Association of αvβ3 integrin expression with the metastatic potential and migratory and chemotactic ability of human osteosarcoma cells

Introduction. Expression of adhesion molecules such as α _v β ₃ integrin has been associated with the metastatic potential of tumor cells. The purpose of this study was to determine whether α _v β ₃ expression correlated with the metastatic potential of human osteosarcoma cells. Materials and methods. We developed a series of sublines (LM2–LM7) from human osteosarcoma SAOS parental cells, with progressively increasing potential to form lung metastases in nude mice after intravenous injection. SAOS parental and LM2 cells were poorly metastatic, but LM7 cells resulted in visible metastatic lung nodules by 6–8 weeks. We quantified α _v β ₃ integrin expression using flow cytometry. Results. α _v β ₃ expression correlated with the metastatic potential of the cells, with LM7 cells showing the highest expression. LM7 cell adhesion to vitronectin decreased after treatment with echistatin, a RGD-containing peptide antagonist of α _v β ₃. LM7 cells demonstrated higher chemotactic activity than SAOS cells to a homogenate made from lung tissue. This chemotactic activity was also inhibited by echistatin. These data indicated that α _v β ₃ was critical for the migration of LM7 cells to the lung homogenate. Chemotaxis to a liver homogenate was the same for LM7 and SAOS cells. Migration of LM7 cells through lung endothelial cells was higher than that through liver endothelial cells, and echistatin again inhibited this migration. Conclusions. α _v β ₃ integrin expression may play a role in the metastatic potential of osteosarcoma cells by enhancing the ability of the cells to migrate specifically to the lung. α _v β ₃ integrin may therefore be a potential new target for osteosarcoma.This revised version was published online in August 2005 with a corrected cover date.     

Long-term potentiation in hippocampus of rats is enhanced by endogenous acetylcholine in a way that is independent of N-methyl- -aspartate receptors

By using extracellular recordings of field potential, the exact pathway by which the endogenous ACh influencing the induction of long-term potentiation (LTP) in CA1 area was analysed in slices of rat hippocampus. The results showed that: (1) the application of (−) huperzine A, an AChE inhibitor extracted from Chinese herb Qian Ceng Ta (Huperzia Serrata), could enhance the induction of LTP, while this drug showed little effect on the second components of multiple population spikes that were recorded in Mg²⁺-free medium and had proven to be N-methyl- -aspartate (NMDA) receptor-mediated response; and (2) scopolamine, a muscarinic receptor antagonist, could significantly suppressed the induction of LTP, while most of the suppressive effect of scopolamine was blocked when slices were pretreated by bicuculline, a γ-aminobutyric acid (GABA_A) receptor antagonist. These results suggest that endogenous ACh potentiates the induction of LTP through the inhibition of GABAergic interneurons that modulate pyramidal neurons, but not through the activation of NMDA receptors located on pyramidal neurons.     

Rhythmic variation in γ-aminobutyric acidA-receptor subunit composition in the circadian system and median eminence of Syrian hamsters

Temporal changes in the level of expression of γ-aminobutyric acid (GABA)_A receptor subunits α2, α5, β1 and β3 were characterized by Western blot analysis in the hamster suprachiasmatic nuclei, retina and median eminence. A nocturnal maximum in the level of GABA_A receptor β1 subunit at midday and midnight (12:00 and 00.00 h) was found in the suprachiasmatic nucleus (SCN), the retina and the median eminence of Syrian hamsters. α2 and β3 subunit levels peaked during the day in the median eminence. Finally, retinal α5 levels were maximal during the night. β1 temporal changes in the SCN and median eminence, as well as α2 variations in the median eminence were maintained under constant dark conditions, suggesting an endogenous control, while the other variations were only observed under light–dark cycle conditions.     

Increased Expression of Intercellular Adhesion Molecule-1 and Mucosal Adhesion Molecule α4β7 Integrin in Small Intestinal Mucosa of Adult Patients with Food Allergy

