Chromatography studies on bio-affinity of nine ligands of α1-adrenoceptor to α1D subtypes overexpressed in cell membrane

To improve selectivity and specificity of cell membrane chromatography (CMC), the chromatography affinities of nine ligands of α₁-adrenergic receptor(AR)to α_1D-AR subtype were investigated. The human embryonic kidney (HEK) 293 cells expressed by cDNA of α_1D-AR subtypes were cultured and cell membrane stationary phase (CMSP) was prepared. Then the interactions between ligands and α_1D-AR in CMSP were investigated using CMC. The affinity rank order to α_1D-AR subtype obtained from CMC for the nine α₁-adrenoceceptor ligands is: prazosin, BMY7378, phentolamine, oxymetazoline, 5-methylurapidil, norepinephrine, phenyle-phrine, methoxamine, RS-17053. The affinity rank order is similar and correlates well with that obtained from others’ radioligand binding assays (RBA). CMSP prepared by transfected HEK293 cells with α_1D-adrenoceptor cDNA and CMC method could be used to evaluate affinities of drug-receptor and drug-receptor subtypes and to screen drugs selective to α_1D-AR.     

Parameters of blood plasma proteolysis and phenotypes of α1-proteinase inhibitor in children with duodenal ulcer

The aim of this study was to determine correlation between parameters of proteolysis and phenotypes of α₁-proteinase inhibitor in blood plasma of children with duodenal ulcer. Activation of kininogenesis, pepsin-and trypsin like proteinases was accompanied by the decrease in activity of α₂-macroglobulin and the increase in activity of acid-stable inhibitors. Three subtypes of normal phenotype of α₁-proteinase inhibitor (M₁M₃, M₁M₁, M₂M₂) were determined. Activation of proteolysis was more pronounced in the individuals with subtype M₂M₂. In M₂M₂ activity of α₁-proteinase inhibitor was two times lower than in control, in M₁M₁ it insignificantly differed from control group and in the M₁M₃ subtype it was 1.9 times higher than in control. Low activity of α₁-proteinase inhibitor seen at the M₂M₂ subtype was accompanied by higher activity of acid-stable inhibitors; this may be regarded as the protective compensatory reaction of the body. Determination of the α₁-proteinase inhibitor phenotypes may be used for evaluation of a state of proteolysis and as a basis for employment of polyvalent proteinase inhibitors for therapy of ulcer.     

Localization ofα 2-macroglobulin in human primary sarcomas and synthesis in established cell linesin human primary sarcomas and synthesis in established cell lines

Summary The presence ofα ₂-macroglobulin was detected with the avidin-biotin technique in more than 20-yr-old paraffin blocks from human sarcomas.α ₂M was found mainly in the cytoplasm of the tumor cells, and almost all tumor cells were positive. This serum glycoprotein, which is a major plasma proteinase inhibitor with a wide specificity, was also shown to be synthesized and secreted by all three cell lines derived from primary sarcomas but was not detected in cultures of the autologous skin fibroblasts. For the detection ofα ₂M in situ and in vitro an antiserum to tumor-associatedα ₂-macroglobulin was used. Our work was supported by grant no. 55-B86-21XB, from the Swedish Cancer Society.     

Thyroid hormone receptor α<Subscript>1</Subscript>–β<Subscript>1</Subscript> expression in epididymal epithelium from euthyroid and hypothyroid rats

The objectives of the present work were to assess whether epithelial cells from the different segments of epididymis express TRα₁–β₁ isoforms, to depict its subcellular immunolocalization and to evaluate changes in their expression in rats experimentally submitted to a hypothyroid state by injection of 131I. In euthyroid and hypothyroid groups, TR protein was expressed in epididymal epithelial cells, mainly in the cytoplasmic compartment while only a few one showed a staining in the nucleus as well. A similar TR immunostaining pattern was detected in the different segments of the epididymis. In hypothyroid rats, the number of TR-immunoreactive epithelial cells as well as the intensity of the cytoplasmic staining significantly increased in all sections analyzed. In consonance to the immunocytochemical analysis, the expression of TRα₁–β₁ isoforms, assessed by Western blot revealed significantly higher levels of TR in cytosol compared to the nuclear fractions. Furthermore, TR expression of both α₁ and β₁ isoforms and their mRNA levels were increased by the hypothyroid state. The immuno-electron-microscopy showed specific reaction for TR in principal cells associated with eucromatin, cytosolic matrix and mitochondria. The differences in expression levels assessed in control and thyroidectomized rats ascertain a specific function of TH on this organ.     

