Aplidiasterols A and B, two new cytotoxic 9,11-secosterols from the Mediterranean ascidian Aplidium conicum

Two novel 9,11-secosterols, aplidiasterols A (3β,6β,11-trihydroxy-9,11-seco-5α-cholest-7-en-9-one, 1) and B (3β,5α,6β,11-tetrahydroxy-9,11-secocholest-7-en-9-one, 2), along with the known secosterols 3 and 4, were isolated from the Mediterranean ascidian Aplidium conicum and their structures were determined by spectroscopic data. Aplidiasterols A and B were found to be cytotoxic against rat glioma (C6) and murine monocyte/macrophage (J774) tumor cells in vitro. Compounds 1-4 represent the first example of secosterols isolated from tunicates.     

Different sensitivity to apoptosis in cells of monocytic or lymphocytic origin chronically infected with human immunodeficiency virus type-1

Apoptotic death of CD4+ T lymphocytes is a major cause of the immunodeficiency caused by human immunodeficiency virus (HIV), but it is still unclear how this process precisely occurs. To characterize a potentially useful cellular model, we have analyzed the tendency of chronically HIV-infected CD4+ human cell lines of different origin to undergo apoptosis. We studied ACH-2 and U1 lines, derived from the CD4+ T-cell A301 and the promonocytic U937 cell lines, respectively, and induced apoptosis via several stimuli that trigger different pathways. Their capacity to regulate plasma membrane CD95 expression and to produce soluble CD95 was also analyzed. Using staurosporine, TNF-alpha plus cycloheximide, and gamma-radiations, we observed that ACH-2 were more sensitive to programmed cell death than A301, while U1 were less sensitive than U937. Both infected cell types had a lower sensitivity to CD95-induced apoptosis; the analysis of changes in mitochondrial membrane potential corroborated these observations. Plasma membrane CD95 was similarly regulated in all cell types, which, however, presented a different capacity to produce soluble CD95 molecules. Our in vitro results may offer a new perspective for developing further studies on the pathogenesis of HIV infection. A chronically infected cell line of lymphocytic origin is more susceptible to apoptosis than its parental cell type, while infected monocytic cells are less sensitive than their uninfected counterpart. Thus, it is possible to hypothesize that one of the reasons by which circulating monocytes survive and represent a viral reservoir is the capacity of HIV to decrease the sensitivity to apoptosis of this cell type. However, further studies on ex-vivo collected fresh cells, as well as on other cell lines, are urgently needed to confirm such hypothesis.     

Desialylated N-CAM and chromatin-derived acidic peptide effects in the hypothalamus

Chromatin-derived acidic peptides (ACPs) have been shown to acutely modulate hypothalamic catecholamine release. To investigate whether this effect is mediated through membrane polysialylated neural-cell adhesion molecule (PSA-N-CAM), we pretreated rat hypothalamic synaptosomes with neuraminidase enzyme, which partially cleaves sialic acid residues from N-CAM, and perfused them with ACP-1 (Asp-Asp-Ser-Asp-Glu-Glu-Asn) or a more lipophilic derivative, ACP-2 ([Ala-Ile-Ser-Pro]-Asp-Asp-Ser-Asp-Glu-Glu-Asn). We have found that neuraminidase completely abolish the inhibitory effect of ACP-1 on dopamine release, while the inhibitory activity of ACP-1 on norepinephrine release is partially lost. On the other hand, ACP-2 inhibition of dopamine release is not modified by neuraminidase pretreatment.     

Different subcellular localization and phosphoinositides binding of insulin receptor substrate protein pleckstrin homology domains

Insulin evokes diverse biological effects through receptor-mediated tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins. Here, we show that, in vitro, the IRS-1, -2 and -3 pleckstrin homology (PH) domains bind with different specificities to the 3-phosphorylated phosphoinositides. In fact, the IRS-1 PH domain binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3), the IRS-2 PH domain to phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), and the IRS-3 PH domain to phosphatidylinositol 3-phosphate. When expressed in NIH-IR fibroblasts and L6 myocytes, the IRS-1 and -2 PH domains tagged with green fluorescent protein (GFP) are localized exclusively in the cytoplasm. Stimulation with insulin causes a translocation of the GFP-IRS-1 and -2 PH domains to the plasma membrane within 3-5 min. This translocation is blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin and LY294002, suggesting that this event is PI 3-K dependent. Interestingly, platelet-derived growth factor (PDGF) did not induce translocation of the IRS-1 and -2 PH domains to the plasma membrane, indicating the existence of specificity for insulin. In contrast, the GFP-IRS-3 PH domain is constitutively localized to the plasma membrane. These results reveal a differential regulation of the IRS PH domains and a novel positive feedback loop in which PI 3-K functions as both an upstream regulator and a downstream effector of IRS-1 and -2 signaling.     

Implication of tissue transglutaminase and desmoplakin in cell adhesion mechanism in human epidermis

The distribution patterns of both tissue and keratinocyte transglutaminases (TGase), as well as that of desmoplakin (DP), have been immunohistochemically investigated in human skin cultured in the absence or presence of cystamine and enalapril, two acantholytic agents. In the control samples, tissue TGase is predominantly expressed in lower layers of the epidermis and is located intercellularly. Conversely, in tissues cultured with cystamine or enalapril, a diffuse cytoplasmatic staining was observed. Similarly, DP, detected on the cell membrane in the control, shifts into the cytosol of the keratinocytes following treatment. The distribution pattern of the keratinocyte enzyme in the acantholytic epidermis was identical to that observed in the normal one. Since cystamine and enalapril are TGase inhibitors and DP was shown to act as a TGase substrate in vitro, we suggest that DP and tissue enzyme may participate in cell adhesion at the intraepidermal level.     

Effect of deamidation on folding of ribonuclease A

The folding of ribonuclease A (RNase A) has been extensively studied by characterizing the disulfide containing intermediates using different experimental conditions and analytical techniques. So far, some aspects still remain unclear such as the role of the loop 65-72 in the folding pathway. We have studied the oxidative folding of a RNase A derivative containing at position 67 the substitution Asn --> isoAsp where the local structure of the loop 65-72 has been modified keeping intact the C65-C72 disulfide bond. By comparing the folding behavior of this mutant to that of the wild-type protein, we found that the deamidation significantly decreases the folding rate and alters the folding pathway of RNase A. Results presented here shed light on the role of the 65-72 region in the folding process of RNase A and also clarifies the effect of the deamidation on the folding/unfolding processes. On a more general ground, this study represents the first characterization of the intermediates produced along the folding of a deamidated protein.