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     

Polarographische Untersuchung cytostatischer Chinonderivate

Zusammenfassung Bayer E 39 zeigt ein ungewöhnliches Polarogramm insofern, als es durch eine reversible Stufe (_1/2=–0,23 V gegen NCE bei Ph 7) und eine besonders ausgesprochene katalytische Welle (–1 V) charakterisiert ist. Das cytostatisch inaktive Produkt einer Hydrolyse von E 39 wird in einem Zwei-Elektronenschritt bei _1/2=–0,45 V bei Ph 7 reduziert.

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Summary 2,5-Bis-(ethyleneimino)-3,6-bis-(n-propoxy)-benzoquinone-(1,4) (Bayer E 39), a particularly remarkable member in the series of quinone derivatives exhibiting a cytostatic action, has been characterized polarographically. Its unusual polarogram is formed by a reversible step (_1/2=–0,23 V against NCE at Ph 7) and a particularly distinct catalytic wave (–1 V).The two-electronic step which is likewise reversible yet not followed by a catalytic wave of the product of its hydrolysis 2,5-bis-(-chloroethyleneamino)-3,6-bis-(n-propoxy)-benzoquinone-(1,4) (?) irreversibly formed in acid solution and which has lost the remarkable cytostatic activity of the initial substance, lies more negative by _1/2=–0,22 V at _1/2=–0,45 V (Ph 7) which corresponds to a shifting of the absorption band of long wave length by =60 m.It is further to be noticed that, according to the electrophoretic and chromatographic studies of Ch. Scholtissek, the Ph-dependent reaction of Bayer E 39 (and A 139) with nucleic acids makes obvious that an interaction between the substrate and the cytostatically active quinones takes place in an acid medium only.

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Mit 6 Textabbildungen     

Isolierung von freien Nucleotiden aus verschiedenen Geweben

Zusammenfassung In Erweiterung früherer Mitteilungen wird über den Gehalt des Flexner-Jobling-Carcinoms der Ratte an freien Nucleosidmono- und-polyphosphorsäureestern berichtet.Die vorliegenden Untersuchungen wurden zum Teil dankenswerter-weise durch denAnna-Fuller-Fund und dieDeutsche Forschungsgemeinschaft unterstützt.In der Arbeit werden folgenden Abkürzungen verwendet Ad Adenosin - AMP Adenosin-5-monophosphat - ADP Adenosin-5-diphosphat - ATP Adenosin-5-triphosphat - GMP Guanosin-5-monophosphat - GDP Guanosin-5-diphosphat - GTP Guanosin-5-triphosphat - CMP Cytidin-5-monophosphat - CDP Cytidin-5-diphosphat - CTP Cytidin-5-triphosphat - UMP Uridin-5-monophosphat - UDP Uridin-5-diphosphat - UTP Uridin-5-triphosphat - UDPA Uridin-5-diphosphat-N-acetylglucosamin - UDPG Uridin-5-diphosphat-glucose - UDPGa Uridin-5-diphosphat-galaktose - UDPGl Uridin-5-diphosphat-glucuronsäure - IMP Inosin-5-monophosphat - DPN Diphosphopyridinnucleotid - HS Harnsäure - RNS Ribonucleinsäure - DNS Desoxyribonucleinsäure - TPN Triphosphopyridinnucleotid Mit 19 TextabbildungenDer überwiegende Teil der dieser Arbeit zugrunde liegenden Versuche wurde 1952/53 im McArdle Memorial Laboratory, University of Wisconsin durchgeführt.     

