Nerve growth factor protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a phosphatidylinositol 3-kinase-dependent manner

The survival signal elicited by the phosphatidylinositol 3-kinase (PI3K)/Akt1 pathway has been correlated with inactivation of pro-apoptotic proteins and attenuation of the general stress-induced increase in reactive oxygen species (ROS). However, the mechanisms by which this pathway regulates intracellular ROS levels remain largely unexplored. In this study, we demonstrate that nerve growth factor (NGF) prevents the accumulation of ROS in dopaminergic PC12 cells challenged with the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves PI3K/Akt-dependent induction of the stress response protein heme oxygenase-1 (HO-1). The effect of NGF was mimicked by induction of HO-1 expression with CoCl(2); by treatment with bilirubin, an end product of heme catabolism; and by infection with a retroviral expression vector for human HO-1. The relevance of HO-1 in NGF-induced ROS reduction was further demonstrated by the evidence that cells treated with the HO-1 inhibitor tin-protoporphyrin or infected with a retroviral expression vector for antisense HO-1 exhibited enhanced ROS release in response to 6-OHDA, despite the presence of the neurotrophin. Inhibition of PI3K prevented NGF induction of HO-1 mRNA and protein and partially reversed its protective effect against 6-OHDA-induced ROS release. By contrast, cells transfected with a membrane-targeted active version of Akt1 exhibited increased HO-1 expression, even in the absence of NGF, and displayed a greatly attenuated production of ROS and apoptosis in response to 6-OHDA. These observations indicate that the PI3K/Akt pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1.     

Compromised influenza virus-specific CD8(+)-T-cell memory in CD4(+)-T-cell-deficient mice

The primary influenza A virus-specific CD8(+)-T-cell responses measured by tetramer staining of spleen, lymph node, and bronchoalveolar lavage (BAL) lymphocyte populations were similar in magnitude for conventional I-A(b+/+) and CD4(+)-T-cell-deficient I-A(b-/-) mice. Comparable levels of virus-specific cytotoxic-T-lymphocyte activity were detected in the inflammatory exudate recovered by BAL following challenge. However, both the size of the memory T-cell pool and the magnitude of the recall response in the lymphoid tissues (but not the BAL specimens) were significantly diminished in mice lacking the CD4(+) subset. Also, the rate of virus elimination from the infected respiratory tract slowed at low virus loads following challenge of na?ve and previously immunized I-A(b-/-) mice. Thus, though the capacity to mediate the CD8(+)-T-cell effector function is broadly preserved in the absence of concurrent CD4(+)-T-cell help, both the maintenance and recall of memory are compromised and the clearance of residual virus is delayed. These findings are consistent with mathematical models that predict virus-host dynamics in this, and other, models of infection.     

Drug release from Kollicoat SR 30D-coated nonpareil beads: evaluation of coating level,plasticizer type,and curing condition

A newly available polyvinylacetate aqueous dispersion, Kollicoat SR 30D, was evaluated with respect to its ability to modulate the in vitro release of a highly water-soluble model compound (diphenhydramine hydrochloride) from nonpareil-based systems. Kollicoat SR 30D premixed with a selected plasticizer (10% wt/wt propylene glycol, 2.5% triethyl citrate, or 2.5% dibutyl sebacute), talc, and red #30 lake dye was coated onto the drug beads in an Aeromatic Strea I fluid-bed drier with a Wurster insert using bottom spray. With propylene glycol as the plasticizer, increases in polymer coating level retarded drug release from beads in a stepwise fashion along with apparent permeability, indicating a consistent release mechanisms. Stability studies at 40°C/75% RH revealed gradual decreases in dissolution rate, and additional curing studies further confirmed the dependence of release kinetics on curing condition. Furthermore, the type of plasticizer was found to play a key role. Unplasticized formulations exhibited the fastest dissolution, followed by formulations plasticized with triethyl citrate, propylene glycol, and dibutyl sebacate. All 4 formulations (unplasticized and plasticized), nevertheless, revealed a marked difference between uncured and cured dissolution profiles. Kollicoat SR 30D has, thereby, been demonstrated to effectively retard drug release from nonpareilbased systems. However, selected plasticizer type and subsequent curing condition play important roles in controlling drug release from such a system.     

Effect of extracellular matrix proteins on in vitro testosterone production by rat Leydig cells

The aim of this study was to detect the effect of extracellular matrix (ECM) proteins on rat Leydig cell shape, adhesion, expression of integrin subunits and testosterone production, in vitro. Leydig cells isolated from adult rats were cultured on plates uncoated or coated with different concentrations of laminin-1, fibronectin, or type IV collagen in the presence or absence of hCG for 3 or 24 hr. A significant increase of cell adhesion and of alpha3, alpha5, and beta1 integrin subunit expression was observed when cells were cultured on ECM proteins, compared to those grown on uncoated plates. Leydig cells cultured on glass coverslips coated with ECM proteins for 24 hr exhibited elongated shapes with long cell processes (spreading), while cells cultured on uncoated plates showed few cell processes. A significant decrease in testosterone production was observed when basal and hCG-stimulated Leydig cells were cultured for 3 or 24 hr on plates coated with type IV collagen (12 and 24 microg/cm(2)) compared to uncoated plates. A significant though a slighter decrease in testosterone production was also observed in cells cultured on plates coated with fibronectin (12 and 24 microg/cm(2)), compared to uncoated plates. Laminin-1 did not modify testosterone production under basal or hCG stimulated conditions. These results suggest that ECM proteins are able to modulate Leydig cell steroidogenesis, in vitro.     

