Genomic and proteomic analysis of the effects of cannabinoids on normal human astrocytes.

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.

Substrate recognition of isopeptidase: specific cleavage of the epsilon-(alpha-glycyl)lysine linkage in ubiquitin-protein conjugates.

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.

Intestinal cell apoptosis and Bcl-2 expression.

Programmed cell death (apoptosis) is a normal characteristic of cells with a limited life span like the enterocyte and the usual mode of death for proliferative crypt cells subjected to radiation or chemotherapy. The Bcl-2 proto-oncogene is considered a major regulator of apoptosis. We investigated the relationship of enterocyte apoptosis and Bcl-2 expression in rat intestine and tissue culture cells. Fragmentation of DNA and levels of Bcl-2 transcripts were evaluated in rat enterocyte fractions of the crypt-to-villus axis of differentiation and in IEC tissue culture cells. A low percentage of isolated nuclei from each enterocyte fraction showed features of DNA fragmentation, including crypt cells. Detectable DNA fragmentation was seen in IEC cells only when cells were subjected to long-term confluent culture conditions. Bcl-2 mRNA was not detected in isolated rat intestinal cells but was detected in IEC cells where its level increased with serum deprivation and long-term culture. We conclude that increased Bcl-2 expression may be important in rescue of proliferative enterocytes subjected to stress.

Galectin-1 suppresses methamphetamine induced neuroinflammation in human brain microvascular endothelial cells: Neuroprotective role in maintaining blood brain barrier integrity.

Methamphetamine (Meth) abuse can lead to the breakdown of the blood-brain barrier (BBB) integrity leading to compromised CNS function. The role of Galectins in the angiogenesis process in tumor-associated endothelial cells (EC) is well established; however no data are available on the expression of Galectins in normal human brain microvascular endothelial cells and their potential role in maintaining BBB integrity. We evaluated the basal gene/protein expression levels of Galectin-1, -3 and -9 in normal primary human brain microvascular endothelial cells (BMVEC) that constitute the BBB and examined whether Meth altered Galectin expression in these cells, and if Galectin-1 treatment impacted the integrity of an in-vitro BBB. Our results showed that BMVEC expressed significantly higher levels of Galectin-1 as compared to Galectin-3 and -9. Meth treatment increased Galectin-1 expression in BMVEC. Meth induced decrease in TJ proteins ZO-1, Claudin-3 and adhesion molecule ICAM-1 was reversed by Galectin-1. Our data suggests that Galectin-1 is involved in BBB remodeling and can increase levels of TJ proteins ZO-1 and Claudin-3 and adhesion molecule ICAM-1 which helps maintain BBB tightness thus playing a neuroprotective role. Galectin-1 is thus an important regulator of immune balance from neurodegeneration to neuroprotection, which makes it an important therapeutic agent/target in the treatment of drug addiction and other neurological conditions.

Nanotherapeutic approach for opiate addiction using DARPP-32 gene silencing in an animal model of opiate addiction.

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.

Rat intestinal crypt-cell replication factor with homology to early S-phase proteins required for cell division.

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.

Substrate specificity and other properties of the beta-D-galactosidase from Aspergillus niger.

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.

Modified assay-procedure for guanosine diphosphate-L-fucose: 2-acetamido-2-deoxy-beta-D-glucopyranoside-(1 leads to 4)-alpha-L-fucosyltransferase with the aid of synthetic phenyl 2-acetamido-2-deoxy-4-O-alpha-L-fucopyranosyl-3-O-beta-D-galactopy-ranosyl -beta-D-glucopyranoside as a reference compound.

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.

Histone kinase activities in normal and transformed mouse cells.

Crude extracts from replicating normal and transformed cells were assayed for protein kinase activities specific for different sites in purified Hl histone in vitro. Extracts from normal cells favored the NH2-terminal region while extracts from transformed cells favored the COOH-terminal region. Analysis of phosphopeptides demonstrated that histone kinases from both normal and transformed cells catalyzed the phosphorylation of a number of sites in common, and these were typical of sites phosphorylated in replicating cells. The preference for the NH2-terminal region by extracts from normal cells was due to the extensive phosphorylation of a site previously shown to be phosphorylated by cyclic AMP-dependent protein kinase. This activity was very low in transformed cells.

Polyphenol oxidase produced during encystation of Acanthamoeba castellanii.

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.

The identification of genes specifically expressed in epithelial cells of the rat intestinal crypts.

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 greater than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of vitamin D on rat intestinal plasma membrane CA-pump mRNA.

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.

Rat intestinal basement membrane synthesis. Epithelial versus nonepithelial contributions.

