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Galectins and prostate specific membrane antigen (PSMA) are glycoproteins that are functionally implicated in prostate cancer (CaP). We undertook this study to analyze the “PSMA-galectin pattern” of the human CaP microenvironment with the overarching goal of selecting novel-molecular targets for prognostic and therapeutic purposes. We examined CaP cells and biopsy samples representing different stages of the disease and found that PSMA, Gal-1, Gal-3, and Gal-8 are the most abundantly expressed glycoproteins. In contrast, other galectins such as Gal-2, 4–7, 9–13, were uniformly expressed at lower levels across all cell lines. However, biopsy samples showed markedly higher expression of PSMA, Gal-1 and Gal-3. Independently PSA and Gleason score at diagnosis correlated with the expression of PSMA, Gal-3. Additionally, the combined index of PSMA and Gal-3 expression positively correlated with Gleason score and was a better predictor of tumor aggressiveness. Together, our results recognize a tightly regulated “PSMA-galectin- pattern” that accompanies disease in CaP and highlight a major role for the combined PSMA and Gal-3 inhibitors along with standard chemotherapy for prostate cancer treatment. Inhibitor combination studies show enzalutamide (ENZ), 2-phosphonomethyl pentanedioic acid (2-PMPA), and GB1107 as highly cytotoxic for LNCaP and LNCaP-KD cells, while Docetaxel (DOC) + GB1107 show greater efficacy in PC-3 cells. Overall, 2-PMPA and GB1107 demonstrate synergistic cytotoxic effects with ENZ and DOC in various CaP cell lines.

Interleukin-8 (IL-8) is a pro-angiogenic cytokine associated with aggressive prostate cancer (CaP). We detected high levels of IL-8 in sera from patients with CaP compared with healthy controls and patients with benign prostatic hypertrophy. This study examines the role of IL-8 in the pathogenesis of metastatic prostate cancer. We developed a biocompatible, cationic polylactide (CPLA) nanocarrier to complex with and efficiently deliver IL-8 small interfering RNA (siRNA) to CaP cells in vitro and in vivo. CPLA IL-8 siRNA nanocomplexes (nanoplexes) protect siRNA from rapid degradation, are non-toxic, have a prolonged lifetime in circulation, and their net positive charge facilitates penetration of cell membranes and subsequent intracellular trafficking. Administration of CPLA IL-8 siRNA nanoplexes to immunodeficient mice bearing human CaP tumours produced significant antitumour activities with no adverse effects. Systemic (intravenous) or local intra-tumour administration of IL-8 siRNA nanoplexes resulted in significant inhibition of CaP growth. Magnetic resonance imaging and ultrasonography of experimental animals demonstrated reduction of tumour perfusion in vivo following nanoplex treatment. Staining of tumour sections for CD31 confirmed significant damage to tumour neovasculature after nanoplex therapy. These studies demonstrate the efficacy of IL-8 siRNA nanotherapy for advanced, treatment-resistant human CaP.

Single nucleotide polymorphisms (SNPs) can play a direct or indirect role in phenotypic expression in food allergy pathogenesis. Our goal was to quantitate the expression of SNPs in relevant cytokines that were expressed in food allergic patients. SNPs in cytokine genes IL-4 and IL-10 are known to be important in IgE generation and regulation. We examined IL-4 (C-590T), IL-4Rα (1652A/G) and IL-10 (C-627A) SNPs using real-time PCR followed by restriction fragment length polymorphism (RFLP) analysis. Our results show that the AA, AG and GG genotypes for IL-4Rα (1652A/G) polymorphisms were statistically different in radioallergosorbent test (RAST) positive versus negative patients, and although no statistically significant differences were observed between genotypes in the IL-4 (C-590T) and IL-10 (C-627A) SNPs, we observed a significant decrease in IL-4 (C-590T) gene expression and increase in IL-4Rα (1652A/G) and IL-10 (C-627A) gene expression between RAST(+) versus RAST(-) patients, respectively. We also observed significant modulation in the protein expression of IL-4 and IL-10 in the serum samples of the RAST(+) patients as compared to the RAST(-) patients indicating that changes in SNP expression resulted in altered phenotypic response in these patients.

