Plasma acidic glycohydrolases in insulin-dependent diabetes mellitus

Summary We assayed plasma activities of -galactosidase, -hexosaminidase, -mannosidase, -fucosidase and -galactosidase involved in degradation of the glycoprotein molecule in 110 insulin-dependent diabetics aged 3-1/2 to 19 years and compared them to a group of normal youngsters. We correlated the plasma enzyme activities with the duration, control and sequelae of insulin-dependent diabetes. Insulin-dependent diabetics had a significantly higher plasma activity of -hexosaminidase and -mannosidase (p<0.01) and a significantly lower plasma activity of -fucosidase and -galactosidase (p<0.01). Of the 5 enzymes studied, only plasma -hexosaminidase correlated with fasting and postprandial blood sugar (p<0.01), cholesterol and triglycerides (p<0.05). Additionally, poor control of diabetes was also associated with a significantly higher plasma -hexosaminidase activity (p<0.01). Proteinuria or an abnormal Addis count suggestive of renal involvement was associated with various changes in plasma acidic hydrolases. These changes may be related to insulin deficiency rather than hyperglycemia and may be genetically determined.Deceased on August 2, 1981.     

Growth suppression of transformed cells by a human placental extract not related to transforming growth factor Β

Summary We have examined whether human placental extracts contain tumour-growth-inhibitory factors. One fraction (EAP) from such extracts inhibited growth, in soft agar, ofHa-ras-transformed BALB/c 3T3 cells and human squamous lung carcinoma A-2182 cells. However, this fraction had no effect on the anchorage-dependent growth of these cells, although there was a slight mitogenic activity on nontransformed cells. These data together with those on plating efficiency indicated no significant cytotoxicity of EAP on transformed cell lines. Although this fraction contained transforming growth factor (TGF), this cannot account for its inhibitory activity, since (a) pure TGF does not inhibit anchoragedependent growth of Ha-ras-transformed BALB/c 3T3 cells, (b) EAP retains its inhibitory activity in the presence of antibodies against TGF and (c) the inhibitory activity did not copurify with TGF. Partial characterization of our inhibitory factor suggests that the inhibitory factor is a new tumour-growth-inhibitory factor.Abbreviations SDS sodium dodecyl sulphate - TGF or transforming growth factor or - TIF1 or 2 transformed inhibitory factor 1 or 2 - A-2182 human squamous lung carcinoma cell line - BALB/c 3T3 1-1 mouse fibroblast cell line, clone A31 1-1 - BALB/c 3T3 Ha-ras, BALB/c 3T3 1-1 cells transformed by Ha-ras oncogene - CHO Chinese hamster ovary; MEM, minimum essential medium This research was supported by a training grant from Groupement d'Etude et de Recherche sur le Placenta.     

Comparison of mRNA contents of interleukin-1Β and nitric oxide synthase in pancreatic islets isolated from female and male nonobese diabetic mice

Summary Interleukin-1 (IL-1) has been suggested to mediate beta-cell destruction in insulin-dependent diabetes mellitus (IDDM) by inducing nitric oxide production. In this study, we assessed the levels of IL-1 and the inducible form of nitric oxide synthase (iNOS), using a semi-quantitative polymerase chain reaction assay, and performed determinations of nitrite accumulation and IL-1 bioactivity, on pancreatic islets isolated from 5- and 16-week-old female and male nonobese diabetic (NOD) mice and from nondiabetes prone NMRI mice. NOD mouse islets contained notable amounts of IL-1 mRNA. At 5 weeks of age, but not at 16 weeks, the values were higher in islets isolated from NOD females compared to males. The IL-1 bioactivity showed differences roughly reflecting the mRNA levels in the NOD mouse islets. In the NMRI mouse islets the IL-1 bioactivity was very low. The expression of iNOS mRNA increased in both male and female islets between 5 and 16 weeks of age. Immunocytochemistry of pancreatic sections indicated the presence of macrophages especially in the peri-insular area of the NOD mice which suggests that IL-1 was produced by macrophages. The levels of IL-1 activity and mRNA in freshly isolated islets from NOD 5-week-old females did not correlate to the iNOS mRNA content or to the nitrite production. However, after incubation with IL-1 in vitro, both NOD and NMRI islets responded with a marked increase in nitric oxide production. It is concluded that the presence of IL-1 in isolated NOD mouse islets, via an induction of iNOS expression and nitric oxide production, cannot explain the gender difference in diabetes incidence in NOD mice.Abbreviations BB BioBreeding - GAPDH glyceraldehyde-3-phosphate dehydrogenase - IDDM insulin-dependent diabetes mellitus - iNOS inducible form of nitric oxide synthase - IL-1 interleukin-1 - IL-1ra interleukin-1 receptor antagonist protein - IL-6 interleukin-6 - NMRI Naval Marine Research Institute - NO nitric oxide - NOD nonobese diabetic - PAP peroxidase-antiperoxidase - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - TNF tumour necrosis factor - OD optical density     