Clinical usefulness of free light chain concentration as a tumor marker in multiple myeloma

Monoclonal immunoglobulin, as a marker for monoclonal gammopathy, is evaluated by protein electrophoresis (PEP) and immunofixation electrophoresis (IFE). However, PEP and IFE are not satisfactory in sensitivity, objectivity, and facility. Recently, a highly sensitive, automated immunoassay for measurement of free light chain (FLC) concentrations in serum and urine has been developed for the identification and monitoring of patients with monoclonal gammopathy. To explore the clinical usefulness of measurement of FLC concentrations, we measured the and FLC concentrations and calculated the / FLC ratios for three groups [multiple myeloma (MM), other diseases, and control] and compared the results of the FLC assay with the results of PEP or IFE. The concentrations of serum and FLCs and the / FLC ratios for the MM group and non-MM groups were distinct. In the MM group, some sera and urine samples had no evidence of M protein on PEP and IFE, but FLC assay showed abnormal concentrations of FLCs and abnormal / FLC ratios in most cases. As compared with the PEP, the / FLC ratio revealed higher sensitivity in all diagnostic ranges with different cutoff values. Particularly, when the cutoff value 2.0 for / FLC ratio was used, specificity and positive predictive value were largely improved than when the cutoff values 1.2 and 1.5 were used. These findings indicated that FLC assay enables to detect myeloma patients with very low M protein due to early stage or after therapy and to distinguish patients with monoclonal increase of FLC from patients with polyclonal increase of FLC due to other conditions, particularly using / FLC ratio 0.3–2.0 as a diagnostic range. Despite some technical limitations of the assay, the incorporation of / FLC ratios with FLC concentrations is useful in the detection of M protein, particularly with negative serum or urine IFE results, and differentiation of monoclonal gammopathies from patients with polyclonal increase in FLC due to other conditions.     

Expression and prognostic roles of integrins and interleukin-1 receptor type I in patients with ductal adenocarcinoma of the pancreas

To Investigate the prognostic indicator, we examined the expression of ₆- and ₅- integrin and interleukin-1 receptor type I (IL-1RI) immunohistochemically, and analyzed the correlation between immunohistochemical findings and clinicopathological factors in pancreatic cancer. In patients with a strongly expressing ₆- integrin subunit or weakly expressing ₅₁-integrin in pancreatic cancer tissues there was a significant association with advanced TNM stage (P = 0.027 and 0.014, respectively), presence of liver metastases (P = 0.032 and 0.002, respectively), and poor prognosis (P = 0.0155 and 0.0056, respectively). In patients with a weakly expressing ₆ integrin subunit or weakly expressing ₅₁-integrin in noncancerous pancreatic tissues there was a significant association with poor prognosis (P = 0.0324 and 0.0396, respectively). Multivariate analysis demonstrated that strong expression of ₆- and weak expression of ₅₁-integrin were found to be independent prognosticators in pancreatic cancer patients. Our present results indicate that ₆₁- and ₅₁-integrin expression can be a significant prognostic indicator in pancreatic cancer.     

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.     