Galectin-1, an adhesion molecule, is expressed in macrophages and implicated in human immunodeficiency virus (HIV-1) viral adsorption. In this study, we investigated the effects of methamphetamine on galectin-1 production in human monocyte derived macrophages (MDM) and the role of galectin-1 in methamphetamine potentiation of HIV-1 infection. Herein we show that levels of galectin-1 gene and protein expression are significantly increased by meth-amphetamine. Furthermore, concomitant incubation of MDM with galectin-1 and methamphetamine facilitates HIV-1 infection compared to galectin-1 alone or methamphetamine alone. We utilized a nanotechnology approach that uses gold nanorod (GNR)-galectin-1 siRNA complexes (nanoplexes) to inhibit gene expression for galectin-1. Nanoplexes significantly silenced gene expression for galectin-1 and reversed the effects of methamphetamine on galectin-1 gene expression. Moreover, the effects of methamphetamine on HIV-1 infection were attenuated in the presence of the nanoplex in MDM.

Morphine is a widely abused, addictive drug that modulates immune function. Macrophages are a primary reservoir of HIV-1; therefore, they play a role in the development of this disease, as well as impact the overall course of disease progression. Galectin-1 is a member of a family of β-galactoside–binding lectins that are soluble adhesion molecules and that mediate direct cell–pathogen interactions during HIV-1 viral adhesion. Because the drug abuse epidemic and the HIV-1 epidemic are closely interrelated, we propose that increased expression of galectin-1 induced by morphine may modulate HIV-1 infection of human monocyte-derived macrophages (MDMs). In this article, we show that galectin-1 gene and protein expression are potentiated by incubation with morphine. Confirming previous studies, morphine alone or galectin-1 alone enhance HIV-1 infection of MDMs. Concomitant incubation with exogenous galectin-1 and morphine potentiated HIV-1 infection of MDMs. We used a nanotechnology approach that uses gold nanorod–galectin-1 small interfering RNA complexes (nanoplexes) to inhibit gene expression for galectin-1. We found that nanoplexes silenced gene expression for galectin-1, and they reversed the effects of morphine on galectin-1 expression. Furthermore, the effects of morphine on HIV-1 infection were reduced in the presence of the nanoplex.

Phytochemicals are dietary phytoestrogens that may play a role in prostate cancer prevention. Forty percent of Americans use complementary and alternative medicines (CAM) for disease prevention and therapy. Ashwagandha (Withania somnifera) contains flavonoids and active ingredients like alkaloids and steroidal lactones which are called ‘Withanolides’. We hypothesize that the immunomodulatory and anti-inflammatory properties of Ashwagandha might contribute to its overall effectiveness as an anti-carcinogenic agent. The goal of our study was gain insight into the general biological and molecular functions and immunomodulatory processes that are altered as a result of Ashwagandha treatment in prostate cancer cells, and to identify the key signaling mechanisms that are involved in the regulation of these physiological effects using genomic microarray analysis in conjunction with quantitative real-time PCR and western blot analysis. Ashwagandha treatment significantly downregulated the gene and protein expression of proinflammatory cytokines IL-6, IL-1β, chemokine IL-8, Hsp70 and STAT-2, while a reciprocal upregulation was observed in gene and protein expression of p38 MAPK, PI3K, caspase 6, Cyclin D and c-myc. Furthermore, Ashwagandha treatment significantly modulated the JAK-STAT pathway which regulates both the apoptosis process as well as the MAP kinase signaling. These studies outline several functionally important classes of genes, which are associated with immune response, signal transduction, cell signaling, transcriptional regulation, apoptosis and cell cycle regulation and provide insight into the molecular signaling mechanisms that are modulated by Ashwagandha, thereby highlighting the use of this bioflavanoid as effective chemopreventive agent relevant to prostate cancer progression.