Cytokine-induced apoptotic cell death in a mouse pancreatic beta-cell line: inhibition by Bcl-2

Summary Cytokines are thought to contribute to the induction of pancreatic beta-cell destruction in insulin-dependent diabetes mellitus. The molecular mechanisms that underlie beta-cell death were investigated by studying cytokine-induced cell death in beta-cell lines. A combination of three cytokines (interleukin-1, tumour necrosis factor-, and interferon-) induced apoptotic cell death in the mouse pancreatic beta-cell line TC1, as judged from the appearance of cells with hypodiploid nuclei and oligonucleosomal DNA fragmentation. The same treatment also induced apoptosis in the mouse pancreatic alpha-cell line TC1 and the NOD/Lt mouse beta-cell line NIT-1, although to a lesser extent than in TC1 cells. The abundance of endogenous Bcl-2 in TC1 cells was lower than that in the other two cell lines. Overexpression of human Bcl-2 in TC1 cells partially protected them from cytokine-induced cell death. These results suggest that apoptosis may be responsible, at least in part, for cytokine-induced beta-cell destruction and that Bcl-2 prevents apoptosis in pancreatic islet cells.Abbreviations IDDM Insulin-dependent diabetes mellitus - IL interleukin - TNF tumour necrosis factor - IFN interferon - FBS fetal bovine serum - ATA aurintricarboxylic acid - CHX cycloheximide - PI propidium iodide     

Effect of T-cell receptor VΒ-specific monoclonal antibodies on cyclophosphamide-induced diabetes mellitus in non-obese diabetic mice

Summary The expression of specific T-cell receptor gene segments by T lymphocytes appears to be critically important for the induction of several experimental autoimmune diseases mediated by these cells. We examined whether this situation also applied to non-obese diabetic mice by using various T-cell receptor V-specific monoclonal antibodies. No significant age- or sex-related differences were observed in V usage by peripheral and splenic T lymphocytes. CD8⁺ T lymphocytes among the islet-derived mononuclear cells isolated from 20-week-old female non-obese diabetic mice showed heterogeneity of their V gene usage. In order to examine the role of T lymphocyte subsets expressing specific T-cell receptor V gene segments in the development of diabetes mellitus, T-cell receptor V-specific monoclonal antibodies were administered to 10-week-old male non-obese diabetic mice treated with cyclophosphamide. None of the antibodies used could significantly diminish the incidence of cyclophosphamide-induced diabetes and the severity of insulitis [anti-V3 (11 of 22 mice became diabetic, 50%), anti-V5 (9 of 14, 64%), anti-V8 (9 of 21, 43%), anti-V11 (12 of 23, 52%), anti-V14 (7 of 12, 58%), and anti-V5 + anti-V11 (6 of 12, 50%)] when compared with control mice (12 of 21, 57%). In addition, there were no significant differences in T-cell receptor V usage between diabetic and non-diabetic cyclophosphamide-treated mice. These results suggest that five T-lymphocyte subsets expressing different T-cell receptor V gene segments, considered to be candidates involved in the pathogenesis of autoimmune diabetes, do not individually contribute to the development of cyclophosphamide-induced diabetes in non-obese diabetic mice.     

