KirrelL, a member of the Ig-domain superfamily of adhesion proteins, is essential for fusion of primary mesenchyme cells in the sea urchin embryo.

In the sea urchin embryo, primary mesenchyme cells (PMCs) adhere to one another and fuse via filopodia, forming cable-like structures within which skeletal rods are deposited. Although this process was first described more than a century ago, molecules that participate in PMC adhesion and fusion have not been identified. Here we show that KirrelL, a PMC-specific, Ig domain-containing transmembrane protein, is essential for PMC fusion, probably by mediating filopodial adhesions that are a pre-requisite for subsequent membrane fusion. We show that KirrelL is not required for PMC specification, migration, or for direct filopodial contacts between PMCs. In the absence of KirrelL, however, filopodial contacts do not result in fusion. kirrelL is a member of a family of closely related, intronless genes that likely arose through an echinoid-specific gene expansion, possibly via retrotransposition. Our findings are significant in that they establish a direct linkage between the transcriptional network deployed in the PMC lineage and an effector molecule required for a critically important PMC morphogenetic process. In addition, our results point to a conserved role for Ig domain-containing adhesion proteins in facilitating cell fusion in both muscle and non-muscle cell lineages during animal development.

COPS5 (Jab1) protein increases ß site processing of amyloid precursor protein and amyloid ß peptide generation by stabilizing RanBP9 protein levels.

Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid ß peptide (Aß) in the brain is central to the pathogenesis of Alzheimer's disease (AD). Therefore, the identification of molecules that regulate Aß generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aß generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aß levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aß generation, followed by increased soluble APP-ß (sAPP-ß) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aß generation, implying that endogenous COPS5 regulates Aß generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aß generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aß generation by stabilizing RanBP9 protein levels.

Striking reduction of amyloid plaque burden in an Alzheimer's mouse model after chronic administration of carmustine.

BACKGROUND: Currently available therapies for Alzheimer's disease (AD) do not treat the underlying cause of AD. Anecdotal observations in nursing homes from multiple studies strongly suggest an inverse relationship between cancer and AD. Therefore, we reasoned that oncology drugs may be effective against AD. METHODS: We screened a library of all the FDA-approved oncology drugs and identified bis-chloroethylnitrosourea (BCNU or carmustine) as an effective amyloid beta (Aß) reducing compound. To quantify Aß levels, Chinese hamster ovary (CHO) cells stably expressing amyloid precursor protein 751WT (APP751WT) called 7WD10 cells were exposed to different concentrations of BCNU for 48 hours and the conditioned media were collected. To detect Aß the conditioned media were immunoprecipitated with Ab9 antibody and subjected to immunoblot detection. Amyloid plaques were quantified in the brains of a mouse model of AD after chronic exposure to BCNU by thoflavin S staining. RESULTS: BCNU decreased normalized levels of Aß starting from 5 µM by 39% (P < 0.05), 10 µM by 51% (P < 0.01) and 20 µM by 63% (P < 0.01) in CHO cells compared to a control group treated with butyl amine, a structural derivative of BCNU. Interestingly, soluble amyloid precursor protein α (sAPPα) levels were increased to 167% (P < 0.01) at 0.5 µM, 186% (P < 0.05) at 1 µM, 204% (P < 0.01) at 5 µM and 152% (P < 0.05) at 10 µM compared to untreated cells. We also tested the effects of 12 structural derivatives of BCNU on Aß levels, but none of them were as potent as BCNU. BCNU treatment at 5 µM led to an accumulation of immature APP at the cell surface resulting in an increased ratio of surface to total APP by 184% for immature APP, but no change in mature APP. It is also remarkable that BCNU reduced Aß generation independent of secretases which were not altered up to 40 µM. Interestingly, levels of transforming growth factor beta (TGFß) were increased at 5 µM (43%, P < 0.05), 10 µM (73%, P < 0.01) and 20 µM (92%, P < 0.001). Most significantly, cell culture results were confirmed in vivo after chronic administration of BCNU at 0.5 mg/kg which led to the reduction of Aß40 by 75% and amyloid plaque burden by 81%. Conversely, the levels of sAPPα were increased by 45%. CONCLUSIONS: BCNU reduces Aß generation and plaque burden at non-toxic concentrations possibly through altered intracellular trafficking and processing of APP. Taken together these data provided unequivocal evidence that BCNU is a potent secretase-sparing anti-Aß drug. See related commentary article here http://www.biomedcentral.com/1741-7015/11/82.