The matrix-degrading metalloproteinases (MMPs), particularly MMP-9, are involved in the neuroinflammation processes leading to disrupting of the blood brain barrier (BBB), thereby exacerbating neurological diseases such as HIV-1 AIDS dementia and cerebral ischemia. Nanoparticles have been proposed to act as non-viral gene delivery vectors and have great potential for therapeutic applications in several disease states. In this study, we evaluated the specificity and efficiency of quantum dot (QD) complexed with MMP-9-siRNA (nanoplex) in downregulating the expression of MMP-9 gene in brain microvascular endothelial cells (BMVEC) that constitute the BBB. We hypothesize that silencing MMP-9 gene expression in BMVECs and other cells such as leukocytes may help prevent breakdown of the BBB and inhibit subsequent invasion of the central nervous system (CNS) by infected and inflammatory cells. Our results show that silencing of MMP-9 gene expression resulted in the up-regulation of extracellular matrix (ECM) proteins like collagen I, IV, V and a decrease in endothelial permeability, as reflected by reduction of transendothelial resistance across the BBB in a well validated in-vitro BBB model. MMP-9 gene silencing also resulted in an increase in expression of the gene tissue inhibitor of metalloproteinase-1 (TIMP-1). This indicates the importance of a balance between the levels of MMP-9 and its natural inhibitor TIMP-1 in maintaining the basement membrane integrity. These studies promise the application of a novel nanoparticle based siRNA delivery system in modulating the MMP-9 activity in BMVECs and other MMP-9 producing cells. This will prevent neuroinflammation and maintain the integrity of the BBB.

We used proteomic analyses to assess how drug abuse modulates immunologic responses to infections with the human immunodeficiency virus type 1 (HIV-1).Two-dimensional difference gel electrophoresis was utilized to determine changes in the proteome of peripheral blood mononuclear cells (PBMC) isolated from HIV-1-positive donors that occurred after treatment with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins. We further isolated specific subpopulations of PBMC to determine which subpopulations were selectively affected by treatment with drugs of abuse. Monocytes, B cells, and T cells were positively or negatively selected from PBMC isolated from HIV-1-positive donors.Our results demonstrate that cocaine and methamphetamine modulate gene expression primarily in monocytes and T cells, the primary targets of HIV-1 infection. Proteomic data were validated with quantitative, real-time polymerase chain reaction. These studies elucidate the molecular mechanisms underlying the effects of drugs of abuse on HIV-1 infections. Several functionally relevant classes of proteins were identified as potential mediators of HIV-1 pathogenesis and disease progression associated with drug abuse.

Drug abuse has become a global health concern. Understanding how drug abuse modulates the immune system and how the immune system responds to pathogens associated with drug abuse, such hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1), can be assessed by an integrated approach comparing proteomic analyses and quantitation of gene expression. Two-dimensional (2D) difference gel electrophoresis was used to determine the molecular mechanisms underlying the proteomic changes that alter normal biological processes when monocyte-derived mature dendritic cells were treated with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins including those that modulate apoptosis, protein folding, protein kinase activity, and metabolism and proteins that function as intracellular signal transduction molecules. Proteomic data were validated using a combination of quantitative, real-time PCR and Western blot analyses. These studies will help to identify the molecular mechanisms, including the expression of several functionally important classes of proteins that have emerged as potential mediators of pathogenesis. These proteins may predispose immunocompetent cells, including dendritic cells, to infection with viruses such as HCV and HIV-1, which are associated with drug abuse.

