Search

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.

We synthesized and characterized curcumin-stabilized silver nanoparticles (Cur-AgNP) and found them to be 45 nm by dynamic light scattering with a maximum absorbance at 406 nm. We evaluated Cur-AgNP for immunomodulatory activities and their potential as an antiretroviral agent. The antiretroviral effects of Cur-AgNP were determined in ACH-2 cells latently infected with human immunodeficiency virus (HIV)-1. ACH-2 cells, 200,000/ml, were treated with Cur-AgNP for 24-48 h. Expression of HIV-1 LTR and p24, the pro-inflammatory cytokines, IL-1β, TNF-α, and NF-κB was quantitated. Treatment of ACH-2 cells latently infected with HIV-1 with Cur-AgNP produced no toxic effects but significantly inhibited the expression of HIV-1 LTR (-73%, P < 0.01) and p24 (-57%, P < 0.05), IL-1βα (-61%, P < 0.01), TNF-αα (-54%, P < 0.05), IL-6 (-68%, P < 0.01), and NF-κB (-79%, P < 0.0001) as compared to untreated controls. Thus, Cur-AgNP have therapeutic potential as direct antiretroviral agents, as well as having immunomodulatory activities inhibiting the expression of pro-inflammatory mediators induced by infection with HIV-1. Experimental controls, such as curcumin alone, and conventional silver nanoparticles capped with citric acid, produced no similar biological effects. We conclude that treatment of HIV-1 infected cells with Cur-AgNP significantly reduced replication of HIV by inhibition of NF-κB nuclear translocation and the downstream expression of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6. Subsequent in vivo studies with Cur-AgNP using a humanized mouse model of HIV infection are underway.

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.

Inhibition of Matrix metalloproteinase-9 (MMP-9) activity using delivery of short interfering RNA (siRNA) molecules to brain microvascular endothelial cells (BMVECs) that constitute the BBB may have a significant impact on reducing the BBB permeability. Gold nano rods (GNRs) can electrostatically bind with MMP-9 siRNA to form a nanoplex and the uptake of this nanoplex by BMVEC cells can result in suppression of MMP-9 expression. The current study explores if this GNR-MMP-9 siRNA nanoplex gene silencing modulates the expression of tight junction (TJ) proteins in the BMVEC. The endothelial TJ's of the BBB play a critical role in controlling cellular traffic into the central nervous system. We hypothesize that silencing of the MMP-9 gene expression in BMVEC will increase the expression of TJ proteins thereby decrease endothelial permeability. Our results showed a significant increase in the gene and protein expression of TJ proteins: ZO-1, Occludin and Claudin-5 in BMVEC cells that were transfected with the GNRs-siRNA-MMP-9 nanoplex suggesting that BBB disruption, which results from loss of TJ function due to MMP-9 activation during neuroinflammation can be prevented by silencing MMP-9 expression.

Matrix metallaprotinase-9 (MMP-9) is zinc-containing proteinase whose expression and trafficking are frequently altered in cancer. MMP-9 in the plasma membrane and the secreted forms are thought to contribute to the invasive and metastatic properties of malignant tumors. We have manipulated the expression of MMP-9 in prostate tumor cell line LNCaP and measured their capacity to invade through a basement membrane matrix. Stable expression of human MMP-9 in a poorly metastatic LNCaP prostate cancer cell line produced a 2-3-fold increase in MMP-9 activity and a comparable increase in invasiveness. Transient transfection of LNCaP stable clone expressing MMP-9 with MMP-9 antisense oligonucleotide (ASODN) produced 55-90% less MMP-9 than control cells and were proportionately less invasive. In contrast, manipulating MMP-9 levels had no effect on cell migration across an uncoated membrane. A standard MMP-9 inhibitor at a concentration ranging from 1-10 nM, caused a nearly quantitative inhibition of extracellular MMP-9 activity and had significant effect on basement membrane invasion. Collectively, these results confirm the role of MMP-9 in tissue remodeling associated with prostate tumor invasion.

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.