The mechanisms of adverse reactions to foods in the gastrointestinal tract are poorly understood. Previous studies of other atopic diseases and animal models suggest that adhesion molecules and mucosal lymphocytes may be implicated in the pathogenesis of food allergy (FA). The aim of our study was to investigate the expression of adhesion molecules and mucosal lymphocytes in duodena of patients with food allergies and of controls. Ten patients with FA to cereals (wheat, oats, and rye) or cow's milk and 9 control patients were included in the study. Quantitative analysis and immunohistochemical stainings for two pairs of adhesion molecules (intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-1 (LFA-1), α₄β₇ integrin, and mucosal addressin cell adhesion molecule (MAdCAM-1) and lymphocyte markers on endoscopic duodenal biopsy specimens were performed. The villous structure and density of LFA-1-positive cells were normal in every biopsy specimen, but the patients had significantly more α₄β₇⁺ cells in the intraepithelial space (P = 0.01). The expression of ICAM-1 in the lamina propria of patients with FA was also substantially increased (P = 0.003); however, staining with MAdCAM showed no intergroup difference. Moreover, we found significantly increased CD4⁺ and HLA-DR⁺ cells in the lamina propria of patients, in comparison to the controls, P = 0.05 and P = 0.04, respectively. The densities of CD3, CD8, HLA-DP, T cell receptor αβ⁺ and γδ⁺ cells and IgA-, IgA₁-, and IgA₂-containing cells did not differ in the two groups studied. Our results suggest that the increased expression of ICAM-1 and α₄β₇ integrin may play an important role in the pathogenesis of food hypersensitivity and with the elevation of CD4- and HLA-DR-positive cells reflect a stage of inflammation in the structurally normal intestines.     

Activation of glycogen synthase kinase-3 inhibits protein phosphatase-2A and the underlying mechanisms

The activity of protein phosphatase-2A (PP-2A) is significantly suppressed in the brain of Alzheimer's disease (AD) patients, but the mechanism is not understood. Here, we found an in vivo association of glycogen synthase kinase 3β (GSK-3β) with inhibitor-2 of PP-2A (I₂^PP-2A). The activation of GSK-3 resulted in accumulation of I₂^PP-2A with concomitant suppression of PP-2A activity and increases of tau phosphorylation in HEK293, N2a and PC12 cells, while inhibition of GSK-3 caused decreases of I₂^PP-2A with increased PP-2A activity and decreased tau phosphorylation. A positive correlation between GSK-3β and I₂^PP-2A (R = 0.9158) and a negative correlation between GSK-3β and PP-2A (R = −0.9166) were detected. GSK-3 activation did not affect I₂^PP-2A mRNA level, while it increased the mRNA level of a heterogeneous ribonucleoprotein A18 (hnRNP A18). The activation of GSK-3 increased the expression and the activity of proteasome system. It suggests that activation of GSK-3 inhibits PP-2A through up-regulation of I₂^PP-2A with hnRNP A18-involved mechanism.     

MicroELISA detection of thymosin α1 released in thymic organ cultures

Using a polyclonal specific rabbit anti-thymosin α₁ a highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed to measure thymosin α₁. Production of thymosin α₁ was detected in both thymic organ cultures and in mouse serum. The method is rapid (5 h), reproducible and easy to perform.     

Anti-inflammatory effect of β2-agonists: Inhibition of TNF-α release from human mast cells

β₂-Agonists inhibit the release of preformed mediators such as histamine and newly synthesized mediators such as prostaglandin D₂ from mast cells. However, although mast cells have been identified as an important source of several cytokines including tumor necrosis factor-α (TNF-α), there is no information about their regulation by β₂-agonists. Thus given the importance of TNF-α in inflammation and the widespread use of β₂-agonists, we investigated the effect of long-acting (salmeterol) and short-acting (salbutamol) β₂-agonists on the secretion of TNF-α from human skin mast cells. Treatment of mast cells with salmeterol or salbutamol (100 nmol/L) inhibited the IgE-dependent release of TNF-α (82% and 74%, respectively). Moreover, 2-hour treatment with salmeterol, isoproterenol, or salbutamol inhibited mast cell cytotoxicity against a TNF-α–sensitive cell line, WEHI-164, with an IC₅₀ of 71, 50, and 29 nmol/L, respectively. Specificity for β-adrenergic receptors was shown with propranolol. The inhibitory effect of β₂-agonists was observed after only 20 minutes of treatment but was lost by 24 hours after removal of salbutamol and isoproterenol (7% and 11% inhibition remaining, respectively). In contrast, the inhibition of TNF-α release was increased 1 hour after removal of salmeterol and remained significant 24 hours later. Furthermore, β₂-agonists did not show tachyphylaxis for the inhibition of TNF-α release. Thus selective β₂-agonists demonstrate anti-inflammatory activity by inhibiting the release of TNF-α from mast cells stimulated through their IgE receptor or by a tumor target cell. This inhibitory effect of β-agonists may be important in their mode of action in the treatment of allergic diseases. (J Allergy Clin Immunol 1997;100:825-31.)     