BACKGROUND Human and animal studies have suggested that diet-derived flavonoids, in particular quercetin may play a beneficial role by preventing or inhibiting oncogenesis, but the underlying mechanism remains unclear. The aim of this study is to evaluate the effect(s) of quercetin on normal and malignant prostate cells and to identify the target(s) of quercetin's action. METHODOLOGY We addressed this question using cells in culture and investigated whether quercetin affects key biological processes responsible for tumor cell properties such as cell proliferation and apoptosis and also studied the effect of quercetin on the proteome of prostate cancer cells using difference gel electrophoresis (DIGE) to assess changes in the expression of relevant proteins. RESULTS Our findings demonstrate that quercetin treatment of prostate cancer cells results in decreased cell proliferation and viability. Furthermore, we demonstrate that quercetin promotes cancer cell apoptosis by down-regulating the levels of heat shock protein (Hsp) 90. Depletion of Hsp90 by quercetin results in decreased cell viability, levels of surrogate markers of Hsp90 inhibition (intracellular and secreted), induced apoptosis and activation of caspases in cancer cells but not in normal prostate epithelial cells. Knockdown of Hsp90 by short interfering RNA also resulted in induction apoptosis similar to quercetin in cancer cells as indicated by annexin V staining. CONCLUSION Our results demonstrate that quercetin down-regulates the expression of Hsp90 which, in turn, induces inhibition of growth and cell death in prostate cancer cells while exerting no quantifiable effect on normal prostate epithelial cells. Prostate © 2008 Wiley-Liss, Inc.

The pathogenesis of human immunodeficiency virus (HIV) associated encephalopathy is attributed to infiltration of the central nervous system (CNS) by HIV-1 infected mononuclear cells that transmigrate across the blood brain barrier (BBB). The endothelial tight junctions (TJ) of the blood brain barrier (BBB) play a critical role in controlling cellular traffic into the CNS. Neuropathogenesis of HIV-1 is exacerbated by drugs of abuse such as methamphetamine (Meth) which are capable of dysregulating BBB function. HIV-1 viral proteins like gp120 are both neurotoxic and cytotoxic and have been implicated in the development of HIV-1 dementia (HAD). We hypothesize that gp120 in synergy with Meth can alter BBB permeability via the modulation of tight junction expression. We investigated the effect of Meth and/or gp120 on the basal expression of TJ proteins ZO-1, JAM-2, Occludin, Claudin-3 and Claudin-5, using in vitro cultures of the primary brain microvascular endothelial cells (BMVEC). Further, the functional effects of TJ modulation were assessed using an in vitro BBB model, that allowed measurement of BBB permeability using TEER measurements and transendothelial migration of immunocompetent cells. Our results show that both Meth and gp120 individually and in combination, modulated TJ expression, and these effects involved Rho-A activation. Further, both Meth and gp120 alone and in combination significantly decreased transendothelial resistance across the in vitro BBB and the enhanced transendothelial migration of immunocompetent cells across the BBB. An understanding of the mechanisms of BBB breakdown that lead to neurotoxicity is crucial to the development of therapeutic modalities for Meth abusing HAD patients.

Delta-9-tetrahydrocannabinol (Delta(9)-THC), the main psychoactive component of marijuana, is known to dysregulate various immune responses. Cannabinoid (CB)-1 and -2 receptors are expressed mainly on cells of the central nervous system (CNS) and the immune system. The CNS is the primary target of cannabinoids and astrocytes are known to play a role in various immune responses. Thus we undertook this investigation to determine the global molecular effects of cannabinoids on normal human astrocytes (NHA) using genomic and proteomic analyses. NHA were treated with Delta(9)-THC and assayed using gene microarrays and two-dimensional (2D) difference gel electrophoresis (DIGE) coupled with mass spectrometry (MS) to elucidate their genomic and proteomic profiles respectively. Our results show that the expression of more than 20 translated protein gene products from NHA was differentially dysregulated by treatment with Delta(9)-THC compared to untreated, control NHA.