Inborn errors of bile acid metabolism

Summary Cholesterol is converted to cholic acid and chenodeoxycholic acid by a series of reactions involving modifications to the steroid nucleus and oxidation of the side chain. These reactions can be affected by a number of inborn errors of metabolism. When this happens unusual bile acids or bile alcohols are synthesized; these can be identified using gas chromatography-mass spectrometry and fast atom bombardment mass spectrometry techniques. Two defects affecting the modifications to the steroid nucleus have been described; both present with cholestatic liver disease of neonatal onset. The better characterized of the two — 3-hydroxy-5-C27-steroid dehydrogenase deficiency — leads to excretion of 3-7-dihydroxy-5-cholenoic acid and 3,7,12-trihydroxy-5-cholenoic acid in the urine. The liver disease improves dramatically on treatment with chenodeoxycholic acid. Deficient activity of 3-oxo-4-steroid 5-reductase is thought to be the cause of familial liver disease in some infants who excrete 7-hydroxy-3-oxo-4-cholenoic acid and 7,12-dihydroxy-3-oxo-4-cholenoic acid in the urine. However, diagnosis of this disorder is problematical; a similar pattern of metabolite excretion can occur as a result of liver damage caused by viruses or inborn errors of pathways unrelated to bile acid synthesis. Defective side chain oxidation in patients with cerebrotendinous xanthomatosis (CTX) leads to synthesis of bile alcohols such as 5-cholestane-3,7,12,25-tetrol and 5-cholestane-3,7,12,23,25-pentol. Patients with CTX do not have cholestatic liver disease. Their major problems (neurological disease, atherosclerosis and xanthomata) are caused by accumulation of cholestanol and cholesterol in the tissues. Bile acid precursors are probably diverted into synthesis of cholestanol. Chenodeoxycholic acid suppresses the production of abnormal metabolites from cholesterol (by inhibition of cholesterol 7-hydroxylase) and leads to improvement in the neurological disease. Defective side chain oxidation also occurs in peroxisomal disorders but this time it leads to accumulation of C27 bile acids such as 3,7,12-trihydroxy-5-cholestanoic acid (trihydroxycoprostanic acid, THCA). This compound is readily detected in the bile and plasma of patients with defects of peroxisome biogenesis. In patients with defects of a single peroxisomal-oxidation enzyme (the 3-hydroxyacyl-CoA component of the bifunctional protein or the thiolase), the major C27 bile acid in bile may be 3,7,12,24-tetrahydroxy-5-cholestanoic acid (varanic acid). In addition to the above inborn errors, others which are less well characterized undoubtedly exist, as do defects of bile acid transport across membranes.     

High glucose and hydrogen peroxide increase c-Myc and haeme-oxygenase 1 mRNA levels in rat pancreatic islets without activating NFκB

Aims/hypothesis Hyperglycaemia and the pro-inflammatory cytokine IL-1 induce similar alterations of beta cell gene expression, including up-regulation of c-Myc and haeme-oxygenase 1. These effects of hyperglycaemia may result from nuclear factor-kappa B (NFB) activation by oxidative stress. To test this hypothesis, we compared the effects of IL-1, high glucose, and hydrogen peroxide, on NFB DNA binding activity and target gene mRNA levels in cultured rat islets.Methods Rat islets were pre-cultured for 1 week in serum-free RPMI medium containing 10 mmol/l glucose, and further cultured in glucose concentrations of 5–30 mmol/l plus various test substances. Islet NFB activity was measured by ELISA and gene mRNA expression was measured by RT-PCR.Results IL-1 consistently increased islet NFB activity and c-Myc, haeme-oxygenase 1, inducible nitric oxide synthase (iNOS), Fas, and inhibitor of NFB alpha (IB) mRNA levels. In comparison, 1- to 7-day culture in 30 mmol/l instead of 10 mmol/l glucose stimulated islet c-Myc and haeme-oxygenase 1 expression without affecting NFB activity or iNOS and IB mRNA levels. Fas mRNA levels only increased after 1 week in 30 mmol/l glucose. Overnight exposure to hydrogen peroxide mimicked the effects of 30 mmol/l glucose on haeme-oxygenase 1 and c-Myc mRNA levels without activating NFB. On the other hand, the antioxidant N-acetyl-l-cysteine inhibited the stimulation of haeme-oxygenase 1 and c-Myc expression by 30 mmol/l glucose and/or hydrogen peroxide.Conclusions/interpretation In contrast to IL-1, high glucose and hydrogen peroxide do not activate NFB in cultured rat islets. It is suggested that the stimulation of islet c-Myc and haeme-oxygenase 1 expression by 30 mmol/l glucose results from activation of a distinct, probably oxidative-stress-dependent signalling pathway.     