Primary dermatofibrosarcoma protuberans of orbit--a rare entity.

Primary Dermatofibrosarcoma Protuberance (DFSP) is a rare neoplasm of dermal origin. Though it is a locally aggressive tumor with high recurrence rate, however distant metastasis can also occur. Orbital DFSP is an uncommon phenomenon. It has been reported due to distant metastasis or invasion from adjacent structures but Primary Orbital DFSP is a unique entity in itself. Herein we report a rare case of primary DFSP of the orbit in a 70-year- old lady who underwent orbital exenteration. Histopathology examination (HPE) revealed spindle cells arranged in storiform pattern and immunohistochemistry (IHC) revealed CD34 positive and S100 negative.

Molecular mechanism of insulin resistance.

Free fatty acids are known to play a key role in promoting loss of insulin sensitivity,thereby causing insulin resistance and type 2 diabetes.However,the underlying mechanism involved is still unclear.In searching for the cause of the mechanism,it has been found that palmitate inhibits insulin receptor (IR)gene expression,leading to a reduced amount of IR protein in insulin target cells. PDK1-independent phosphorylation of PKC(eta) causes this reduction in insulin receptor gene expression.One of the pathways through which fatty acid can induce insulin resistance in insulin target cells is suggested by these studies.We provide an overview of this important area,emphasizing the current status.

Involvement of novel PKC isoforms in FFA induced defects in insulin signaling.

Involvement of novel PKCs (nPKCs) in the negative regulation of insulin-signaling pathway is a current interest of many workers investigating the cause for insulin resistance and type 2 diabetes. Free fatty acids (FFAs) are recently shown to be the major players in inducing insulin resistance in insulin target cells. They are also found to be involved in activating nPKCs associated with the impairment of insulin sensitivity. In this overview, we describe PKC delta, theta and epsilon linked to the FFA induced damage of insulin-signaling molecules.

Effects of Cilnidipine on Heart Rate and Uric Acid Metabolism in Patients With Essential Hypertension.

BACKGROUND: The relation between hypertension and hyperuricemia has been established by epidemiological studies. Calcium channel blockers are one of the first-line drugs for newly diagnosed patients with essential hypertension. Cilnidipine is a new calcium channel blocker acting by blocking both L- and N-type calcium channels. The aim of this study was to compare the effectiveness of amlodipine and cilnidipine in patients with essential hypertension and their effects on heart rate and serum uric acid levels. METHODS: Out of 100 enrolled patients, 92 completed the study. They were randomly assigned to amlodipine (N = 47) and cilnidipine (N = 45) groups. Cilnidipine was started at 10 mg/day and then adjusted to 5 - 20 mg/day, and amlodipine was started at 5 mg/day and then adjusted to 2.5 - 10 mg/day. RESULTS: After 24 weeks of study, patients in cilnidipine groups showed significant reduction in heart rate and serum uric acid levels from baseline (P = 0.00). CONCLUSION: In clinical setting where both hypertension and hyperuricemia exist, cilnidipine can be a promising drug of choice.

Comparative analysis of use of porous orbital implant with mucus membrane graft and dermis fat graft as a primary procedure in reconstruction of severely contracted socket.