In the US, the increase in methamphetamine (METH) use has been associated with increased human immunodeficiency virus (HIV-1) infection. Dendritic cells (DC) are the first line of defense against HIV-1. DC play a critical role in harboring HIV-1 and facilitate the infection of neighboring T cells. However, the role of METH on HIV-1 infectivity and the expression of the proteome of immature dendritic cells (IDC) has not been elucidated. We hypothesize that METH modulates the expression of a number of proteins by IDC that foster the immunopathogenesis of HIV-1 infection. We utilized LTR amplification, p24 antigen assay and the proteomic method of difference gel electrophoresis (DIGE) combined with protein identification through high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to analyze the effects of METH on HIV-1 infectivity (HIV-1 IIIB; CXCR4-tropic, X4 strain) and the proteomic profile of IDC. Our results demonstrate that METH potentiates HIV-1 replication in IDC. Furthermore, METH significantly differentially regulates the expression of several proteins including CXCR3, protein disulfide isomerase, procathepsin B, peroxiredoxin and galectin-1. Identification of unique, METH-induced proteins may help to develop novel markers for diagnostic, preventive and therapeutic targeting in METH using subjects.

The US is experiencing an epidemic of cocaine use entangled with HIV-1 infection. Normal human astrocytes (NHA) are susceptible to HIV-1 infection. We utilized LTR-R/U5 amplification, p24 antigen assay and the proteomic method of difference gel electrophoresis (DIGE) combined with protein identification through HPLC-MS/MS to investigate the effect of cocaine on HIV-1 infectivity and the proteomic profile of NHA, respectively. Data demonstrate that cocaine significantly upregulates HIV-1 infection in NHA as measured by LTR-R/U5 amplification and p24 antigen assay. Further, our results show for the first time that cocaine differentially regulates the expression of a number of proteins by NHA that may play a role in the neuropathogenesis of HIV-1 disease.

BACKGROUND Many viral and neoplastic diseases are resistant to interferon-α/β (IFN-α/β) therapy or develop resistance during the course of IFN treatment. In patients with viral diseases, the authors identified four IFN inhibitors, of which the most important, most likely is a free IFN receptor of type 1 appearing in the circulation that captures and neutralizes IFN-α/β. METHODS Ninety-one cancer patients and 25 healthy individuals were studied. Free circulating IFN receptor-α/β type 1 was studied. The patients were ages 35–75 years. The diagnoses were 24 cases of colon carcinoma, 7 cases of prostate carcinoma, 16 cases of breast carcinoma, 8 cases of ovarian carcinoma, 9 cases of uterine carcinoma, 5 cases of lung carcinoma, 3 cases of astrocytoma, 4 cases of transitional cell carcinoma of the bladder, 1 case of osteosarcoma, 3 cases of multiple myeloma, 4 cases of Hodgkin disease, 2 cases of non-Hodgkin lymphoma, 3 cases of myelodysplastic syndrome, and 2 disseminated tumors of unknown origin. RESULTS All patients were found to have increased free IFN receptor-α/β type 1 in the circulation, with the highest levels reported in patients with adenocarcinoma. CONCLUSIONS High IFN inhibitory activity in patients with cancer may be a significant factor in their increased susceptibility to progressive disease, infectious complications, and resistance to IFN therapy. Ongoing studies are being performed with the objective of overcoming this inhibitory activity. Cancer 2003;98:2730–3. © 2003 American Cancer Society.

Both antisense oligonucleotides (ASODN) and small interfering RNA (siRNA) have enormous potential to selectively silence specific cancer-related genes and could therefore be developed to be important therapeutic anti-cancer drugs. The use of nanotechnology may allow for significant advancement of the therapeutic potential of ASODN and siRNA, due to improved pharmacokinetics, bio-distribution and tissue specific targeted therapy. In this mini-review, we have discussed the advantages of using a nanocarrier such as a multimodal quantum rod (QR) complexed with siRNA for gene delivery. Comparisons are made between ASODN and siRNA therapeutic efficacies in the context of cancer and the enormous application potential of nanotechnology in oncotherapy is discussed. We have shown that a QR-interleukin-8 (IL-8) siRNA nanoplex can effectively silence IL-8 gene expression in the PC-3 prostate cancer cells with no significant toxicity. Thus, nanocarriers such as QRs can help translate the potent effects of ASODN/siRNA into a clinically viable anti-cancer therapy. Drug delivery for cancer therapy, with the aid of nanotechnology is one of the major translational aspects of nanomedicine, and efficient delivery of chemotherapy drugs and gene therapy drugs or their co-delivery continue to be a major focus of nanomedicine research.