Comparative and combined effects of transforming growth factors α and β, interleukin-1 and interferon-γ on rheumatoid synovial cell proliferation,glycolysis and prostaglandin E production

Summary Enhanced cell proliferation, glycolysis and prostaglandin E production are all characteristic features of rheumatoid synovial tissue. The interrelationships of these three cellular parameters have been examined using rheumatoid synovial fibroblasts and their responses to specific cytokines in vitro. Transforming growth factor (TGF) caused a more than threefold increase in synovial cell proliferation whilst transforming growth factor (TGF), interleukin-1 (IL-1) and interferon- (IFN-) produced only marginal changes. The combined addition of IL-1 with TGF resulted in an enhanced proliferative response comparable with that produced by TGF. Glycolysis, estimated by glucose utilisation and measurements of the glycolytic regulatory metabolite fructose 2,6-bisphosphate was significantly stimulated by TGF, IL-1 and IFN-, but less so by TGF. Prostaglandin E production was significantly increased by IL-1 to an extent much greater than that produced by TGF or TGF, although the combined addition of IL-1 with either TGF or resulted in a synergistic increase in PGE production, a response partly diminished by the addition of IFN-. These findings suggest that the extent to which a cytokine stimulates glycolysis is not consistently related to its mitogenicity, and that cytokine combinations which stimulate high levels of PGE production (a growth inhibitor) will not necessarily be associated with a reduced rate of cellular proliferation in cultured, adherent, rheumatoid synovial fibroblasts.     

Selective A2A adenosine agonist ATL-146e attenuates acute lethal liver injury in mice

Background d-Galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver injury is an experimental model of fulminant hepatic failure in which tumor necrosis factor- (TNF-) plays a pivotal role. We examined the effects of a highly selective adenosine A_2A receptor agonist (ATL-146e) on GalN/LPS-induced fulminant hepatic failure.Methods Mice were given an intraperitoneal dose of GalN (800mg/g body weight)/LPS (100ng/g body weight) with and without ATL-146e (0.01µg/kg) treatment. Liver injury was assessed biochemically and histologically. Also, TNF- levels in the serum were determined.Results The serum liver enzyme (ALT) level in vehicle-treated mice was 20960 ± 2800IU/ml and was reduced by 63% to 7800 ± 1670IU/ml by treatment with 0.01µg/kg per minute ATL146e, P < 0.05. Treatment with ATL-146e significantly reduced serum TNF- and greatly reduced inflammation assessed by histopathologic examination compared with control mice treated with GalN/LPS. ATL-146e also reduced lethality at 12h from 65% to 13%.Conclusion The present findings suggest that the highly selective adenosine A_2A receptor agonist (ATL-146e) prevents endotoxin-induced lethal liver injury by suppression of TNF- secretion.     

Hepatic expression of interferon-α in chronic hepatitis B virus infection

Hepatic expression of interferon- (IFN-) was examined by immunohistochemistry in 90 Chinese patients (M/F 67:23, age: 14–69) with a spectrum of hepatitis B virus (HBV)-related chronic liver diseases. Immunoreactive IFN- was detected in sinusoidal cells in 79 patients (88%) and in mononuclear cells in 59 patients (65.6%). Patients with active liver diseases (chronic active hepatitis, active cirrhosis,N=55) had a higher level of IFN- expression compared to patients with inactive histology (N=35; sinusoidal cells,P<0.01; mononuclear cells,P<0.01). Cytoplasmic HBsAg, nuclear HBcAg, and cytoplasmic HBcAg were detected in 79 (88%), 42 (47%), and 23 (27%) patients respectively. Expression of IFN- in mononuclear cells correlated with the expression of cytoplasmic HBcAg (P<0.05) but not with nuclear HBcAg or cytoplasmic HBsAg. When the patients were divided into four different phases according to the natural history of chronic HBV infection, patients in the active liver disease phase had higher IFN- expression compared to the immunotolerant and late phase patients (P<0.01). Using double immunohistochemical staining, both IFN- and cytoplasmic HBcAg were frequently detected near inflammatory infiltrates but no correlation existed between the hepatic expression of HBsAg and IFN-. These data indicate that IFN- is expressed in the liver in HBV-related active liver diseases and that the reported suboptimal production of IFN- by PBMC in HBV-related chronic active liver diseases may be due to a redistribution of the IFN--producing mononuclear cells into the liver, the site of inflammation.     