Detection of allelic loss within the β1-interferon gene in childhood acute lymphoblastic leukemia using differential PCR

Summary Deletion of the short arm of chromosome 9p involving the 1-interferon (IFN) gene has been implicated in the process of malignant transformation in lymphomas and acute lymphoblastic leukemias. Since cytogenetic analysis is frequently unsuccessful in clinical samples, we used a recently described differential PCR technique to detect losses within the 1-IFN gene in 86 acute leukemias. Using differential PCR, no 1-IFN deletion was detected in 44 acute myeloid leukemia (AML) and eight control samples. However, five of 42 acute lymphoblastic leukemia (ALL) probes (12%) exhibited loss of the 1-IFN gene (three common ALL, two T-ALL). Cytogenetic analysis was performed independently in three of these five cases and revealed abnormalities of chromosome 9p in two samples. Two of five T-ALL cases exhibited a loss within the 1-IFN gene, compared with 3/29 c-ALLs, suggesting a predominance of IFN gene loss in T-ALLs. These data indicate that PCR can be used for rapid detection of gene dosage phenomena in clinical leukemia samples.     

Regulatory role of eicosanoids in extracellular matrix overproduction induced by long-term exposure to high glucose in cultured rat mesangial cells

Summary Accumulation of extracellular matrix in the mesangium and altered renal eicosanoid synthesis are two prominent features of diabetic glomerular disease. We investigated the relationship between eicosanoid and extracellular matrix production in rat mesangial cells cultured under high glucose vs normal glucose conditions. Long-term exposure of rat mesangial cells to high glucose, but not to iso-osmolar mannitol, significantly increased extracellular matrix accumulation and gene expression and transforming growth factor- (TGF-) mRNA levels, and decreased prostaglandin (PG) E₂ synthesis without affecting production of either thromboxane (TX) B₂ or PGF₂, with respect to cells incubated in normal glucose. Addition of exogenous PGE₂ resulted in a dose-dependent reduction of matrix protein and mRNA levels and TGF- gene expression in cells cultured in either normal or high glucose conditions, whereas exposure to exogenous PGF₂ produced a significant increment in matrix production and matrix and TGF- gene expression in cells grown in normal glucose, but only a slight increase in those cultured in high glucose. Stimulation of endogenous endoperoxide metabolism towards PGE₂ and PGF₂ synthesis with FCE-22,178, a drug originally developed as TXA₂ synthase inhibitor, resulted in a dose-dependent decrease in matrix accumulation and matrix and TGF- gene expression which was suppressed by co-incubation with the cyclo-oxygenase inhibitor feno-profen blocking the FCE-22,178-enhanced PG production. In both cell lines, the rate of synthesis of TXA₂ was very low and the selective blockade of its synthesis (by two other TXA₂ synthase inhibitors, OKY-046 and Ridogrel) or action (by the TXA₂ receptor antagonist BM-13,177) did not alter matrix production or TGF- mRNA levels. These results suggest that the cyclo-oxygenase pathway is involved in the regulation of matrix changes induced by high glucose in rat mesangial cells; the reduced production of PGE₂ may enhance the synthesis or potentiate the effect of stimulators of ECM formation such as TGF-, whereas TXA₂ does not appear to be involved. These data also indicate that glucose-enhanced mesangial matrix accumulation may be prevented by exogenous PGE₂ or by drugs capable of increasing endogenous PGE₂ synthesis.Abbreviations AGE Advanced glycosylation end-products - ECM extracellular matrix - PG prostaglandin - RMC rat mesangial cells - TGF- transforming growth factor- - TX thromboxane - Cox cyclo-oxygenase     

Association of Trp64Arg mutation of the β3-adrenergic-receptor with NIDDM and body weight gain