The advent of highly active antiretroviral therapy (HAART) has significantly improved the prognosis for human immunodeficiency virus (HIV)-infected patients, however the adverse side effects associated with prolonged HAART therapy use continue. Although systemic viral load can be undetectable, the virus remains sequestered in anatomically privileged sites within the body. Nanotechnology-based delivery systems are being developed to target the virus within different tissue compartments and are being evaluated for their safety and efficacy. The current review outlines the various nanomaterials that are becoming increasingly used in biomedical applications by virtue of their robustness, safety, multimodality, and multifunctionality. Nanotechnology can revolutionize the field of HIV medicine by not only improving diagnosis, but also by improving delivery of antiretrovirals to targeted regions in the body and by significantly enhancing the efficacy of the currently available antiretroviral medications. Published Version

Monocytes/macrophages are a primary source of human immunodeficiency virus (HIV-1) in the central nervous system (CNS). Macrophages infected with HIV-1 produce a plethora of factors, including matrix metalloproteinase-9 (MMP-9) that may contribute to the development of HIV-1-associated neurocognitive disorders (HAND). MMP-9 plays a pivotal role in the turnover of the extracellular matrix (ECM) and functions to remodel cellular architecture. We have investigated the role of methamphetamine and HIV-1 gp120 in the regulation of lipopolysaccaride (LPS) induced-MMP-9 production in monocyte-derived macrophages (MDM). Here, we show that LPS-induced MMP-9 gene expression and protein secretion are potentiated by incubation with methamphetamine alone and gp120 alone. Further, concomitant incubation with gp120 and methamphetamine potentiated LPS-induced MMP-9 expression and biological activity in MDM. Collectively methamphetamine and gp120 effects on MMPs may modulate remodeling of the extracellular environment enhancing migration of monocytes/macrophages to the CNS.

HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. We used a well-validated siRNA (si510) which targets the poly A/TAR (transactivator of the HIV-1 LTR) site and suppresses viral replication. Nanotechnology holds much potential for impact in the field of HIV-1 therapeutics, and nanoparticles such as quantum rods (QRs) can be easily functionalized to incorporate siRNA forming stable nanoplexes that can be used for gene silencing. We evaluated the efficacy of the QR-si510 HIV-1 siRNA nanoplex in suppressing viral replication in the HIV-1-infected monocytic cell line THP-1 by measuring p24 antigen levels and gene expression levels of HIV-1 LTR. Our results suggest that the QR-si510 HIV-1 siRNA nanoplex is not only effective in delivering siRNA, but also in suppressing HIV-1 viral replication for a longer time period. HIV-1 nanotherapeutics can thus enhance systemic bioavailability and offer multifunctionality.

Proteomic profiles of RAST+ subjects with severe food allergies and RAST− subjects were compared using 2D-DIGE analysis to obtain candidate biomarkers specific to food allergies. Our analysis highlighted 52 proteins that were differentially expressed between the RAST+ and RAST− groups of which 37 were successfully identified that include chondroitin sulfates, zinc finger proteins, C-type lectins, retinoic acid binding proteins, heat shock proteins, myosin, cytokines, mast cell expressed proteins, and MAP kinases. Biological network analysis tool Metacore revealed that most of these regulated proteins play a role in immune tolerance, hypersensitivity and modulate cytokine patterns inducing a Th2 response that typically results in IgE-mediated allergic response which has a direct or indirect biological link to food allergy. Identifying unique biomarkers associated with certain allergic phenotypes and potentially cross-reactive proteins through bioinformatics analyses will provide enormous insight into the mechanisms that underlie allergic response in patients with food allergies.