Novel cell lines derived from transgenic mice expressing recombinant human proteins

We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human α₁-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of anonc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human α₁-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.     

The distribution of α1-antichymotrypsin and amyloid production in the brain in Alzheimer’s disease

In this immunohistopathological study α₁-antichymotrypsin, which is barely demonstrable in the normal brain, was found in amyloid fibrils, endothelial cells and the cytoplasm of astroglial cells in brains from patients with Alzheimer’s disease. Amyloid precursors stained with methenamine silver were arrayed mainly along the membranes, and amyloid fibrils, which stained densely with anti-α₁-antichymotrypsin, were in direct contact with the fibrous structures connecting with the membranes of vascular feet or astrocytic processes. From the above findings, α₁-antichymotrypsin seems to play a role in the production of amyloid fibrils in Alzheimer’s disease.     

A mutant α-amylase with only part of the catalytic domain and its structural implication

A truncated mutant α-amylase, Xa-S2, was obtained from Xanthomonas campestris wild type α-amylases (Xa-WT) through random mutagenesis that contained 167 amino acid residues (approx 65% shorter than that of Xa-WT). Secondary structure prediction implied that Xa-S2, would be unable to form the whole (β/α)₈-barrel catalytic domain and did not have the three conserved catalytic residues of wild type α-amylase, but it still displays the starch-hydrolyzing activity. Xa-S2 was prepared, characterized and compared to the recombinant wild-type enzymes. The K _m for starch was 32 mg/ml; activity was optimal at pH 6.2 and 30°C. In contrast, the K _m for starch of Xa-WT was 8 mg/ml and optimal enzyme activity was at pH 6.0–6.2 and 45–50°C. Our results suggested that Xa-S2 is a new amylase with a minimal catalytic domain for hydrolyzing substrates with of α-1,4-glucosidic bonds. T. Ke and X. D. Ma contributed equally to this work     

Assessment of the effect of candidate anti-inflammatory treatments on the interaction between meningococci and inflammatory cellsin vitro in a whole blood model

A wide range of immunomodulating agents are now available which may be of benefit in reducing inflammatory cell activation in meningococcal sepsis. In order to facilitate selection of candidate anti-inflammatory agents for clinical trials, we have used an in vitro whole blood model to evaluate the effects on meningococcal induced neutrophil and monocyte activation, of dexamethasone, prostacyclin, pentoxifylline and a human IgM anti-lipid A monoclonal antibody (HA-1A). Known concentrations of heat and penicillin killed meningococci were added to whole blood and the time course of cellular activation was determined. Using elastase-α ₁-antitrypsin (elastase-α ₁-AT) and TNFα production as markers of neutrophil and monocyte activation respectively, plasma levels of elastase-α ₁-AT and TNFα were found to increase in a dose-dependant manner. Elastase-α ₁-AT was detected early, with most release occurring between 15–30 min whereas TNFα was detected later, between 120–180 min. Dexamethasone, prostacyclin and pentoxifylline caused a dose dependant inhibition of TNFα release but had no effect on elastase release. HA-1A had no effect on either TNFα or elastase release. This model may be useful in determining the sequence of inflammatory cell activation and in selecting candidate anti-inflammatory agents for evaluation in clinical trials.     

Transforming growth factor-β1 modulates the effect of 1α,25-dihydroxyvitamin D3 on leukemic cells

Summary The human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-β1 (TGF-β1) were studied by examining cell surface antigens and macrophage-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-β1 with 1α,25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] induced more Leu-M3 (CD14)-positive cells (approximately 80%) than 5×10⁻⁸ M 1α,25(OH)₂D₃ alone did (30 to 50%), although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-α (TNF-α) with TGF-β1 and 1α,25(OH)₂D₃ was found to potentiate the expression of these surface antigens. Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CD11b) antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells after the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-β1 and 1α,25(OH)₂D₃ might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-β superfamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.     