Mesenchymal-epithelial interactions play an important role during tissue differentiation and morphogenesis. The basement membrane, which separates these compartments, appears to be critical to these interactions by providing a substratum for cell adhesion, promoting cell polarity and the differentiated phenotype. Unlike other epithelia, gut enterocytes adhere to, and migrate along a thin basement membrane as they differentiate along the crypt-villus axis with a turnover rate of 48 to 72 hours (rat). The relative importance of the enterocytes or of the mesenchymal cells of the lamina propria to the maintenance of the basement membrane is unknown. As indirect indicators of basement membrane biosynthesis, we have measured, by filter hybridization with labeled cDNA probes, the relative abundance of mRNAs for laminin and collagen IV chains in enterocyte fractions representing the crypt-villus gradient of differentiation and in cells of the underlying lamina propria. In confirmation of a gradient, mRNA for histone H2B was present as a decreasing gradient from crypt to villus, the crypt fraction containing the mitotically active enterocytes being most enriched for this transcript and, in contrast, the mRNA for beta-actin was present as an increasing gradient from crypt to villus, paralleling the abundance of microvillus core structures. The mRNAs for alpha 1(IV) and alpha 2(IV) collagen and laminin B1 and B2 chains were most abundant in the lamina propria. Little, if any, collagen IV mRNA was detectable in the enterocyte fractions. In contrast, laminin B1 and B2 mRNAs were enriched in crypt enterocytes but the steady-state level of these transcripts decreased in the superficial villus enterocyte fractions. These data suggest that the components of the intestinal basement membrane are synthesized by both mesenchymal and entodermal-derived cells. Alterations in the intestinal basement membrane structure and in cell adhesion during enterocyte differentiation may be partly mediated by changes in laminin synthesis by the enterocyte.

Changes in ribosomal protein and ribosomal RNA synthesis during rat intestinal differentiation.

Subtraction hybridization studies, used to identify genes involved in the control of enterocyte proliferation and/or differentiation, allowed detection of a clone shown to have homologies with rat, chicken, and human acidic ribosomal phosphoprotein P1. Since increases in P1 transcript have been associated with intestinal malignancy, we explored the relationship of P1 and other ribosomal proteins to normal intestinal proliferation and differentiation. Male rats were used to prepare enterocytes as isolated cell fractions representative of the crypt to villus axis of differentiation. Total RNA was extracted from pooled cell fractions and evaluated for mRNA and rRNA steady-state levels. Nuclei were prepared from isolated enterocytes, and nuclear runoff studies were performed to estimate rates of nascent transcription. The P1 complementary DNA from the crypt cell library detected a mRNA of 650 base pairs which showed approximately 8-fold greater steady-state levels in crypt than in villus cells. Similar crypt specificity was also noted for mRNAs coding for elongation factor EF-12 and for ribosomal proteins P0, P1, and S6 (using clones from Y-L. Chan and I. G. Wool). In contrast, 28S rRNA steady-state levels did not differ between villus and crypt, indicating that ribosomal content had remained constant. In situ hybridization studies confirmed the predominant crypt localization of P1 mRNA. Nascent transcription rate studies showed that the proportion of newly synthesized P1 mRNA to total RNA was the same for the villus and crypt, suggesting that the lower content of villus P1 mRNA may be due to increased degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity of chromatin constituents for isopeptidase.

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.

Laminin receptor expression in rat intestine and liver during development and differentiation.

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 (alpha 6, beta 1, and beta 4) 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.

Heat shock protein 70 expression in native and heterotopically transplanted rat hearts.

Heat shock proteins (hsp) are intracellular proteins that are rapidly synthesized in response to a variety of stress factors. Recent studies in rats have shown that these proteins can elicit a lymphocyte response during cardiac allograft rejection. We studied the expression of the inducible (i) and constitutive (c) forms of hsp70 in rat cardiac allograft and isograft recipients to evaluate their utility as indicators of transplant rejection. Heterotopic transplantation of rat hearts was performed, using Lewis to Lewis isografts and ACI to Lewis allografts. Sham-operated rats were used as controls. Transplanted isograft, allograft, and native hearts of the transplant recipients and their livers and spleens were harvested at 5 days posttransplant and analyzed for hsp70 (i) and (c) expression by Western blots. Seven animals were studied in each group. Isografts at 3 and 60 days and allografts at 8 days were also studied. Quantification of band densities was carried out by laser densitometry. Physiological function of the native hearts of the transplant recipients was studied using Langendorff preparations. High levels of hsp70 (i) were noted in the transplanted and native hearts of the transplant recipients but not in their livers or spleens or in the hearts of the sham-operated control animals. Myocardial function of the native hearts of the transplant recipients was not significantly different from that of the controls. Significantly higher levels of hsp70 (c) were present in mild and severely rejecting allografts compared with controls and nonrejecting isografts. In the rat model of heterotopic cardiac transplantation, high levels of hsp70 (i) in the native hearts of the allograft and isograft recipients suggest a transplant-related, cardiac-specific stress process, not previously described. Heat shock protein 70 (c) expression is significantly increased during early and late allograft rejection and may serve as an indicator of transplant rejection.