Summary A possible pathogenic mutation in the 3-adrenergic-receptor gene (Trp64Arg) has been reported to be associated with an earlier age of onset of non-insulin-dependent diabetes mellitus (NIDDM) and clinical features of the insulin resistance syndrome in Pima Indian, Finnish and French subjects. Since marked heterogeneity has been reported in the association of mutations of candidate genes with NIDDM between Japanese and other ethnic groups, we investigated the association of Trp64Arg with NIDDM in Japanese subjects. The allele frequency of the mutation (Arg) was slightly, but not significantly, higher in NIDDM than in control subjects (70 out of 342 alleles [20.5%] vs 40 out of 248 [16.1%], respectively, p>0.2). When our data were combined with those of Pima Indian and Finnish subjects, however, the Arg/Arg genotype was significantly associated with NIDDM as compared with the other two genotypes (p<0.005, relative risk [RR] 2.13, 95% confidence interval [CI] 1.28–3.55). The Arg allele was also associated with NIDDM (p<0.05, RR 1.27, 95% CI 1.06–1.52). Japanese subjects homozygous for the mutation had a significantly higher body mass index (mean ± SD25.5±3.9 kg/ m²) than heterozygotes (22.6±4.1, p<0.05) and normal homozygotes (22.8±3.8, p<0.05). NIDDM patients homozygous for the mutation tended to have an earlier age of onset of NIDDM than those with other genotypes. These data suggest that the Trp64Arg mutation not only contributes to weight gain and age-at-onset of NIDDM but is also associated with susceptibility to NIDDM.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - 3-AR 3-adrenergic-receptor - Trp64Arg a mutation in the 3-adrenergic-receptor gene causing a Trp to Arg change at codon 64 - BMI body mass index - ADRB3 3-adrenergic receptor gene - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - RR relative risk - CI confidence interval     

Rapid detection of −α 4.2 deletion of α-thalassemia-2 by polymerase chain reaction

Summary We sequenced part of the X boxes of-thalassemia-1 of Southeast Asia type (- -SEA) with– ^4.2,– ^3.7,– ^G-Taichung, and ^CS. We found the X box of– ^3.7 belonged to the X box of 2 globin gene and the X box of ^cs contained X boxes of both al and2 globin gene, whereas the X box of– ^4.2 and– ^G-Taichung was a hybrid of X boxes of 2 and 1 globin gene. We also found there are two types of– ^4.2 deletion; type 1 is a common type of– ^4.2 deletion and type 2 is linkage to– ^G-Taichung. We used a combination of two methods, the amplification refractory mutation system (ARMS) and the amplified created restriction sites (ACRS), to amplify the hybrids of X boxes specifically. The upstream primer for X box of2 globin gene was designed following the standard ARMS procedure to amplify the X segment of the-globin gene. The downstream primer was designed according to the ACRS method to check the specificity of PCR products. Using this approach, we can diagnose the different types of– ^4.2 deletion. This kind of approach can also be used to amplify the specific region from the cluster of highly homologous genes.     

Effect of a diet rich in linseed oil on complex viscosity and blood pressure in spontaneously hypertensive rats (SHR)

Summary The influence of a diet rich in linseed oil (10 % in weight) with a content of 61.2 % of -linolenic fatty acid on blood pressure and complex blood viscosity was investigated in spontaneously hypertensive rats. A decrease in blood pressure by 59 mm Hg was found compared to the age-matched, untreated control group. The viscous () and elastic () components of viscosity were also reduced at various shear rates ( ). The same applies to the aggregation index, which is a measure of the aggregation tendency of red blood cells (RBC).Of course, the effects on blood viscosity cannot explain the observed degree of blood-pressure lowering. Nevertheless, a decrease in the viscosity can improve the flow conditions in the microcirculation, which may lead to a better oxygen supply.Supported by the Alfred-Teufel-Stiftung.This study is part of the unpublished doctoral thesis of M. Schäch     

