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.     

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.     

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.     

Treatment of human hepatocellular carcinoma by fibroblast-mediated human interferon α gene therapy in combination with adoptive chemoimmunotherapy

The therapeutic effect of the fibroblast-mediated human interferon (IFN) gene therapy in combination with interleukin-2 (IL-2) activated killer cells (AK)/doxorubicin (i.e., adoptive chemoimmunotherapy) on nude mice bearing the human hepatocellular carcinoma (HCC) was investigated. A fibroblast cell clone (NIH3T3-IFN⁺) secreting 1024 U/ml human IFN was obtained from 14 positive clones by BMGNeo-INF DNA transfection, G418-resistant selection, limiting dilution and assay of IFN activity. After i.p. implantation of NIH3T3-IFN⁺ encapsulated into collagen, serum human IFN activity could be detected from 12 h to day 15 with a peak at 72 h. AK were prepared from human peripheral mononuclear cells costimulated in vitro by IL-2 and inactivated human SMMC 7721 HCC cells. When the NIH3T3-IFN⁺ cells were i.p. implanted into the HCC-bearing nude mice, the grown of HCC was inhibited and the survival time of the mice was extended. The growth of HCC was inhibited more obviously when AK was i.v. injected and IL-2 was i.p. injected after the NIH3T3-IFN⁺ cells had been implanted. The best therapeutic effect was achieved when NIH3T3-IFN⁺ cells were used in combination with IL-2/AK/doxorubicin. All these results suggested that the fibroblast-mediated human IFN gene therapy could be used to treat the human hepatocellular carcinoma effectively and that when used in combination with IL-2-based adoptive chemoimmunotherapy, the therapeutic effect would be better.Abbreviations IFN interferon - HCC hepatocellular carcinoma - IL-2 interleukin-2 - AK activated killer cells - Dox doxorubicin This research was supported by the National Natural Sciences Foundation of China (grant 39421009)     

Interferonγ and tumour necrosis factorα induce a synergistic antiproliferative response in human Ewing's sarcoma cells in vitro

Three human cell lines derived from Ewing's sarcoma (RM-82, VH-64, and WE-68) were investigated to establish the influence of recombinant human interferon (rhIFN) and tumour necrosis factor (rhTNF) on cell proliferation and survival and to characterize IFN and TNF receptor expression. Incorporation of [³H]thymidine into cells was inhibited by rhIFN after 24 h of incubation. Half-maximal inhibition was observed with 10–80 U/ml rhIFN. A maximal effect (50%–70% inhibition of cell proliferation) was achieved by treatment of cells with 250 U/ml rhIFN. The influence of rhTNF on proliferation was found to differ among cell lines and varied with the concentration and the duration of exposure of cells to this cytokine. In WE-68 and VH-64 cells [³H]thymidine incorporation was not affected by rhTNF up to 2000 U/ml after 96 h of incubation, where-as in RM-82 cells the incorporation was inhibited by 35% after 48 h of incubation with 100 U/ml rhTNF. However, all cell lines showed a synergistic antiproliferative response to the combination of rhIFN and rhTNF after 24 h of incubation. The human recombinant cytokines interleukin(IL)-1, IL-1, IL-2, IL-3, IL-4, IL-6 and granulocyte/macrophagecolony-stimulating factor, tested alone and in combination with rhIFN and rhTNF, had no influence on cell proliferation. Binding studies in the cell lines with¹²⁵I-rhIFN revealed a dissociation constant (K _d ) of 160–306 pM and approximately 8000–13500 receptors/cell. Binding experiments with¹²⁵I-rhTNF indicated 430–1250 receptors/cell withK _d ranging from 13 pM to 162 pM. These data indicate that, among various cytokines, only IFN and TNF are capable of potently reducing Ewing's sarcoma cell growth in vitro. Our data suggest that IFN alone or in combination with TNF may be useful in the design of novel strategies in Ewing's sarcoma therapy.     