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.     

The antigenicity of human hemoglobins

Summary The correlation of the antigenicities among native hemoglobins and their subunit chains were investigated by the absorption of antisera and the combination of urea added immunoelectrophoresis with double diffusion. Alphachain showed identity with Hb-F but partial identity with -chain and Hb-A. Beta-chain showed identity with Hb-A but -chain and Hb-F showed partial identity with this chain. Gamma-chain showed identity only with Hb-F and its antigenicity was considered as being different from those of - or -chains.The lines of -, -and -chains were reconfirmed from the facts that the appearance of them depended always on the existence of anti-Hb-A or anti-Hb-F antibodies in the absorbed antisera and the minor component lines of

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Zusammenfassung Die Zusammenhänge der Antigenität zwischen nativen Hämoglobinen und deren Unterketten wurden mit der Absorption der Antiseren und der Kombination der Harnstoff-Immunelektrophorese und Doppeldiffusion untersucht. Die -Kette zeigte Identität mit Hb-F, aber nur partielle Identität mit der -Kette und Hb-A. Die -Kette war in ihrer Antigenität mit Hb-A identisch, die -Kette und Hb-F waren teilweise identisch mit der -Kette. Die -Kette zeigte die Identität mit Hb-F; es wird angenommen, daß ihre Antigenität verschieden von der -oder -Ketten ist.Für das Auftreten der Linien der -, - und -Ketten müssen Anti-Hb-A-oder Anti-Hb-F-Antikörper in den absorbierten Antiseren vorhanden sein, außerdem fusionieren die schwächeren Linien der Doppeldiffusion nicht mit irgendwelchen Linien der Unterketten. Auch gereinigte - oder -Ketten wurden zur Feststellung ihrer Linien benutzt.

     

New tumor necrosis factor-α-inducing protein released from<Emphasis Type="Italic"> Helicobacter pylori</Emphasis> for gastric cancer progression

Purpose To investigate the association between Helicobacter pylori infection and its inflammatory reaction in gastritis, gastric ulcer, and gastric cancer, a new tumor necrosis factor- (TNF-)-inducing protein of H. pylori was studied.Methods The HP0596 gene of H. pylori was identified as the TNF--inducing protein (Tip) gene from genome sequence of H. pylori strain 26695. Using recombinant Tip (rTip) and deleted Tip (rdel-Tip) proteins, the latter of which lacks six amino acids containing two cysteines in the N-terminal domain, we examined their activities in TNF- gene expression and NF-B activation in both Bhas 42 (v-H-ras transfected BALB/3T3) cells and mouse gastric epithelial cell line MGT-40, and in vitro transformation of Bhas 42 cells.Results Tip protein as a homodimer form (38 kDa) was found in both extracts and culture medium of various H. pylori strains. rTip significantly induced TNF- gene expression and NF-B activation in both Bhas 42 cells and MGT-40, and induced in vitro transformation of Bhas 42 cells. However, rdel-Tip did not. Treatment with MG-132, a proteasome inhibitor, inhibited translocation of NF-B p65, and abrogated TNF- induction induced by Tip protein.Conclusion Tip is a new carcinogenic factor released from H. pylori mediated through NF-B activation.     

Mechanical properties and imaging characteristics of remanufactured intravascular ultrasound catheters