Prostaglandin F2α can modulate the growth and the differentiation of bovine corneal epithelial cells cultured in vitro

The effects of PGF_2α on the growth, morphology, morphometry and keratinization pattern of bovine corneal epithelial cells cultured in vitro were studied. The cells were grown with a basal medium or, in the presence of keratocytes and/or their products, using a keratocyte-conditioning medium. Cell growth was evaluated by MTT assay. Daily treatments with exogenous PGF_2α at concentrations equal to or lower than 10⁻⁶ M induced significant increases in cell proliferation when the epithelial cells were cultured on a keratocyte feeder-layer or with the conditioning medium. No variations were observed in cultures grown with the basal medium. 10⁻⁵ M PGF_2α induced a decrease in cell growth under all culturing conditions. PGF_2α did not affect cell morphology and modified only nuclear dimensions among the cells grown under different culturing conditions. No variations of any parameters were observed between cells cultured on feeder-layer, with conditioning or basal medium and the corresponding cultures supplemented with the autacoid. Moreover, PGF_2α induced only the disappearance of 43 kDa keratin in cells grown on basal medium, while the keratin pattern of epithelial cells cultured on feeder-layer or with the conditioning medium was not modified by the autacoid. From these data we can suppose that a cooperation could exist between PGF_2α and fibroblasts and their products for the modulation of cell growth. Finally, it was observed that the autacoid had no effect on cell morphology and morphometry, except for nuclear dimensions, despite the presence of other prostaglandins, such as PGE₂.     

Immunohistochemical detection of estrogen receptor α in articular chondrocytes from cows, pigs and humans: in situ and in vitro results

Clinical observations suggest that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis, but only little is known about the influence of these hormones on articular cartilage cells. The effect of estradiol is mediated by estrogen receptors α and β. The goal of the present study was to search for estrogen receptor α in articular tissue from cows, pigs and humans by immunohistochemistry to form a basis for in vitro studies. In addition, we also tried to detect estrogen receptor α in cultivated articular chondrocytes from cows and bulls under certain culture conditions. Estrogen receptor α is detected by the use of antibody 13H2 in articular chondrocytes from cows, bulls, pigs and humans. Chondrocytes are physiologically exposed to reduced oxygen tension. In isolated articular chondrocytes from cows and bulls incubated either with 21% O₂ or with 5% O₂ positive cells were also found. These positive results therefore encourage testing the influence of estradiol on cultivated articular cartilage cells in these species under different culture conditions.     

Effect of bilateral destruction of the subretrofacial area on receptor mechanisms of neuroprotective effect of GABAergic agents

Both GBA_A (muscimol, gaboxadol, and isonipecotate) and GABA_B (baclofen) receptor agonists produce marked neuroprotective effect during total brain ischemia. The antagonists of GABA_A receptors bicuculline and picrotoxin attenuate the effect of muscimol, and the GABA_B receptor antagonists hydroxysaclofen and aminovaleriate decrease the effect of baclofen. The GABAergic substances protect the brain via GABA receptors of both types. The effect of the GABA agonists is central in nature. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 2, pp. 162–164, February, 1998.     

New insights on the role of gephyrin in regulating both phasic and tonic GABAergic inhibition in rat hippocampal neurons in culture

Gephyrin is a tubulin-binding protein that acts as a scaffold for clustering glycine and GABA_A receptors at postsynaptic sites. In this study, the role of gephyrin on GABA_A receptor function was assessed at the post-translational level, using gephyrin-specific single chain antibody fragments (scFv-gephyrin). When expressed in cultured rat hippocampal neurons as a fusion protein containing a nuclear localization signal, scFv-gephyrin were able to remove endogenous gephyrin from GABA_A receptor clusters. Immunocytochemical experiments revealed a significant reduction in the number of synaptic γ2-subunit containing GABA_A receptors and a significant decrease in the density of the GABAergic presynaptic marker vesicular GABA transporter (VGAT). These effects were associated with a slow down of the onset kinetics, a reduction in the amplitude and in the frequency of miniature inhibitory postsynaptic currents (mIPSCs). The quantitative analysis of current responses to ultrafast application of GABA suggested that changes in onset kinetics resulted from modifications in the microscopic gating of GABA_A receptors and in particular from a reduced entry into the desensitized state. In addition, hampering gephyrin function with scFv-gephyrin induced a significant reduction in GABA_A receptor-mediated tonic conductance. This effect was probably dependent on the decrease in GABAergic innervation and in GABA release from presynaptic nerve terminals. These results indicate that gephyrin is essential not only for maintaining synaptic GABA_A receptor clusters in the right position but also for regulating both phasic and tonic inhibition.     