Objective The continuous discovery of biomarkers and their evolving use for the diagnosis and guidance of therapy for patients with cancer has increased awareness of the need to triage biospecimens properly. On occasion, cytology samples are the only type of biospecimen available for analysis. Often, the current approach for these latter specimens is cytopathology-centric, with cells limited to examination by bright field microscopy. When specimens are paucicellular, there is often insufficient material for ancillary testing. Therefore, a need exists to develop an alternative approach that allows for the multiplexed analysis of cells when they are limited in number. In recent previous publications, we demonstrated that clinically derived cells from tissue are suitable for evaluation in a microfluidic device. In our current endeavour, we seek to expand upon those findings and determine if those same cells can be recovered for further analysis. Methods A microfluidic channel was designed, fabricated and tested using cytology specimens generated from tissue specimens. The cytological features of the cells tested were examined prior to entering the channel; they were then compared to similar cells while in the channel, and upon recovery from the channel. Recovery of DNA and proteins were also tested. Results The morphology of the tested cells was not compromised in either the channel or upon recovery. More importantly, the integrity of the cells remained intact, with the recovery of proteins and high molecular weight DNA possible. Conclusions We developed and tested an alternative approach to the processing of cytopathology specimens that enables multiplexed evaluation. Using microfluidics, cytological examination of biopecimens can be performed, but in contrast to existing approaches, the same cells examined can be recovered for downstream analysis.

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.

Opiates act on the dopaminergic system of the brain and perturb 32 kDa dopamine and adenosine 3′, 5′-monophosphate-regulated phosphoprotein (DARPP-32) function. The DARPP-32 mediated inhibition of protein phosphatase-1 (PP-1) and modulation of transcriptional factor CREB is critical to the changes in neuronal plasticity that result in behavioral responses during drug abuse. To investigate the role of DARPP-32 mediated signaling on withdrawal behavior in a rat model of opiate addiction, we used intracerebral administration of gold nanorods (GNR) complexed to DARPP-32 siRNA to silence DARPP-32 gene expression and measure its effects on the opiate withdrawal syndrome. We hypothesized that DARPP-32 siRNA will suppress the neurochemical changes underlying the withdrawal syndrome and therefore prevent conditioned place aversion by suppressing or removing the constellation of negative effects associated with withdrawal, during the conditioning procedure. Our results showed that opiate addicted animals treated with GNR-DARPP-32 siRNA nanoplex showed lack of condition place aversive behavior consequent to the downregulation of secondary effectors such as PP-1 and CREB which modify transcriptional gene regulation and consequently neuronal plasticity. Thus, nanotechnology based delivery systems could allow sustained knockdown of DARPP-32 gene expression which could be developed into a therapeutic intervention for treating drug addiction by altering reward and motivational systems and interfere with conditioned responses.

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 effects of vitamin D on steady-state levels of rat intestinal Ca-pump mRNA were examined in RNA extracted from isolated cell fractions of the crypt-to-villus gradient of differentiation. Northern blots revealed three different size mRNAs. Vitamin D deficient animals showed a decrease in these Ca-pump mRNAs, which increased markedly after 1,25-(OH)2D3 repletion, particularly for the villus cell. The data suggest that one of the effects of 1,25-(OH)2D3 may be to modulate enterocyte Ca-pump mRNA and that this effect is partly dependent on the stage of cell differentiation.

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.

Human immunodeficiency virus (HIV)-1 patients who abuse opiates are at a greater risk of developing neurological complications of AIDS. Alterations in blood-brain barrier (BBB) integrity are associated with cytoskeletal disorganization and disruption of tight junction (TJ) integrity. We hypothesize that opiates in combination with HIV-1 viral proteins can modulate TJ expression in primary brain microvascular endothelial cells (BMVEC), thereby compromising BBB integrity and exacerbating HIV-1 neuropathogenesis. We investigated the effect of morphine and/or tat on the expression of TJ proteins ZO-1, JAM-2, Occludin and P-glycoprotein and the functional effects of TJ modulation in BMVEC. Morphine and/or tat, via the activation of pro-inflammatory cytokines, intracellular Ca(2+) release, and activation of myosin light chain kinase, modulated TJ expression resulting in decreased transendothelial electric resistance and enhanced transendothelial migration across the BBB. These studies may lead to the development of novel anti-HIV-1 therapeutics that target specific TJ proteins, thus preventing TJ disruption in opiate using HIV-1 patients.