Rho GTPases mediated integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> activation in sphingosine-1-phosphate stimulated chemotaxis of endothelial cells

Integrins, a family of transmembrane heterodimeric polypeptides, mediate various biological responses including cell adhesion and migration. In this report, we show that sphingosine-1-phosphate (S1P) activates integrin α_vβ₃ in endothelial cells (ECs) via the sphingosine-1-phosphate receptor subtype 1 (S1P1)-mediated signaling pathway. S1P treatment results in the activation of integrin α_vβ₃ in the lamellipodia region of ECs, suggesting that integrin α_vβ₃ plays a critical role in the S1P-stimulated chemotactic response of ECs. Indeed, S1P treatment induces the association of focal adhesion kinase (FAK) and cytoskeletal proteins with integrin α_vβ₃, the ligation of α_v and β₃ subunits, as well as enhances endothelial migration on vitronectin-coated substrata. Knockdown endothelial S1P1 receptor, treatments with pertussis toxin or dominant-negative-Rho family GTPases abrogates the S1P-induced integrin α_vβ₃ activation in ECs. Consequently, these treatments markedly inhibit the S1P-induced endothelial migratory response on vitronectin-coated substrata. Collectively, these data indicate that the S1P-mediated signaling via the S1P1/G_i/Rho GTPases pathway activates integrin α_vβ₃, which is indispensable for S1P-stimulated chemotactic response of ECs.     

Computational studies of the binding site of α<Subscript>1A</Subscript>-adrenoceptor antagonists

Aimed at achieving a good understanding of the 3-dimensional structures of human α_1A-adrenoceptor (α_1A-AR), we have successfully developed its homology model based on the crystal structure of β₂-AR. Subsequent structural refinements were performed to mimic the receptor’s natural membrane environment by using molecular mechanics (MM) and molecular dynamics (MD) simulations in the GBSW implicit membrane model. Through molecular docking and further simulations, possible binding modes of subtype-selective α_1A-AR antagonists, Silodosin, RWJ-69736 and (+)SNAP-7915, were examined. Results of the modeling and docking studies are qualitatively consistent with available experimental data from mutagenesis studies. The homology model built should be very useful for designing more potent subtype-selective α_1A-AR antagonists and for guiding further mutagenesis studies. Figure The superposition of β₂-AR crystal structure (gold ribbons) and α_1A-AR homology model (blue ribbons)     

Distinct glycoforms of human α1-acid glycoprotein have comparable synthesis rates: a [13C]valine-labelling study in healthy humans

Various α₁-acid glycoprotein (AGP) glycoforms are present in plasma differing in extent of branching and/or fucosylation of their 5 N-linked glycans, as well as in concentration. It is assumed that hepatic synthesis determines the relative occurrence of the AGP-glycoforms in plasma, but experimental evidence is lacking. In this study, we have investigated the contribution of fractional synthesis rates to the plasma concentration of AGP-glycoforms that differed in relative occurrence in healthy human plasma. During a [¹³C]valine infusion, AGP was isolated from the plasma of healthy volunteers. Four AGP-glycoforms, differing strongly in plasma concentration were obtained by sequential affinity chromatography over concanavalin-A- and Aleuria aurantia-agarose columns. The incorporation of the [¹³C]valine tracer into the AGP-glycoforms was measured by gas chromatography combustion isotope ratio mass spectrometry. The mean fractional synthesis rates of the four AGP-glycoforms did not differ significantly between each other as well between individuals. The results indicated a renewal of about 15%/day of the plasma pools of each of the AGP-glycoforms. This is in support to the assumption that the differences in plasma concentration of the AGP-glycoforms are a reflection of the state of the hepatic glycosylation process. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Design of specific antagonists of the fibrinogen receptor

Summary The aim of this work was to elucidate, using molecular modeling, structural and electrostatic common parameters of several selective fibrinogen receptor antagonists. From this theoretical pattern, we are currently designing and synthesizing original non-peptidic and selective carbohydrate-based antagonists of the fibrinogen receptor.     