α1-Antitrypsin Nonsense Mutation Associated with a Retained Truncated Protein and Reduced mRNA

α₁-Antitrypsin (α1AT) provides the major protection in the lung against neutrophil elastase-mediated proteolysis. Inheritance of α1AT deficiency alleles is associated with an increased risk of emphysema and liver disease. α1AT null alleles cause the total absence of serum α1AT and represent the ultimate in a continuum of alleles associated with α1AT deficiency. The molecular mechanisms responsible for absence of serum α1AT include splicing abnormalities, deletion of α1AT coding exons, and premature stop codons. We identified an Italian individual with asthma, emphysema, and a very low level of serum α1AT. DNA sequencing demonstrated theMprocidadeficiency allele and a novel null allele,QOtrastevere(c654 G → A, W194Z), a nonsense mutation near the intron 2 (IVS2) splice acceptor site. To determine the molecular basis ofQOtrastevereand specifically to evaluate whether this nonsense mutation interfered with mRNA processing by altered splicing, we used a Chinese hamster ovary cell line permanently transfected withQOtrastevereor normal M α1AT with and without IVS2. Northern blot analysis demonstrated that the normal M construct, with or without IVS2, expressed α1AT mRNA of a similar size. The nonsense mutation was associated with moderately reduced α1AT mRNA regardless of the presence or absence of IVS2. Reduction in α1AT mRNA regardless of the opportunity for splicing supports a translational-translocation model as the cause of reduced α1AT mRNA rather than the nuclear scanning model. Pulse–chase studies followed by immunoprecipitation demonstrated an endoplasmic reticulum-retained 31 kDaQOtrastevereα1AT, which was rapidly degraded. Although mRNA content was moderately reduced, retention and rapid intracellular degradation of the truncated form are the major mechanisms for the absence of secreted α1AT.     

GM1 gangliosidosis: review of clinical, molecular, and therapeutic aspects

GM₁ gangliosidosis is a lysosomal storage disorder due to deficiency of the β-galactosidase enzyme. This deficiency results in accumulation of GM₁ gangliosides and related glycoconjugates in the lysosomes leading to lysosomal swelling, cellular damage, and organ dysfunction. The disease is lethal in the infantile and juvenile forms. To date, up to 102 mutations distributed along the β-galactosidase gene (GLB1) have been reported. This review gives an overview of the clinical and molecular findings in patients with GM₁ gangliosidosis. Furthermore, it describes therapeutic approaches which are currently under investigation in animal models of the disease.     

CD1d-restricted mouse Vα14 and human Vα24 T cells: lymphocytes of innate immunity

Mouse V α 14 T cells and their human homologs, V α 24 T cells, are prominent subsets of CD1d-restricted T cells. Here we discuss their striking similarities to B-1 B cells andγδ T cells and propose that these immune cells mediate various innate strategies in response to endogenous or exogenous danger signals.     

Effect of IFN-α on CC1<Subscript>4</Subscript>-Induced Fibrosis of the Liver and Immune Status in Mice of Different Age

In young, adult, and old mice fibrosis was induced by administration of CC1₄ and treated with IFN-α. Liver fibrosis was evaluated by morphometry of argyrophilic fibers, immune status by the splenocyte proliferative response. Minimum immunosuppression and maximum antifibrotic effect were observed in young mice, while adult mice exhibited pronounced immunotoxicity and weak response to interferon therapy.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 3, pp. 303–306, March, 2005     

Subcellular localization of GABAA/benzodiazepine receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus

The monoclonal antibody bd-17, which recognizes the β₂ and β₃-subunits of GABA_A/benzodiazepine receptors, was used to determine the cellular and subcellular localization of receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus. In numerous dendrites, very strong immunostaining was present in the cytoplasm and on the postsynaptic dendritic membrane of synaptic junctions. The extrasynaptic portion of the dendritic membrane also very often showed [β₂ + β₃]-like immunoreactivity. However, due to methodological limitations, it could not be stated with certainty whether presynaptic β₂- and β₃-subunits of GABA_A/benzodiazepine receptors actually occur in this mesencephalic visual structure. In conclusion, these results strongly suggest that synaptic and non-synaptic GABA_A/benzodiazepine receptors are present in the superficial gray layer of the rat superior colliculus. These receptors may modulate neuronal cell activity in different ways, depending on their location.     

Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development

Gamma‐aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABA_A and metabotropic GABA_B receptors. During the development of the nervous system, GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation, and activity‐dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABA_A and GABA_B receptors during early development of Xenopus laevis. Using in situ hybridization and qRT‐PCR, GABA_A α2 was detected as a maternal mRNA. All other α‐subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern, suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. Developmental Dynamics 240:862–873, 2011. © 2011 Wiley‐Liss, Inc.     

Ethanol modulates mammalian circadian clock phase resetting through extrasynaptic gaba receptor activation

Ethanol modulates the actions of multiple neurotransmitter systems, including GABA. However, its enhancing effects on GABA signaling typically are seen only at high concentrations. In contrast, although GABA is a prominent neurotransmitter in the circadian clock of the suprachiasmatic nucleus (SCN), we see ethanol modulation of clock phase resetting at low concentrations (<50 mM). A possible explanation is that ethanol enhances GABAergic signaling in the SCN through activating GABA_A receptors that contain the δ subunit (GABA_Aδ receptors), which are sensitive to low ethanol concentrations. Therefore, we investigated whether ethanol acts on GABA_Aδ receptors in the SCN. Here we show that acute application of the GABA_Aδ receptor antagonist, RO15-4513, to mouse hypothalamic slices containing the SCN prevents ethanol inhibition of nighttime glutamate-induced (photic-like) phase delays of the circadian clock. Diazepam, which enhances activity of GABA_A receptors containing the γ subunit (GABA_Aγ receptors), does not modulate these phase shifts. Moreover, we find that RO15-4513 prevents ethanol enhancement of daytime serotonergic (non-photic) phase advances of the circadian clock. Furthermore, diazepam phase-advances the SCN circadian clock when applied alone in the daytime, while ethanol has no effect by itself at that time. These data support the hypothesis that ethanol acts on GABA_Aδ receptors in the SCN to modulate photic and non-photic circadian clock phase resetting. They also reveal distinct modulatory roles of different GABA_A receptor subtypes in circadian clock phase regulation.     

PCR-Based Methods for Identifying Genetic Variations in Human α1B- and β2-Adrenergic Receptors

α- and β-adrenergic receptors belong to the superfamily of G-protein-coupled, seven transmembrane domain receptors and regulate a variety of cellular processes. Previous studies have demonstrated that changes in the amino acid sequence can result in substantial changes in the function of the receptors and it has been suggested that there may be an association between different disease states and the altered structure of α- and β-adrenergic receptors. Accordingly, we have developed a simple PCR method for the identification of polymorphisms in the coding sequences of the human β₂-adrenergic receptor and the α_1B-adrenergic receptor. This method may be useful for screening individual patients or at-risk populations for endocrine-metabolic disorders, as well as for asthma, cardiovascular disorders, and neuropsychiatric diseases.     

Higher Risk of Mismatch Repair-Deficient Colorectal Cancer in α1-Antitrypsin Deficiency Carriers and Cigarette Smokers

Microsatellite instability (MSI) is a genomic alteration observed in 15–30% of colorectal cancer (CRC). Two MSI phenotypes have been defined for CRC: MSI-H is characterized by MSI at ≥30% of the examined loci and MSI-L by MSI at 1–30% of the loci. An absence of MSI at any examined loci has been defined as a microsatellite stable (MSS) phenotype. Current data suggest the majority of MSI tumors are the result of defective DNA mismatch repair (MMR). In this study, we have determined the α₁-antitrypsin deficiency carrier (α₁ATD-ht) status of 161 CRC patients whose MSI phenotype and protein expression states had previously been determined. Cases were selected to enrich a larger number of MSI-H cases. Among 51 CRC patients with MSI-H tumors, the α₁ATD-ht rate was 21.6%; among 110 patients with MSI-L/MSS tumors, the rate was 9.1% (MSI-H vs MSI-L/MSS, P = 0.02); and among the 191 population-based controls the α₁ATD-ht rate was 9.4% (MSI-H vs controls, P = 0.02). The estimated relative risk of having MSI-H CRC among α₁ATD-ht was 3.1 after adjusting for age, gender, and smoking history. The risk of having MSI-H CRC among current and past smokers was 6.6 and 2.7, respectively. Patients who were α₁ATD-ht and smoked had a 20-fold increased risk of developing an MSI-H CRC compared to nonsmokers who were homozygous normal at the α₁ATD locus. Our findings suggest an etiologic link between α₁ATD alleles and development of CRC with defective MMR, and a synergistic effect between smoking and α₁ATD allele in the development of MSI-H CRC.