Intravascular ultrasound (IVUS) as a routine device in interventional cardiology is handicapped by its high price. 19 factory-made, remanufactured IVUS catheters which consist of sterilized, used phased-array IVUS transducers inserted into a new catheter shaft were compared with 23 new IVUS catheters. 3 mechanical and 4 imaging characteristics were assessed on a 5 point scale (1 = unacceptable, 5 = excellent). Mechanical as well as imaging properties of remanufactured IVUS catheter were comparable to new catheters with excellent ratings for each of the evaluated characteristics in 38 to 94% of remanufactured catheters and 50 to 96% of new catheters. The initial failure rate for remanufactured IVUS catheters was 31.6% vs. 4.3% for new catheters (P < 0.05). Overall failure rate was 47.3% for remanufactured catheters vs. 8.7% for new catheters (P < 0.05). The failure was due to an electronic connecting problem occurring during mechanical stress to the IVUS catheter. In conclusion, remanufactured IVUS catheters offer mechanical and imaging characteristics which are comparable to new catheters. Improvements in the remanufacturing process to resolve the high rate of electronic connecting problems may make this a promising approach to substantially lower the price of IVUS catheters.     

Cellular and molecular pharmacology of 4′-epidoxorubicin in HeLa Cells

Summary The effects on cellular DNA and cytotoxicity produced by doxorubicin (Dx) and its epimer 4-epidoxorubicin (4E-Dx) were investigated in cultured HeLa cells. 4E-Dx was 2.3 times more cytotoxic than Dx after 1 h of treatment, but the two anthracyclines were equally cytotoxic on longer-term (24h) drug exposure. The different kinetics of cell lethality were related to pharmacodynamic differences between the two drugs. In fact, cellular uptake and efflux rates of 4E-Dx were faster than those of Dx on 1 h of drug exposure but similar after 24 h of treatment. 4E-Dx caused more protein-concealed strand breaks in DNA (single and double) than did Dx, despite a lower potency for free-radical formation. The degree of strand breakage by 4E-Dx was not a linear function of exposure time and, in fact, the rate of strand-break induction declined continuously with time. In contrast, Dx caused an almost linear increase in DNA single-strand breaks with time during 1 h of drug exposure; this was apparently due to its slower uptake. There was little repair of the DNA single-strand breaks produced by Dx upon postincubation for 5 h in a drug-free medium, whereas DNA lesions caused by 4E-Dx were removed with a t _1/2 of about 1.7h. These findings underline the importance of the cellular pharmacokinetics of anthracyclines in relation to their cytotoxic and DNA-damaging effects.Abbreviations used DX doxorubicin - 4E-Dx 4-epidoxorubicin     

Human Plasma Fibronectin Mediates Adhesion of U937 Cells by RGD and CS1

Fibronectin specifically binds to U937 cells (monocytic cell line) in a dose-dependent manner. The specific receptors for the RGD sequence have been identified as ₅₁ and _IIb3, and that for CS1 has been defined as ₄₁. RGDS, CS1 peptide, and two peptides together showed similar inhibitory activities on this adhesion, while the 29-kD dispase-digested fragment of the C-terminal heparin-binding domain did not. Thus, the adhesion of fibronectin to U937 cells is mainly mediated by RGDS in the cell-binding domain and CS1 in the alternatively spliced region. Flow cytometry using monoclonal antibodies revealed expressions of ₃₁, ₄₁, and ₅₁, and not expression of ₂₁. Adhesion of U937 cells to fibronectin-coated wells is specific and is inhibited by anti-₄₁ and anti- ₅₁ monoclonal antibodies. The IC-50 for anti-₅₁ antibody was almost a log lower than the value for anti-₄₁ antibody. These results demonstrated that interactions of RGDS and CS1 sequence of fibronectin with ₅₁ and ₄₁ on U937 cells were required for the adhesion of U937 cells to fibronectin. These results may provide further information to understand the mechanism(s) of tumor cell adhesion and atherogenesis.     