The importance of microscopic examination in the management of desquamative diseases of the scalp

After determining the usual malassezic biota of the scalp in adult, normal persons, 259 patients with different desquamative diseases were studied by a simple adhesive tape technique. The main purpose of this study was to investigate the utility of this technique to improve the diagnosis and treatment of patients. Most patients with seborrhoeic dermatitis and sebopsoriasis demonstrated large numbers of {it Malassezia} spp. cells corresponding to the so called ``pityrosporosis'. Only 43.6% of patients with pityriasis capitis (dandruff) presented with such a diagnosis. Symptomatic pityrosporosis of the scalp should be treated with imidazolic derivatives or other antifungal substances. Patients with psoriasis of the scalp showed a typical microscopic picture represented by parakeratosic (nucleated) keratinocytes with absence of lipophilic yeasts which should be attributed to the usual dryness of the scales .Microbial epidermitis (eczema) of the scalp revealed another characteristic picture constituted by abundant leukocytes and bacteria without the presence of yeasts. The different microscopic pictures seen with this simple technique for diseases of the scalp, offer an adjunct to make a proper diagnosis and to establish a convenient treatment in cases which are not clinically well defined. This revised version was published online in June 2006 with corrections to the Cover Date.     

Membrane topology of the acyl-lipid desaturase from Bacillus subtilis

The Bacillus subtilis acyl-lipid desaturase (Delta5-Des) is an iron-dependent integral membrane protein, able to selectively introduce double bonds into long chain fatty acids. Structural information on membrane-bound desaturases is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase. The topology of Delta5-Des was determined experimentally in Escherichia coli using a set of nine different fusions of N-terminal fragments of Delta5-Des with the reporter alkaline phosphatase (Delta5-Des-PhoA). The alkaline phosphatase activities of cells expressing the Delta5-Des-PhoA fusions, combined with site-directed mutagenesis of His residues identified in most desaturases, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane. These data, together with surface Lys biotinylation experiments, support a model for Delta5-Des as a polytopic membrane protein with six transmembrane- and one membrane-associated domain, which likely represents a substrate-binding motif. This study provides the first experimental evidence for the topology of a plasma membrane fatty acid desaturase. On the basis of our results and the presently available hydrophobicity profile of many acyl-lipid desaturases, we propose that these enzymes contain a new transmembrane domain that might play a critical role in the desaturation of fatty acids esterified in glycerolipids.     

Linkage mapping of Hsa-1<Superscript>Og</Superscript>, a resistance gene of African rice to the cyst nematode, <Emphasis Type="Italic">Heterodera sacchari</Emphasis>

Inheritance of resistance to cyst nematode (Heterodera sacchari) in Oryza sativa was investigated by inoculation tests with isolate 244 from Congo in segregating populations derived from hybridisation between O. sativa and its African sister cultivated species, O. glaberrima. We found that the resistance was controlled by one major gene, Hsa-1(Og), with codominance of susceptible and resistant alleles. To map Hsa-1(Og) on the rice genome, we pooled the data obtained from segregation of the resistance trait and microsatellite markers in three kinds of progeny: BC(1)F(3), BC(1)F(4), and pseudo-F(2) populations. Hsa-1(Og) was unambiguously located between Cornell University's RM206 and RM254 markers on chromosome 11. Two additional microsatellite markers derived from Monsanto publicly available sequences were found to be tightly linked to the Hsa-1(Og) gene. It is possible that numerous plant resistances to a pathogen in fact exhibit a codominant inheritance, possibly explaining misleading conclusions in several reports on resistance segregation.     

TIMP-2 mediated inhibition of angiogenesis: an MMP-independent mechanism

Tissue inhibitors of metalloproteinases (TIMPs) suppress matrix metalloproteinase (MMP) activity critical for extracellular matrix turnover associated with both physiologic and pathologic tissue remodeling. We demonstrate here that TIMP-2 abrogates angiogenic factor-induced endothelial cell proliferation in vitro and angiogenesis in vivo independent of MMP inhibition. These effects require alpha 3 beta 1 integrin-mediated binding of TIMP-2 to endothelial cells. Further, TIMP-2 induces a decrease in total protein tyrosine phosphatase (PTP) activity associated with beta1 integrin subunits as well as dissociation of the phosphatase SHP-1 from beta1. TIMP-2 treatment also results in a concomitant increase in PTP activity associated with tyrosine kinase receptors FGFR-1 and KDR. Our findings establish an unexpected, MMP-independent mechanism for TIMP-2 inhibition of endothelial cell proliferation in vitro and reveal an important component of the antiangiogenic effect of TIMP2 in vivo.