In previous studies, experimental splenosis was produced in normal rodents and in animals following splenectomy. Splenosis was more in splenectomised than in normal animals. In the present study, in animals into whom splenic fragments were implanted subcutaneously, there were less peritoneal splenic growth areas than in controls. The hypothesis is presented that there is a population size feedback control regulatory mechanism operating in splenic growth and regeneration. If such regulators are present in the circulation, administration of normal plasma should inhibit splenosis and this may serve as a model to isolate such regulators. This was not the case in these experiments.

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.

Chemokines and their receptors have been implicated in the pathogenesis of neuroAIDS. Herein we describe the effects of morphine on the gene expression of beta chemokines and their receptors by primary normal human astrocytes (NHA). Our results show that NHA treated with morphine showed significant downregulation of the gene expression of beta chemokines, MCP-1, and MIP-1 beta, while reciprocally upregulating the expression of their specific receptors, CCR2b, CCR3, and CCR5 as detected by real-time quantitative PCR. These morphine-induced effects on NHA cells were reversed by the opioid mu receptor antagonist, naloxone. Further, our results indicate that morphine-induced effects are mediated via the modulation of MAPK and CREB signaling pathways. These results support our hypothesis that opiates act as co-factors in the neuropathogenesis of HIV infection.

Many viral and neoplastic diseases are resistant to interferon-alpha/beta (IFN-alpha/beta) 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-alpha/beta.Ninety-one cancer patients and 25 healthy individuals were studied. Free circulating IFN receptor-alpha/beta 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.All patients were found to have increased free IFN receptor-alpha/beta type 1 in the circulation, with the highest levels reported in patients with adenocarcinoma.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.

Tumor cell arrest and tumor migration are two of the critical steps in the metastatic cascade. We hypothesized that these steps may be facilitated by the low density lipoprotein (LDL)-induced activation of microvessel endothelial cells (MVEC). The purpose of our study was to investigate the biological effects of an LDL-enriched milieu and the effects of the anticholesterol drug Lovastatin on metastatic behavior. The SW480 and SW620 are primary and metastatic human colonic adenocarcinoma cell lines derived from the same patient. We investigated the effect of LDL on adhesion and migration of the two tumor cell lines across human brain, lung, liver and dermal endothelial monolayers. Adhesion and migration assays were done before and after pretreat-ment of the MVEC or tumor cells with LDL (100 mg/ml) for 24 h. Although metastatic SW620 cells were more adherent to MVEC compared with primary SW480 cells, LDL pretreatment of SW480 and SW620 cells did not affect tumor cell adhesion to MVEC. In contrast, tumor cell migration was significantly increased across endothelial monolayers when MVEC were pretreated with LDL. Transendothelial cell migration was not sig-nificantly affected by pretreatment of the tumor cells with LDL. Lovastatin is an inhibitor of HMG-CoA reduc-tase, the rate-limiting enzyme in cholesterol biosynthesis. It has been shown to have anti-tumor activity in vitro. We investigated the effect of Lovastatin on tumor cell kinetics and tumor cell migration across MVEC. Growth curves and migration assays were done before and after pretreatment of the tumor cells with Lovastatin (30 mg/ml). Migration assays were also done after treatment of unstimulated or LDL-stimulated MVEC (100 mg/ml) for 24 h with Lovastatin. Lovastatin inhibited the in vitro growth of the metastatic SW620 cell line to a greater extent than the invasive SW480E cell line. On the other hand, pretreatment of tumor cells with Lovastatin (30 mg/ml) did not suppress transendothelial tumor cell migration of tumor cells. Finally, Lovastatin given to mice effectively suppressed the number of MCA-26 tumor colonies in the liver of Balb/c mice com-pared with untreated mice. ©Lippincott Williams & Wilkins

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.