Elliptocytosis associated with an abnormal α glycophorin

Summary A case of elliptocytosis associated with an undescribed abnormal glycophorin (GP) is reported. Using immunoblotting techniques, a clear-cut minor band 6 was detected emerging just behind the monomer of GP (band 6) when probed with anti- GP antiserum. It also reacted with anti-peptide C antiserum, suggesting that this new band with a molecular weight of 24 K is related to the structural alteration of GP and not GP. The erythrocyte membrane proteins of the patient exhibited a quite normal pattern, with a normal spectrin/ spectrin ratio, but the reaction with anti-protein 4·1 serum confirmed the increase in proteolytic susceptibility of her protein 4·1. The results of DNA mapping implied that the abnormality may be due to a short deletion of the heterozygote. The significance of deviation involving the GP and protein 4·1 to the elliptocytic change of erythrocyte shape is briefly discussed.This work was supported by grant no. HL-21016 from the National Institutes of Health, Bethesda, MD     

Molecular and linkage analysis of type-1 protein phosphatase catalytic beta-subunit gene: lack of evidence for its major role in insulin resistance in Pima Indians

Summary Insulin resistance is believed to be a prediabetic condition that results from reduced rates of insulin-mediated glycogen synthesis in skeletal muscle. A decrease in activities of skeletal muscle glycogen synthase and of its regulatory enzyme type-1 protein phosphatase (PP 1) have been previously identified in insulin-resistant Pima Indians. Because the PP1 catalytic -subunit is presumed to be the major isoform in the glycogen-bound PP1 complex, we have selected the structural gene for this subunit (PPP1CB) as a candidate for a detailed genetic analysis. We have determined the exon-intron structure of PPP1CB, and have identified a polymorphic (CA)-repeat marker (D2S1237) at this gene. No sequence abnormalities were detected in PPP1CB by Southern blot analysis or by single-stranded conformational polymorphism analysis of all eight coding exons. Using sib-pair linkage analyses, no evidence for linkage was found between the D2S1237 marker at this locus and fasting insulin, insulin-stimulated glucose uptake in vivo, obesity, or non-insulin-dependent diabetes mellitus. Similarly, we have found no evidence for association of D2S1237 with any of these phenotypes. Based on our data we conclude that the structural gene for the PP1 catalytic -subunit does not appear to be a major genetic determinant responsible for the PP1 abnormalities characteristic of insulin resistance in Pima Indians.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - PP1 type-1 protein phosphatase - PPP1CB type-1 protein phosphatase catalytic -subunit gene - SSCP single-stranded conformational polymorphism - PCR polymerase chain reaction     

Effects of the hypoglycaemic drugs repaglinide and glibenclamide on ATP-sensitive potassium-channels and cytosolic calcium levels in Β TC3 cells and rat pancreatic beta cells

Summary The present study demonstrates the action of the hypoglycaemic drugs repaglinide and glibenclamide in cultured newborn rat islet cells and mouse TC3 cells. In cell-attached membrane patches of newborn rat islet cells repaglinide (10 nmol/l) and glibenclamide (20 nmol/l) decrease the open probability of single ATP-sensitive K⁺-channels to approximately 10% of the activity prior to addition of the drugs in short-term experiments (<5 min). The influence of repaglinide and glibenclamide on the ATP-sensitive K⁺ current was studied using the whole-cell patch clamp configuration. A half-maximal steady-state inhibition of the ATP-sensitive K⁺ currents is observed at 89 pmol/l repaglinide and at 47 pmol/l glibenclamide in whole-cell experiments of longer duration (30 min). Applying digital Ca²⁺ imaging on single TC3 cells we found that repaglinide and glibenclamide induced a concentration-dependent increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) with a half-maximal effect at 0.5 nmol/l for both drugs in long-term experiments (30 min). The rise in [Ca²⁺]_i results from Ca²⁺ entry through voltage-dependent L-type Ca²⁺-channels since it is inhibited by verapamil (10 mol/l). The effect of repaglinide and glibenclamide is partly reversible (80%).Abbreviations K⁺ _ATP-channel ATP-sensitive potassium channel - [Ca²⁺]_i intracellular free Ca²⁺ concentration - EGTA ethylene glycol-O,O-bis(2-aminoethyl]-N,N,N,N-tetraacetic acid - IC₅₀ apparent inhibitor constant - repaglinide ((S)-(+)-2-ethoxy-4-[2-((3-methyl-1-[2-(1-piperidinyl) phenyl]-butyl)amino)-2-oxoethyl) benzoic acid - NIDDM non-insulin-dependent diabetes mellitus - TC3 cells transgenic mouse insulinoma tumour cell line     

