acts differentially on mitochondria of tumor and extratumoral origin to release apoptogenic proteins from TT and has a protective effect on ET.     

Defining a pathway of communication from the C-terminal peptide binding domain to the N-terminal ATPase domain in a AAA protein

AAA proteins remodel other proteins to affect a multitude of biological processes. Their power to remodel substrates must lie in their capacity to couple substrate binding to conformational changes via cycles of nucleotide binding and hydrolysis, but these relationships have not yet been deciphered for any member. We report that when one AAA protein, Hsp104, engages polypeptide at the C-terminal peptide-binding region, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2) drives a conformational change in the middle region. This, in turn, drives ATP hydrolysis in the N-terminal ATPase domain (NBD1). This interdomain communication pathway can be blocked by mutation in the middle region or bypassed by antibodies that bind there, demonstrating the crucial role this region plays in transducing signals from one end of the molecule to the other.     

Baylis-Hillman reaction: convenient ascending syntheses and biological evaluation of acyclic deoxy monosaccharides as potential antimycobacterial agents

A series of acyclic deoxy carbohydrate derivatives from easily available carbohydrate enals 1, 2, 3 or 5 were prepared involving the Baylis-Hillman reaction. These newly formed carbohydrate based Baylis-Hillman adducts and their amino derivatives were evaluated for their antimycobacterial activity against Mycobacterium tuberculosis H(37)R(v). Among the compounds evaluated for their antimycobacterial activity, compound (10) showed the desired activity in the range of 3.125 microg/mL.     

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller)

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.     

Histological and ultrastructural regulation in rabbit endometrial explants by estrogen in serum-free culture

A repertoire of hormonal signals including estrogen regulate the growth, differentiation, and functioning of diverse target tissues, including the ovary, the mammary gland, and skeletal tissue. A serum-free culture system derived from rabbit endometrium explants has been devised and is reported here to explore estrogen action in vitro. The system involves aseptically harvesting the uterus from a virgin rabbit, dissecting the endometrium, explanting it into 1- to 2-mm(3) pieces weighing approximately 1-2 mg each, and incubating these pieces in serum-free Medium-199. The culture is carried out for a period of 4 d in a humidified CO(2) incubator at 37 degrees C with 5% CO(2). The effect of extraneously added estrogen (1 microg/ml) was investigated by histological and ultrastructural procedures. It was observed that estrogen could induce specific changes, such as abundant mitochondria, rough endoplasmic reticulum, golgi complex, and intracellular collagen deposition, in both the epithelial and the fibroblast cell components of the explanted tissue. The study, therefore, indicates that the proposed system is an ideal tool for exploring and demonstrating estrogen responsiveness under in vitro conditions.     

The impact of polyene,azole, and DNA analogue antimycotics on the cell surface hydrophobicity of Candida albicans and Candida tropicalis in HIV infection

Oral candidiasis is the most common opportunistic infection in individuals infected with the human immunodeficiency virus. Though Candida albicans is the major aetiological agent, non-albicans species such Candida tropicalis are now emerging as important agents of such infection. The Candida cell surface hydrophobicity (CSH) is considered a critical factor contributing to its colonization potential and virulence. It is also known that brief exposure to sub-cidal concentrations of antifungal agents is a likely scenario in the oral environment where the administered drugs are diluted continuously due to the flushing action of saliva. Hence the objective of the present study was to compare the CSH of 10 isolates each of C. albicans and C. tropicalis from HIV-infected individuals following brief exposure (1hour) of isolates to sub-therapeutic concentrations of nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine. The CSH was assessed by a previously described biphasic aqueous-hydrocarbon assay. The mean percentage reduction of CSH of C. albicans following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was27.33 (p < 0.001), 21.34 (p < 0.05), 11.74 (p > 0.05), 18.4 (p > 0.05) and 14.64 (p > 0.05) respectively. The mean percentage reduction of CSH of C. tropicalis following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was 33.81 (p < 0.01), 28.88 (p < 0.01), 12.6 (p > 0.05), 21.53(p > 0.05) and 17.68 (p > 0.05) respectively. A significant inter-species variation in CSH was observed for nystatin and amphoterecin B. Overall the results reveal that the CSH of C. albicans is affected to a significantly lesser degree compared with C. tropicalis when exposed to the antifungals. These data further illustrate another mode of action of antifungals on Candida leading to a reduction in the CSH and thereby the yeast adherence to host tissues. This revised version was published online in June 2006 with corrections to the Cover Date.