Allelic variants of the genes for chemokine receptors and their natural ligands, the chemokines, and cytokines can affect HIV-1 disease progression. This study investigates the level of expression of the CCR5-Delta32, CCR2b-641, RANTES In1.1C, SDF-1 3'A, IL-10-5'-592A and IL-4-589T alleles in two unique HIV-1 infected patient cohorts that represent the two distinct stages of disease progression, namely rapid progressors (RPs) and long term non-progressors (LTNPs) (n=12/group) were recruited. Quantitation of the gene expression of CCR5-Delta32, CCR2b-641, RANTES In1.1C, SDF-1 3'A, IL-10-5'-592A and IL-4-589T in peripheral blood mononuclear leukocytes (PBML) isolated from patients was performed by real time, quantitative (Q)-PCR using DNA was isolated from PBML. We observed that expression of these HIV-protective alleles was generally greater in the LTNP cohort than the RP cohort. LTNPs expressed more of the protective chemokine, SDF-1alpha than RPs, and no statistically significant difference was observed in RANTES production between the LTNPs and RPs. The LTNPs expressed significantly less amounts of cytokines IL-10 and IL-4 as compared to the RPs. Our results demonstrate that gene polymorphisms for CCR5-Delta32, CCR2b-641, RANTES In1.1C, SDF-1 3'A, IL-10-5'-592A and IL-4-589T may be used as clinical markers to predict progression of HIV-1 infections.

The 32-kDa dopamine- and adenosine 3′,5′-monophosphate-regulated phosphoprotein (DARPP-32) is recognized to be critical to the pathogenesis of drug addiction. Opiates via the μ-receptor act on the dopaminergic system in the brain and modulates the expression of DARPP-32 phosphoprotein which is an important mediator of the activity of the extracellular signal-regulated kinase (ERK) signaling cascades, the activation of which represents an exciting nexus for drug-induced changes in neural long-term synaptic plasticity. Silencing of DARPP-32 using an siRNA against DARPP-32 may provide a novel gene therapy strategy to overcome drug addiction. In this study, we investigated the effect of the opiate (heroin) on D1 receptor (D1R) and DARPP-32 expression and additionally, evaluated the effects of DARPP-32-siRNA gene silencing on protein phosphatase-1 (PP-1), ERK, and cAMP response element-binding (CREB) gene expression in primary normal human astrocytes (NHA) cells in vitro. Our results indicate that heroin significantly upregulated both D1R and DARPP-32 gene expression, and that DARPP-32 silencing in the NHA cells resulted in the significant modulation of the activity of downstream effector molecules such as PP-1, ERK, and CREB which are known to play an important role in opiate abuse-induced changes in long-term neural plasticity. These findings have the potential to facilitate the development of DARPP32 siRNA-based therapeutics against drug addiction.

The basis for personalized medicine is the creation of a repository of knowledge about the genetic alterations involved in disease processes. Integral to achieving this goal is the querying of well-preserved, high-quality human tissue samples. Making these findings relevant involves the interrogation of large numbers of samples. The pace with which changes have occurred versus the potential pace with which changes can occur may be indicative of problems associated with traditional approaches on collecting biospecimens. Therefore, transforming personalized medicine from concept to reality may require an alternative approach in the field of tissue specimen procurement."Exfoliation and Enrichment" (EE), a recently described rapid and cost-effective approach for procuring cells, was utilized and assessed relative to a more traditional but temporally and economically comparable approach. Material from the same tumor sample, one collected by EE but the other frozen, were procured, the DNA extracted, and the samples analyzed at the global and gene-specific level by array comparative genomic hybridization and quantitative polymerase chain reaction, respectively.Both approaches resulted in the rapid procurement and retrieval of well-preserved cells and nucleic acids. The presence of "contaminating" normal cells in the more traditional approach masked the significance of genetic gains and losses, findings that were more readily apparent from the material derived by the EE method.The EE approach represents a cost-effective alternative to traditional cell-procurement methods that results in the generation of superior genomic data.