Elevated serum levels of macrophage-derived cytokines precede and accompany the onset of IDDM

Summary To determine whether cytokines could have a role in the development of insulin-dependent diabetes mellitus (IDDM), we measured serum levels of cytokines derived from T helper 1 (interleukin-2 and interferon-), T helper 2 (interleukin-4 and inter-leukin-10) lymphocytes and macrophages (tumour necrosis factor-, interleukin-1 and interleukin-1 ) in patients before and after the onset of IDDM. Recently diagnosed IDDM patients had significantly higher levels of interleukin-2, interferon-, tumour necrosis factor- and interleukin-1 than patients with either long-standing IDDM, non-insulin-dependent diabetes (NIDDM), Graves' disease, or control subjects (p<0.05 for all). Compared with control subjects, patients with long-standing IDDM and those with NIDDM had higher interleukin-2 and tumour necrosis factor- levels (p<0.01 for all). Interleukin-4 and interleukin-10 were detectable in sera of patients with Graves' disease only, while interleukin-1 was not detectable in the serum of any control or test subject. To investigate whether high cytokine levels precede the onset of IDDM, we studied 28 non-diabetic identical co-twins of patients with IDDM, followed-up prospectively for up to 6 years after the diagnosis of the index. Levels of tumour necrosis factor- and interleukin-1 were elevated above the normal range more frequently in the eight twins who developed diabetes than in those 20 who did not (p<0.005). Analysis of T helper 1 and T helper 2 profiles of the twin groups did not reveal a clear difference between prediabetic twins and twins remaining non-diabetic. These results support the notion that T helper 1 lymphocytes may play a role in the development of IDDM. This is associated with release of macrophage-derived cytokines, which is also a feature of the prediabetic period. The lack of evidence of a dominant T helper 1 profile of cytokine release before diabetes onset suggests that additional events, activating this arm of the cellular immune response, are required in the immediate prediabetic period.Abbreviations IL Interleukin - IFN- interferon-gamma - T_H T helper - ICA islet-cell antibody - GAD glutamic acid decar-boxylase - IDDM insulin-dependent diabetes mellitus - NIDDM non-insulin-dependent diabetes mellitus - TNF- tumour necrosis factor-alpha - CTLL-16 murine cytotoxic cell line     

Promoting healthy aging in cities: The Healthy Cities project in Europe

Health promotion is a growing field, spreading into various settings. Thispaper focuses on the city as a setting for health promotion and on theHealthy Cities project (HCP) of the Regional Office for Europe of theWorld Health Organization in particular. In the context of the HCP, variousactivities to promote the health of older citizens have been carried out inrecent years. These are illustrated in this paper through case studies.     

A study of betel quid carcinogenesis

Summary Betel quid chewing is strongly associated with cancer of the oral cavity, especially when tobacco is added to the quid. It is our working hypothesis that, during chewing, Areca-derived N-nitrosamines are formed and, in the presence of tobacco, Nicotiana-specific N-nitrosamines are formed as well and further that these agents may contribute to the high risk of oral cancer in betel-quid chewers. This preliminary report presents our finding of N-nitrosoguvacoline in the saliva of betel-quid chewers (2.2–350 ppb). When the quid contains tobacco, the tobacco-specific N-nitrosamines, N-nitrosonornicotine (1.2–38.3 ppb), N-nitrosoanatabine (3.2–39.5 ppb), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (1.0–2.3 ppb) are also found in the saliva.Abbreviations NG N-nitrosoguvacoline - MNPA 3-(methylnitrosamino)propionaldehyde - MNPN 3-(methylnitrosamino)propionitrile - NNN N-nitrosonornicotine - NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone - NAT N-nitrosoanatabine - TSNA tobacco-specific nitrosamines Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthdayThe study is supported by U.S. National Cancer Institute Grant CA-29580     

