The pathogenicity of human immunodeficiency virus (HIV) type 1 Nef in CD4C/HIV transgenic mice is abolished by mutation of its SH3-binding domain, and disease development is delayed in the absence of Hck

The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important determinant of AIDS pathogenesis. We have previously reported that HIV-1 Nef is responsible for the induction of a severe AIDS-like disease in CD4C/HIV transgenic (Tg) mice. To understand the molecular mechanisms of this Nef-induced disease, we generated Tg mice expressing a mutated Nef protein in which the SH3 ligand-binding domain (P(72)XXP(75)XXP(78)) was mutated to A(72)XXA(75)XXQ(78). This mutation completely abolished the pathogenic potential of Nef, although a partial downregulation of the CD4 cell surface expression was still observed in these Tg mice. We also studied whether Hck, one of the effectors previously found to bind to this PXXP motif of Nef, was involved in disease development. Breeding of Tg mice expressing wild-type Nef on an hck(-/-) (knockout) background did not abolish any of the pathological phenotypes. However, the latency of disease development was prolonged. These data indicate that an intact PXXP domain is essential for inducing an AIDS-like disease in CD4C/HIV Tg mice and suggest that interaction of a cellular effector(s) with this domain is required for the induction of this multiorgan disease. Our findings indicate that Hck is an important, but not an essential, effector of Nef and suggest that another factor(s), yet to be identified, may be more critical for disease development.     

Presence of Na-K-ATPase in mitochondria-rich cells in the yolk-sac epithelium of larvae of the teleost Oreochromis mossambicus

The purpose of this study is to provide biochemical evidence for the functions of the mitochondria-rich cell (MR cell) in the yolk-sac epithelium of the developing larvae of tilapia Oreochromis mossambicus. Western blotting with the antibody (6F) raised against avian Na-K-ATPase alpha1 subunit demonstrated the presence of Na-K-ATPase in yolk-sac epithelium of tilapia larvae and about 1. 46-fold more of the enzyme in seawater larvae than in freshwater ones. The yolk-sac MR cells were immunoreacted to the antibody (alpha5) against the alpha subunit of avian Na-K-ATPase and were double-labeled with anthroylouabain and dimethylaminostyrylethyl-pyridiniumiodine, suggesting the existence and activity of Na-K-ATPase in these cells. Binding of 3H-ouabain in the yolk sac of seawater larvae was much higher than in that of freshwater larvae (4.183+/-0.143 pmol/mg protein versus 1.610+/-0. 060 pmol/mg protein or 0.0508+/-0.0053 pmol/yolk sac versus 0. 0188+/-0.0073 pmol/yolk sac). These biochemical results are further evidence that yolk-sac MR cells are responsible for a major role in the osmoregulatory mechanism of early developmental stages before the function of gills is fully developed.     

Strategies for a successful corrective Asian blepharoplasty after previously failed revisions

Asian blepharoplasty, although a common procedure, has a relatively high rate of complications. Subtle imperfections and more serious iatrogenic complications often require immediate attention by the aesthetic surgeon. After attempted correction of the deformities, residual problems or new ones can arise. Blepharoptosis, supratarsal depression, an excessively high or low crease, a short or discontinuous crease, multiple creases, and asymmetric creases are the most commonly encountered complications that require special attention in this group, which has already undergone more than one surgical procedure. Between January of 1996 and December of 2002, 168 Asian blepharoplasty revisions were performed by one surgeon (S. H.-T. Chen); of these, 36 patients (21 percent) had previously undergone failed revisions. This subgroup of patients consisted of six with blepharoptosis, six with asymmetrical eyelid creases, three with supratarsal depressions, three with high creases, two with short creases, and 16 with combinations of these deformities. The results were graded as excellent, good, fair, or poor, based on the symmetry of the eyelids, palpebral fissures, crease heights, lengths, shapes, eyelid fullness, and overall aesthetics of the final outcome. A survey was performed of patient and surgeon satisfaction and factored into the grading system. With an average follow-up period of 16 months (6 to 60 months), 22 patients (61 percent) were found to have excellent results, 10 (28 percent) had good results, two (5.6 percent) had fair results, and two (5.6 percent) had poor results. Corrective procedures after failed revision Asian blepharoplasty require special strategic considerations because of the presence of extensive scarring and inadequate skin, muscle, and preaponeurotic fat and because of the occasional presence of dehiscence of the levator aponeurosis. By using careful preoperative evaluation, accurate measurements, precise preoperative planning, intraoperative fat repositioning or grafting, skin excision or redraping, and proper placement of anchoring sutures, successful outcomes can be achieved. The authors evaluate the outcomes and detail the surgical procedures that were used to achieve successful outcomes in this particularly challenging group of patients.     