Heroin use is postulated to act as a cofactor in the neuropathogenesis of human immunodeficiency virus (HIV-1) infection. Astrocytes, integral components of the CNS, are reported to be susceptible to HIV-1 infection. Upon activation, astrocytes release a number of immunoregulatory products or modulate the expression of a number of proteins that foster the immunopathogenesis of HIV-1 infection. However, the role of heroin on HIV-1 infectivity and the expression of the proteome of normal human astrocytes (NHA) have not been elucidated. We hypothesize that heroin modulates the expression of a number of proteins by NHA that foster the neuoropathogenesis of HIV-1 infection. We utilized LTR amplification and the p24 antigen assay to quantitate the effect of heroin on HIV-1 infectivity while difference gel electrophoresis (DIGE) combined with protein identification through high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to analyze the effects of heroin on the proteomic profile of NHA. Results demonstrate that heroin potentiates HIV-1 replication in NHA. Furthermore, heroin significantly increased protein expression levels for protein kinase C (PKC), reticulocalbin 1 precursor, reticulocalbin 1, tyrosine 3-monooxgenase/tryptophan 5-monooxgenase activation protein, chloride intracellular channel 1, cathepsin D preproprotein, galectin 1 and myosin light chain alkali. Heroin also significantly decreased protein expression for proliferating cell nuclear antigen, proteasome beta 6 subunit, tropomyosin 3, laminin receptor 1, tubulin alpha 6, vimentin, EF hand domain family member D2, Tumor protein D54 (hD54), ATP synthase, H+ transporting, mitochondrial F1 complex and ribosomal protein S14. Identification of unique, heroin-induced proteins may help to develop novel markers for diagnostic, preventative and therapeutic targeting in heroin using subjects.

Cell proliferation requires inhibitory and permissive factors to monitor cell-cycle progression and control DNA replication. The small intestine has a high rate of proliferation and a very low incidence of cancer, suggestive of efficient mechanisms for control of the cell cycle and assuring fidelity of DNA replication. We have isolated a cDNA from a rat crypt-cell library which hybridized to a 3.0-kb mRNA specific for crypt cells, the proliferative cell compartment of the intestine. Its amino-acid sequence indicates that it is a new member of a family of replication proteins found in yeast, Cenorhabditis elegans, mouse and humans. Its transcripts were markedly increased in fetal rat intestine and liver, decreased in long-term confluent and serum-starved tissue culture cells (IEC cells, a cell line derived from rat crypt cells), increased with serum repletion as cells resumed proliferation, and appeared to be species specific. Isolation and functional characterization of small intestinal crypt-cell replication factors should help explain this organ's low incidence of cancer.

Background/Aims: Studies have identified a 67-kilodalton high-affinity laminin receptor (LR) whose expression has also been related to development, differentiation, and neoplastic transformation. The relationship of the 67-kilodalton LR to hepatic and enterocyte development and to enterocyte differentiation was investigated. Methods: LR messenger RNA (mRNA) was identified using a complementary DNA isolated from a rat crypt cell library. LR and integrin (α6, β1, and β4) expression by rat intestinal crypt cells was compared with that of the more differentiated villus cells using Northern blotting. Developmental differences in LR expression were studied in fetal and neonatal rats. The pattern of LR expression in fetal and adult rat intestines was examined further by in situ hybridization. Results: LR mRNA levels were highest in fetal liver and intestine and adult rat crypt cells. LR mRNA levels were 9–10 times greater in crypt than in villus cells. Integrin subunit expression differed little between crypt and villus cells. Nascent transcription studies showed that the proportion of newly transcribed LR mRNA per total RNA synthesized was similar for crypt and villus cells, suggesting posttranscriptional control of LR mRNA levels in villus cells. Conclusions: Increased LR mRNA expression is a feature of the fetal intestine and of the undifferentiated, mitotically active crypt cells.