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.

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.

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.

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.

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)

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)

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.

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.