Undifferentiated embryonic and dedifferentiated tumor cells express genes that are down-regulated or not expressed in differentiated tissue. The progenitor cells of the intestinal crypt are undifferentiated cells that, similarly, should express genes that are not evident in the more differentiated villus cells. Some of these genes may be related to the control of differentiation. We attempted to define crypt-associated genes by constructing a cDNA library from isolated rat intestinal crypt cells and screening for messages that remained after subtractive hybridization using > 20-fold more mRNA from villus than from the crypt cells. This process identified about two percent of the colonies containing transcripts expressed by the crypt cell. Northern blot analysis showed hybridization to messages in a range from 700 to 12,000 base pairs. Six clones out of 136 initial isolates were shown to hybridize to crypt mRNAs at levels four to tenfold greater than to villus mRNAs. Three of these clones showed greater hybridization to mRNA of the distal (ileum) when compared to the proximal end of the adult small bowel. Increased expression in fetal rat intestine was seen for five mRNAs and in fetal liver for four mRNAs when compared to adult. Most of the crypt associated gene probes preferentially bound mRNA from ovary, kidney, and spleen but did not bind mRNA derived from testis, muscle and brain. Cultured mouse teratocarcinoma cells (F9) showed high levels of three of these transcripts. Portions of each insert were sequenced and examined for homology to entries in national computer banks. The genes, with one exception, did not match any reported nucleotide or peptide sequence. These probes may detect mRNAs that are critical to normal differentiation.

The expression of laminin and type IV collagen messenger RNA in rat liver was investigated in relation to liver development. Levels of messenger RNA for laminin chains A, B1 and B2, and alpha 1 and alpha 2 chains of type IV collagen were studied by Northern-blot and dot-blot analysis. Although the expression of messenger RNAs for laminin B1 and B2 chains was higher in 19-day gestational fetal liver when compared with the adult, the highest level was detected in 15-day newborns. This finding coincides with the final establishment of the mature liver. Laminin A messenger RNA was not detected in the 19-day gestational fetus or in the neonatal period. In contrast, alpha 1-(IV) and alpha 2-(IV) collagen messenger RNAs maintained high levels in the early neonate and then decreased gradually after 15 days. Our findings suggest that during late stages of liver ontogeny the main components of liver basement membrane proteins undergo a significant alteration in their synthesis. The data further suggest that laminin may play a role in the final stages of hepatic differentiation.

The structural chromatin protein A24 (uH2A) is a conjugate of histone H2A and a non-histone protein, ubiquitin. Eukaryotic cells contain an enzyme, generically termed isopeptidase, which can cleave A24 stoichiometrically into H2A and ubiquitin in vitro. Isopeptidase, free of proteinase activity, has been partially purified from calf thymus by ion-exchange chromatography, gel filtration and affinity chromatography, and analyzed for its substate specificity. There are three major types of isopeptide bonds besides the epsilon-(alpha-glycyl)lysine bond between H2A and ubiquitin; namely, the disulfide bridge, the aldol and aldimide bonds and the epsilon-(gamma-glutamyl)lysine crosslink. Under conditions where A24 was completely cleaved into H2A and ubiquitin, none of these naturally occurring isopeptide bonds was cleaved by isopeptidase. Furthermore, the bonds formed in vitro by transglutaminase reaction between casein and putrescine, through the gamma-NH2 of glutamine residue and the NH2 of putrescine, were not cleaved by the enzyme. The enzyme also failed to cleave the glycyl-lysyl and other orthodox peptide linkages within proteins. Among various proteins examined, the substrates for isopeptidase reaction were confined to conjugates between ubiquitin and other proteins, formed through epsilon-(alpha-glycyl)lysine bonds. Since ubiquitin released by isopeptidase is re-usable for an ATP-dependent conjugation with other proteins, its carboxyl terminal -Gly-Gly-COOH most likely is preserved intact, and is not blocked. These results suggest that isopeptidase specifically recognizes and cleaves the epsilon-(alpha-glycyl)lysine bond. A possible biological significance of this enzyme is discussed.