Schindler disease: An inherited neuroaxonal dystrophy due to α-N-acetylgalactosaminidase deficiency

Summary The clinical, pathological and biochemical features of a neuroaxonal dystrophy resulting from the deficient activity of lysosomal -N-acetylgalactosaminidase are described. This neurodegenerative disorder was recognized in two brothers who had the typical clinical manifestations and neuropathological lesions observed in patients with Seitelberger disease, the infantile form of neuroaxonal dystrophy. Axonal spheroids were observed histologically in the grey matter, and ultrastructural examination revealed the characteristic formations in dystrophic axons in the myenteric plexus and neocortex. Using a newly synthesized fluorogenic substrate, 4-methylumbelliferyl--N-acetylgalactosaminide, the markedly deficient activity of-N-acetylgalactosaminidase was demonstrated in the affected brothers while their consanguineous parents had intermediate activities, consistent with the autosomal recessive transmission of this disease. No detectable-N-acetylgalactosaminidase was seen in immunoblots using monospecific rabbit antihuman-N-acetylgalactosaminidase antibodies. Abnormally increased amounts of urinary glycopeptides were observed by high resolution thin layer chromatography. Analytical studies identified four of the accumulating urinary compounds, the blood group A trisaccharide GalNAc1 3(Fuc1 2)Gal and threeO-linked glycopeptides, GalNAc1 O-serine and -threonine, NeuNAc2 3Gal1 3(NeuNAc2 6)GalNAc1 O-serine and -threonine, and NeuNAc2 3Gal1 4GlcNAc1 6(NeuNAc2 3Gal1 3)GalNAc1 O-serine and -threonine. Of eight unrelated patients diagnosed as having infantile neuraxonal dystrophy by pathological studies, none had deficient-N-acetylgalactosaminidase activity, emphasizing the biochemical heterogeneity underlying this diagnostic entity. These findings document the first delineation of a metabolic defect in an inherited neuroaxonal dystrophy and suggest that the axonal pathology in this disorder, and perhaps in the other neuroaxonal dystrophies, results from abnormal glycoprotein metabolism involvingO-linked glycopeptides.     

Prospective evaluation of interferon-α in treatment of chronic active Crohn's disease

Several case reports suggested good effects of interferon- in patients with Crohn's disease. In addition, a decreased production of interferon- in Crohn's disease has been shownin vitro. Treatment with interferon- may activate intestinal natural killer cells and down-regulate the overproduction of inflammatory cytokines like interleukin-6 in Crohn's disease. To evaluate the clinical efficacy of interferon-, we treated 12 patients with a chronic active course of Crohn's disease with recombinant human interferon- prospectively for 24 weeks. Prednisolone was continuously tapered and discontinued at week 12. The end point of the study was the prevention of worsening of clinical symptoms defined with the Crohn's disease activity index and was monitored by acute-phase proteins, interleukin-6 serum concentrations, and endoscopy. The biochemical activity of interferon- was measured by 2,5-oligo adenylate serum levels. The end point of the study was reached in four patients (33%). In these patients the final Crohn's disease activity index was above 150, which means that they did not achieve clinical remission. All other patients (66%) did not respond to interferon- and had to be withdrawn prematurely. Interferon- did not show any beneficial effect on interleukin-6 or acute-phase protein concentrations and on endoscopic activity. The 2,5-oligo adenylate levels continuously increased during interferon therapy. Considerable side effects were noted. These results fail to demonstrate a therapeutic role of interferon- in chronic active Crohn's disease.     

Variation of lysosomal enzyme activity with gestational age in chorionic villi

Summary The activities of 14 lysosomal enzymes in chorionic villi at gestational ages of 6–12 weeks were assayed.Arylsulphatases A and B, -glucosidase and -glucuronidase activities increased with advancing gestational age. When compared with the activity in cultured amniotic fluid cells, arylsulphatase A, -galactosidase, -glucosidase, heparan N-sulphatase, -l-iduronidase, -mannosidase, neuraminidase and sphingomyelinase showed significant differences. All except -glucuronidase showed lower activity in chorionic villi than in cultured amniotic fluid cells. Prenatal diagnosis using chorionic villi was possible except for -l-iduronidase.Storage at –20°C up to 42 days did not significantly affect activity. The results emphasize the importance of using fresh or frozen age-matched control tissue for diagnosis.