PURPOSE: The purpose of our study is to present a surgical technique of primary porous orbital ball implantation with overlying mucus membrane graft (MMG) for reconstruction of severely contracted socket and to evaluate prosthesis retention and motility in comparison to dermis fat graft (DFG). STUDY DESIGN: Prospective comparative study. MATERIALS AND METHODS: A total of 24 patients of severe socket contracture (Grade 2-4 Krishna's classification) were subdivided into two groups, 12 patients in each group. In Group I, DFG have been used for reconstruction. In Group II, porous polyethylene implant with MMG has been used as a primary procedure for socket reconstruction. In Group I DFG was carried out in usual procedure. In case of Group II, vascularized scar tissues were separated 360° and were fashioned into four strips. A scleral capped porous polyethylene implant was placed in the intraconal space and four strips of scar tissue were secured to the scleral cap and extended part overlapped the implant to make a twofold barrier between the implant and MMG. Patients were followed-up as per prefixed proforma. Prosthesis motility and retention between the two groups were measured. RESULTS: In Group I, four patients had recurrence of contracture with fall out of prosthesis. In Group II stable reconstruction was achieved in all the patients. In terms of prosthesis motility, maximum in Group I was 39.2% and Group II, was 59.3%. The difference in prosthesis retention (P = 0.001) and motility (P = 0.004) between the two groups was significant. CONCLUSION: Primary socket reconstruction with porous orbital implant and MMG for severe socket contracture is an effective method in terms of prosthesis motility and prosthesis retention.

Chronic cladribine administration increases amyloid beta peptide generation and plaque burden in mice.

BACKGROUND: The clinical uses of 2-chloro-2'-deoxyadenosine (2-CDA) or cladribine which was initially prescribed to patients with hematological and lymphoid cancers is now extended to treat patients with multiple sclerosis (MS). Previous data has shown that 2-CDA has high affinity to the brain and readily passes through the blood brain barrier reaching CSF concentrations 25% of that found in plasma. However, whether long-term administration of 2-CDA can lead to any adverse effects in patients or animal models is not yet clearly known. METHODOLOGY: Here we show that exposure of 2-CDA to CHO cells stably expressing wild-type APP751 increased generation and secretion of amyloid ß peptide (Aß) in to the conditioned medium. Interestingly, increased Aß levels were noticed even at non-toxic concentrations of 2-CDA. Remarkably, chronic treatment of APdE9 mice, a model of Alzheimer's disease with 2-CDA for 60 days increased amyloid plaque burden by more than 1-fold. Increased Aß generation appears to result from increased turnover of APP as revealed by cycloheximide-chase experiments. Additionally, surface labeling of APP with biotin and immunoprecipitation of surface labeled proteins with anti-biotin antibody also indicated increased APP at the cell surface in 2-CDA treated cells compared to controls. Increased turnover of APP by 2-CDA in turn might be a consequence of decreased protein levels of PIN 1, which is known to regulate cis-trans isomerization and phosphorylation of APP. Most importantly, like many other oncology drugs, 2-CDA administration led to significant delay in acquiring a reward-based learning task in a T maze paradigm. CONCLUSIONS: Taken together, these data provide compelling evidence for the first time that chronic 2-CDA administration can increase amyloidogenic processing of APP leading to robustly increased plaque burden which may be responsible for the observed deficits in learning skills. Thus chronic treatment of mice with 2-CDA can have deleterious effects in vivo.

Patterns of gene expression in Drosophila InsP3 receptor mutant larvae reveal a role for InsP3 signaling in carbohydrate and energy metabolism.

BACKGROUND: The Inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an InsP(3) gated intracellular Ca(2+)-release channel. Characterization of Drosophila mutants for the InsP(3)R has demonstrated that InsP(3)-mediated Ca(2+) release is required in Drosophila larvae for growth and viability. METHODOLOGY/PRINCIPAL FINDINGS: To understand the molecular basis of these growth defects a genome wide microarray analysis has been carried out with larval RNA obtained from a strong InsP(3)R mutant combination in which 1504 independent genes were differentially regulated with a log(2) of fold change of 1 or more and P<0.05. This was followed by similar transcript analyses from InsP(3)R mutants where growth defects were either suppressed by introduction of a dominant suppressor or rescued by ectopic expression of an InsP(3)R transgene in the Drosophila insulin like peptide-2 (Dilp2) producing cells. CONCLUSIONS/SIGNIFICANCE: These studies show that expression of transcripts related to carbohydrate and amine metabolism is altered in InsP(3) receptor mutant larvae. Moreover, from a comparative analysis of genes that are regulated in the suppressed and rescued conditions with the mutant condition, it appears that the organism could use different combinations of pathways to restore a 'normal' growth state.