Starting from phenyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-beta-D-glucopyranoside (1), chemical syntheses were developed for phenyl 2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-beta-D-glucopyranoside (4) and phenyl 2-acetamido-2-deoxy-4-O-alpha-L-fucopyranosyl-3-O-beta-D-galactopyranosyl -beta-D-glucopyranoside (8). Thin-layer chromatography in the solvent system 6:4:1:5 (v/v) 2-propanol-ethyl acetate-ammonium hydroxide-water clearly separated the synthetic trisaccharide 8 (RF 0.69) from synthetic disaccharide 4 (RF 0.78), fucose (RF 0.56), and GDP-fucose (which remained at the origin). Based upon this observation, a modified method for the determination of GDP-L-fucose: N-acetylglucosaminide-(1 leads to 4)-alpha-L-fucosyltransferase was developed that employed the synthetic disaccharide 4 as an acceptor, and compound 8 as an authentic reference-compound. This modified assay-procedure can simultaneously monitor possible competing reactions which may interfere with determination of alpha-(1 leads to 4)-L-fucosyltransferase activity; these include phosphorylase and alpha-L-fucosidase activities, and incorporation of alpha-L-[14C]-fucose into endogenous acceptors of enzyme preparations. Thus, the modified assay-procedure should facilitate determination of alpha-(1 leads to 4)-L-fucosyltransferase.

Isopeptidase is a novel eukaryotic enzyme that cleaves a structural chromatin protein, A24, stoichiometrically into H2A and ubiquitin. To understand the rapid turnover of ubiquitin in mitosis as well as the high specific activity of the enzyme associated with metaphase chromosomes, attempts were made to determine chromatin constituents that show high affinity for this enzyme. Endogenous protease-free isopeptidase was prepared from calf thymus and applied to a Sepharose 4B affinity column on which histones, DNA, NHCP and ubiquitin were respectively immobilized. The enzyme proved to bind only histones. To further determine which of the histone fractions is involved, affinity columns with each histone fraction were also used. The enzyme showed affinity for all histone fractions. However, the strength of affinity varied in the order H2A greater than H3 greater than H2B greater than or equal to H4 much greater than H1, being inversely correlated with the ratio of basic/acidic amino acids in these molecules. These results suggest that the turnover of A24 in mitosis is controlled, at least in part, by the affinity of enzyme for histones, and also that such affinity is caused by a mechanism which cannot be explained simply by the electrostatic interaction between negatively charged enzyme molecules and positively charged histones.

beta-D-Galactosidase from Aspergillus niger was purified by conventional techniques, including the repeated use of chromatography on hydroxylapatite. The final preparation represented a 112-fold purification, with a 22% yield. The specific activity of the purified enzyme was 72 mumol of D-galactose released/min/mg of protein, using p-nitrophenyl beta-D-galactopyranoside as the substrate. The substrate specificity of the enzyme was studied by using saccharides having structural linkages similar to those found in naturally occurring glycoconjugates. At substrate concentrations of 5mM, the beta-D-galactosidase efficiently hydrolyzed beta-Gal-1 leads to OC6H4NO2-p, beta-Gal-(1 leads to 3)-Gal, beta-Gal-(1 leads to 3)-beta-Gal-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 3)-alpha-Gal-1 leads to OC6H4NO2-p, at rates of 63, 53, 65, and 29 mumol/min/mg of protein, respectively. Slower hydrolysis was observed for beta-Gal-(1 leads to 4)-beta-Glc, beta-Gal-(1 leads to 4)-beta-GlcNAc-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 6)-beta-GlcNAc-1 leads to OC6H4NO2-p, with rates of 10, 13 and 9 mumol/min/mg of protein, respectively. Poorly hydrolyzed, at rates 1/300th of that of beta-Gal-1 leads to OC6H4NO2-p, were synthetic substrates having D-galactose attached beta-(1 leads to 3)- to either GalNAc or GlcNAc. The Km value for beta-D-galactosidase with beta-Gal-(1 leads to 4)-beta-GlNAc-1 leads to OC6H4NO2-p was approximately 20 times that with beta-Gal-1 leads to OC6H4NO2-p. The beta-D-galactosidase of A. niger has a molecular weight of 300,000, as demonstrated by gel-filtration chromatography. Sodium dodecyl sulfate-poly(acrylamide)-gel electrophoresis indicated a single subunit having a molecular weight of 130,000.

Acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. Enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. Encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. Acanthamoeba polyphenol oxidase has a pH optimum of 6.0 and a Km value of 0.21 mM with dihydroxyphenylalanine. The enzyme does not oxidize tyrosine, and it is inhibited by chloride but not by inhibitors of peroxidase. Its synthesis coincides with encystation, and known inhibitors of polyphenol oxidase prevent encystation. Polyphenol oxidase may have a role in making the cyst resistant to mechanical and chemical breakdown.