Total HLA class I loss in a sarcomatoid renal carcinoma cell line caused by the coexistence of distinct mutations in the two encoding β<Subscript>2</Subscript>-microglobulin genes

In renal cell carcinoma (RCC), HLA class I downregulation has been found in about 40% of the lesions examined. Since only scanty information is available about the molecular basis of these defects, we have investigated the mechanism(s) underlying HLA class I antigen downregulation or loss in six RCC cell lines. Five of them express HLA class I antigens although at various levels; on the other hand, HLA class I antigens are not detectable on the remaining cell line, the RCC52 cell line, belonging to a sarcomatoid subtype, even following incubation with IFN-γ. β₂-microglobulin (β₂ m) was not detected in RCC52 cells. Surprisingly, RCC52 cells harbor two mutations in the β ₂ m genes in exon 1: a single G deletion (delG) in codon 6, which introduces a premature stop at codon 7, and a CT dinucleotide deletion (delCT), which leads to a premature stop at codon 55. Analysis of eight clonal sublines isolated from the RCC52 cell line showed that the two β ₂ m gene mutations are carried separately by RCC52 cell subpopulations. The delG/delCT double mutations were detected in two sublines with a fibroblast-like morphology, while the delCT mutation was detected in the remaining six sublines with an epithelial cell morphology. Furthermore, loss of heterozygosity (LOH) of the β ₂ m gene at STR D15S-209 was found only in the epithelioid subpopulation, indicating loss of one copy of chromosome 15. Immunostaining results of the tumor lesion from which the cell line RCC52 was originated were consistent with the phenotyping/molecular findings of the cultured cells. This is the first example of the coexistence of distinct β ₂ m defects in two different tumor subpopulations of a RCC, where loss of one copy of chromosome 15 occurs in one of the subpopulations with total HLA class I antigen loss. Chin-Hsuan Hsieh, Ya-Jan Hsu and Cheng-Keng Chuang contributed equally to the work.     

Mucin-1-related T cell infiltration in colorectal carcinoma

 Mucins (MUC) are highly glycosylated molecules widely expressed on epithelia of different origins, including colonic mucosa. Altered glycosylation processes in tumour cells result in the exposure of normally cryptic peptide epitopes, which may then be recognized as tumour-specific antigens. Recently, MUC1-specific antibodies were detected in the serum of a broad range of cancer patients, and from different tumours tumour-specific cytotoxic T lymphocytes (CTL) were isolated that recognized MUC1. Absence of HLA restriction in the recognition has been ascribed to the highly repetitive sequence of the polypeptide core, allowing simultaneous recognition of multiple identical epitopes and cross-linking and aggregation of T cell receptor on mucin-specific T cells. We investigated the expression of MUC1 epitopes in 56 cell suspensions from Dukes’ B to D colorectal carcinomas using antibodies that recognize distinct peptide sequences on the glycosylated or deglycosylated MUC1 protein backbone. No relation was observed between MUC1 expression, or the extent of its glycosylation, and Dukes’ stage, tumour location and tumour differentiation, but a positive correlation was detected between the percentages of tumour cells expressing mucin-1 and the numbers of CD3⁺ infiltrating cells. These tumour-infiltrating lymphocytes contained, however, only a few MUC1-specific T lymphocytes, as CTL showing preferential killing of MUC1-expressing target cells were only obtained from one tumour. Since, in addition, the majority of colorectal carcinomas were found to express the fully glycosylated MUC1 glycoprotein, its potential role as a target antigen for T-lymphocyte-mediated immunotherapy in this tumour type is probably limited. Received: 2 April 1996 / Accepted: 28 May 1996     