Immunoblot analysis of the placental form of glutathione S-transferase in protein extracted from paraffin-embedded human glioma tissue

Protein extracted from conventional formalin-fixed and paraffin-embedded tissue sections of human gliomas was examined for immunoblot analysis using antibody against the placental form of glutathioneS-transferase (GST-). Four benign astrocytomas, five anaplastic astrocytomas and four glioblastomas were used in this study. The preliminary study demonstrated that immunoreactivity of GST- was well preserved in normal brain tissue and normal term placenta fixed in acetone, formalin or buffered formalin (pH 7.4). GST- in gliomas fixed in formalin also had a good immunoreactivity and showed clear bands on nitrocellulose membranes processed by the method of Western blotting using anti-GST- antibody. The results of immunoblot analysis for GST- indicate that the intensity of immunoreactivity of benign astrocytoma, anaplastic astrocytoma and glioblastoma increases with the advance of malignancy of these neoplasms. Western blot analysis for GST- can be performed using protein extracted from formalin-fixed and paraffin-embedded tissue sections, and the immunoreactive bands can be analyzed quantitatively by densitometric scanning.Abbreviation GST- placental form of glutathioneS-transferase - SDS-PAGE sodium dodecylsulfate/polyacrylamide gel electrophoresis     

Synergistic cytotoxicity of human recombinant tumour necrosis factor α combined with microtubule effectors

Summary Combinations of human recombinant tumour necrosis factor (rhTNF) with each of four different agents disturbing the microtubule system of the cellular cytoskeleton were tested for synergistic cytotoxic action against murine melanoma B16K and L-M(S) cells. In addition to the known microtubule effectors colchicine, vincristine, and taxol, the influence of the fluorenone-azomethine derivative-diphenylene-N-{p-[bis-(-hydroxyethyl)-amino]-phenyl}-nitrone (DHPN) on the rhTNF cytotoxicity was studied. Applying a novel computerbased isobole method [Suehnel J (1990) Antiviral Res 13:23–40] concentration ranges of synergistic, zero, and antagonistic interaction were found after in vitro combination of rhTNF with each of the drugs tested in a 72-h cytotoxicity assay. In contrast, a 24-h exposure of B16K cells to these combinations still did not inhibit in vitro colony formation to a greater extent than either drug alone. A preliminary in vivo experiment revealed an increased antitumour effect after treatment of established subcutaneous melanoma B16 tumours with a combination of rhTNF and DHPN.Abbreviations rhTNF human recombinant tumour necrosis factor - DHPN -diphenylene-N-{p-[bis-(-hydroxyethyl)-amino]-phenyl}-nitrone     

Metoprolol, a Β-1 selective blocker, can be used safely in coronary artery disease patients with chronic obstructive pulmonary disease

The coexistence of coronary artery disease (CAD) and chronic obstructive pulmonary disease (COPD) is frequent because of common etiological factors. -Blockers remain underutilized in patients with CAD who also have COPD. This study was performed to evaluate the safety of -1 selective blocker agents in CAD patients with COPD. Fifty patients (aged 57.3 ± 10.1 years) were enrolled in this study; 27 patients received metoprolol CR (controlled release), and 23 received metoprolol (conventional). The patients were stratified according to the severity of COPD (21 severe, 21 moderate, and 8 mild), started on metoprolol CR or conventional metoprolol, and titrated up to the maximum tolerated dose. The clinical controls were done during the first week and then at the first and third month. Patients received a mean total daily dose of 92.5 ± 18mg of metoprolol CR or 189 ± 36.7mg of metoprolol. Seven patients could not receive the maximum dose. There was no significant decrease in forced expiratory volume in 1s (FEV₁) in either group (basal vs last FEV₁: 54.5% ± 13.4% vs 54.3% ± 13% in the metoprolol CR group and 49.6% ± 14.5% vs 53.2% ± 12.8% in the metoprolol group). No adverse event was experienced. Metoprolol, a -1 selective blocker, can be used safely at the maximum dose in CAD patients with COPD.