Characterization of a branch of the phylogenetic tree

We use a combination of analytic models and computer simulations to gain insight into the dynamics of evolution. Our results suggest that certain interesting phenomena should eventually emerge from the fossil record. For example, there should be a "tortoise and hare effect": those genera with the smallest species death rate are likely to survive much longer than genera with large species birth and death rates. A complete characterization of the behavior of a branch of the phylogenetic tree corresponding to a genus and accurate mathematical representations of the various stages are obtained. We apply our results to address certain controversial issues that have arisen in paleontology such as the importance of punctuated equilibrium and whether unique Cambrian phyla have survived to the present.     

Peptides trap the human immunodeficiency virus type 1 envelope glycoprotein fusion intermediate at two sites

Human immunodeficiency virus type 1 (HIV-1) entry into target cells requires folding of two heptad-repeat regions (N-HR and C-HR) of gp41 into a trimer of N-HR and C-HR hairpins, which brings viral and target cell membranes together to facilitate membrane fusion. Peptides corresponding to the N-HR and C-HR of gp41 are potent inhibitors of HIV infection. Here we report new findings on the mechanism of inhibition of a N-HR peptide and compare these data with inhibition by a C-HR peptide. Using intact envelope glycoprotein (Env) under fusogenic conditions, we show that the N-HR peptide preferentially binds receptor-activated Env and that CD4 binding is sufficient for triggering conformational changes that allow the peptide to bind Env, results similar to those seen with the C-HR peptide. However, activation by both CD4 and chemokine receptors further enhances Env binding by both peptides. We also show that a nonconservative mutation in the N-HR of gp41 abolishes C-HR peptide but not N-HR peptide binding to gp41. These results indicate that there are two distinct sites in receptor-activated Env that are potential targets for drug or vaccine development.     

Interaction between heme oxygenase-1 and -2 proteins

The three isoforms of heme oxygenase (HO), the rate-limiting enzyme in heme degradation, are the products of different genes that show marked differences in regulation and expression. Why is there redundancy in the heme degradation pathway, and why are there differences in tissue expression of HO isoenzymes are unanswered questions? An interaction between HO-1 and HO-2 is suspected by the co-localization of these enzymes in the lung and regions of the brain. Using multiple models and assays, we demonstrated an interaction between HO-1 and HO-2 at amino acids 0-45 of HO-2 and amino acids 58-80 of HO-1. The latter corresponds to a highly conserved, hydrophilic, and exposed region of the protein. Furthermore, the observed activity of the HO-1.HO-2 complex was lower than that expected from the sum of HO-1- and HO-2-derived activities, suggesting that this interaction serves to limit HO enzymatic activity. We speculate that this HO-1.HO-2 protein interaction may promote non-enzymatic functions of HO.     

Oxidant and antioxidant modulation of chloride channels expressed in human retinal pigment epithelium

Retinal pigment epithelium (RPE)possesses regulated chloride channels that are crucial fortransepithelial fluid and ion transport. At present, little is knownabout the molecular nature of chloride channels in human adult RPE(haRPE) or the effects of oxidative stress on membrane conductanceproperties. In the present study, we assessed ClC channel and cysticfibrosis transmembrane conductance regulator (CFTR) expression andmembrane chloride conductance properties in haRPE cells. ClC-5, ClC-3,ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, andprotein expression was detected with Western blot analysis andimmunofluorescence microscopy. Whole cell recordings of primarycultures of haRPE showed an outwardly rectifying chloride current thatwas inhibited by the oxidant H₂O₂. Theinhibitory effects of H₂O₂ were reduced incultured human RPE cells that were incubated with precursors ofglutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate apossible role for ClC channels in haRPE cells and suggest possibleredox modulation of human RPE chloride conductances.