Alpha-2 macroglobulin ligands and mechanisms of their biotransport

Alpha-2-macroglobulin (α2-MG) is a high-molecular weight glycoprotein with a broad spectrum of regulatory functions. As was shown earlier, covalent binding of α2-MG to proteases results in its conformational transformation, which allows α2-MG to transport additionally certain types of cytokines, attached via non-covalent interactions. Our results have shown that the spectrum of proteins exhibiting additional binding to the transformed α2-MG is rather broad and includes three classes of immunoglobulins, albumin, both types of lipoproteins, plasmin, some cytokines, and even pregnancy-associated alpha-2-glycoprotein (a structural homologue of α2-MG). The main ligands are albumin, IgG, plasmin, and to a lesser extent, lipoproteins. Interaction of native α2-MG with both acidic and weakly alkaline proteases results in neutralization net charge of the formed complex at pH, characteristic for internal media of the body. Addition of LRP (the low density lipoprotein related protein) increased the amount of electrically neutral complexes at pH 7.4. We believe that the transformed α2-MG (possibly in the complex with other effector proteins) employs similar mechanism for quick adsorption on the cell surface; after binding to LRP and repeated neutralization of the net charge at physiological pH, it “falls” through into the cell membrane and realize its regulatory functions.     

Effects of micromolar concentrations of manganese, copper, and zinc on α1-adrenoceptor-mediating contraction in rat aorta

To determine the influences of the Mn, Cu, and Zn on α₁-adrenoceptor (AR)-mediated vasoconstriction, we investigated their effects on vasoconstriction produced by the α₁-AR agonist phenylephrine in isolated rings of rat thoracic aorta. The cumulative concentration-contraction curves for phenylephrine were obtained in the absence and presence of Mn (0.3, 1, 3 μM), Cu (1, 10, 16 μM), and Zn (0.3, 1, 10 μM). Mn, Cu, and Zn each inhibited phenylephrine-mediated contraction in a dose-dependent manner. The maximal phenylephrine-induced contraction was significantly reduced by the pretreatment of the arterial rings with 10 and 16 μM Cu (p<0.05). The results suggest that variations in the plasma concentrations of metal might lead to changes in α₁-AR-mediated constrictive response.     

Mechanisms regulating GABAergic inhibitory transmission in the basolateral amygdala: implications for epilepsy and anxiety disorders

Summary. The amygdala, a temporal lobe structure that is part of the limbic system, has long been recognized for its central role in emotions and emotional behavior. Pathophysiological alterations in neuronal excitability in the amygdala are characteristic features of certain psychiatric illnesses, such as anxiety disorders and depressive disorders. Furthermore, neuronal excitability in the amygdala, and, in particular, excitability of the basolateral nucleus of the amygdala (BLA) plays a pivotal role in the pathogenesis and symptomatology of temporal lobe epilepsy. Here, we describe two recently discovered mechanisms regulating neuronal excitability in the BLA, by modulating GABAergic inhibitory transmission. One of these mechanisms involves the regulation of GABA release via kainate receptors containing the GluR5 subunit (GluR5KRs). In the rat BLA, GluR5KRs are present on both somatodendritic regions and presynaptic terminals of GABAergic interneurons, and regulate GABA release in an agonist concentration-dependent, bidirectional manner. The relevance of the GluR5KR function to epilepsy is suggested by the findings that GluR5KR agonists can induce epileptic activity, whereas GluR5KR antagonists can prevent it. Further support for an important role of GluR5KRs in epilepsy comes from the findings that antagonism of GluR5KRs is a primary mechanism underlying the antiepileptic properties of the anticonvulsant topiramate. Another mechanism regulating neuronal excitability in the BLA by modulating GABAergic synaptic transmission is the facilitation of GABA release via presynaptic α_1A adrenergic receptors. This mechanism may significantly underlie the antiepileptic properties of norepinephrine. Notably, the α_1A adrenoceptor-mediated facilitation of GABA release is severely impaired by stress. This stress-induced impairment in the noradrenergic facilitation of GABA release in the BLA may underlie the hyperexcitability of the amygdala in certain stress-related affective disorders, and may explain the stress-induced exacerbation of seizure activity in epileptic patients.