Fatty acid represses insulin receptor gene expression by impairing HMGA1 through protein kinase Cepsilon.

It is known that free fatty acid (FFA) contributes to the development of insulin resistance and type2 diabetes. However, the underlying mechanism in FFA-induced insulin resistance is still unclear. In the present investigation we have demonstrated that palmitate significantly (p <0.001) inhibited insulin-stimulated phosphorylation of PDK1, the key insulin signaling molecule. Consequently, PDK1 phosphorylation of plasma membrane bound PKCepsilon was also inhibited. Surprisingly, phosphorylation of cytosolic PKCepsilon was greatly stimulated by palmitate; this was then translocated to the nuclear region and associated with the inhibition of insulin receptor (IR) gene transcription. A PKCepsilon translocation inhibitor peptide, epsilonV1, suppressed this inhibitory effect of palmitate, suggesting requirement of phospho-PKCepsilon migration to implement palmitate effect. Experimental evidences indicate that phospho-PKCepsilon adversely affected HMGA1. Since HMGA1 regulates IR promoter activity, expression of IR gene was impaired causing reduction of IR on cell surface and that compromises with insulin sensitivity.

A Lupinoside prevented fatty acid induced inhibition of insulin sensitivity in 3T3 L1 adipocytes.

The decrease in insulin sensitivity to target tissues or insulin resistance leads to type 2 diabetes mellitus, an insidious disease threatening global health. Numerous evidences made free fatty acids (FFAs) responsible for insulin resistance and type 2 diabetes. We demonstrate here that the damage of insulin acitivity by a free fatty acid, palmitate could be prevented by a lupinoside. An incubation of 3T3 L1 adipocytes with a FFA i.e. palmitate inhibited insulin stimulated uptake of (3)H-2 deoxyglucose (2 DOG) significantly. Addition of a lupinoside purified from Pueraria tuberosa, lupinoside PA(4) (LPA(4)) strongly prevented this inhibition. We then examined insulin signaling pathway where palmitate significantly inhibited insulin stimulated phosphorylation of Insulin receptor tyrosine kinase, IRS 1and PI3 kinase, PDK1 and Akt/PKB. LPA(4) rescued this inhibition of signaling molecule by palmitate. Insulin mediated translocation of Glut4, the glucose transporter in insulin target cells, was effectively blocked by palmitate while, LPA(4) waived this block. Administration of LPA(4) to nutritionally induced diabetic rats significantly reduced the increase in plasma glucose. All these indicate LPA(4) to be a potentially therapeutic agent for insulin resistance and type 2 diabetes.

Inhibition of insulin receptor gene expression and insulin signaling by fatty acid: interplay of PKC isoforms therein.

Fatty acids are known to play a key role in promoting the loss of insulin sensitivity causing insulin resistance and type 2 diabetes. However, underlying mechanism involved here is still unclear. Incubation of rat skeletal muscle cells with palmitate followed by I(125)- insulin binding to the plasma membrane receptor preparation demonstrated a two-fold decrease in receptor occupation. In searching the cause for this reduction, we found that palmitate inhibition of insulin receptor (IR) gene expression effecting reduced amount of IR protein in skeletal muscle cells. This was followed by the inhibition of insulin-stimulated IRbeta tyrosine phosphorylation that consequently resulted inhibition of insulin receptor substrate 1 (IRS 1) and IRS 1 associated phosphatidylinositol-3 kinase (PI3 Kinase), phosphoinositide dependent kinase-1 (PDK 1) phosphorylation. PDK 1 dependent phosphorylation of PKCzeta and Akt/PKB were also inhibited by palmitate. Surprisingly, although PKCepsilon phosphorylation is PDK1 dependent, palmitate effected its constitutive phosphorylation independent of PDK1. Time kinetics study showed translocation of palmitate induced phosphorylated PKCepsilon from cell membrane to nuclear region and its possible association with the inhibition of IR gene transcription. Our study suggests one of the pathways through which fatty acid can induce insulin resistance in skeletal muscle cell.