Benzo[a]pyrene-diol-epoxide-induced anchorage-independence in diploid human fibroblasts

Summary Treatment of diploid human fibroblasts with stereoisomeric benzo[a]pyrene anti and syn diol epoxides has been shown to induce anchorage-independent clones of cells with a dose dependence and frequency [(0.5–12)×10^-4] not significantly different from mutations at the hypoxanthine-guanine phosphoribosyltransferase locus [(1–8)×10^-4] in these cells. The majority of the anchorage-independent clones that were picked retained their mutagen-induced, anchorage-independent phenotype through at least 20 generations of expansion in monolayer culture. No variant cells showing extended life-span were detected among survivors in any of the mutagen treatment groups (<1.6×10^-7 frequency). Extensive analysis of a pool of 15 cellular protooncogenes (Ha-ras, Ki-ras, N-ras, mos, fos, fes, myc, abl, sis, myb, erbA, erbB, src, raf, N-myc), using Southern and northern blot analysis, was done to determine whether mutagen-induced rearrangement, amplification or overexpression of any of these genes was responsible for the mutagen-induced, anchorage-independent phenotype. We found no evidence that the genomic arrangement or expression level of any of these genes had been altered, thus indicating that an alternative form of mutation, or an alternative gene not included in this screening was responsible for the mutagen-induced, anchorage-independent phenotype.Abbreviations used s⁶G^r 6-thioguanine-resistant - anti diol epoxide, racemic mixture of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - syn diol epoxide, racemic mixture of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene - DME Dulbecco's modified Eagle medium - hprt hypoxanthineguanine phosphoribosyltransferase     

Lack of α2-Antiplasmin Enhances ADP Induced Platelet Micro-Aggregation through the Presence of Excess Active Plasmin in Mice

Background: The role of 2-antiplasmin (2-AP) on platelet aggregation was investigated using mice deficient in 2-AP (2-AP^–/–) or using wild type mice (2-AP^+/+). Methods: Blood samples were taken from each mouse under anesthesia with ether and platelet rich plasma (PRP) was prepared. Platelet aggregation induced by various doses of ADP (0.3–30 M) was detected using a laser-light scattering (LS) system. Aggregated forms were observed using a scanning electron microscopy (SEM). Results: Dose-dependent platelet aggregation was not different in both types of mice. However, platelet micro-aggregate formation in 2-AP^–/– mice induced by low dose of ADP (1.0 M) markedly increased compared to the situation in wild type mice. Aggregated form detected by SEM showed supported data from LS analysis. When washed platelets of 2-AP^+/+ mice were resuspended in plasma of 2-AP^–/– mice, platelet micro-aggregation was also increased. On the contrary, when washed platelets of 2-AP^–/– mice were suspended in plasma of 2-AP^+/+ mice, platelet micro-aggregation did not change. In separate experiments, tPA (1.0 g/ml) was added to PRP before the stimulation of ADP. tPA had no effect on platelet aggregation in 2-AP^+/+ mice, however platelet micro-aggregation in 2-AP^–/– mice was markedly increased by the treatment with tPA. Moreover, the amount of released ATP from stimulated platelets was increased in 2-AP^–/– mice treated with tPA. Conclusion: Lack of 2-AP increased platelet micro-aggregation, and plasmin plays an important role in the formation of platelet aggregation when 2-AP knockout mice are used. Consequently, the reduction of 2-AP could be a risk factor for the activation of platelets resulting in thrombus formation.     

Changes in expression levels of genes involved in fatty acid metabolism: upregulation of all three members of the PPAR family (α, γ, δ) and the newly described adiponectin receptor 2, but not adiponectin receptor 1 during neonatal cardiac development of the rat

During neonatal cardiac development, the heart changes its substrate preference from glucose to fatty acids. The aim of this study was to investigate the changes in mRNA expression levels of genes involved in the control of cardiac fatty acid metabolism in the transition from neonatal to adult life. Methods mRNA expression levels for peroxisome proliferator activated receptor (PPAR) , and , PPAR co–factor 1 and (PGC–1 and ), 9–cis retinoc–acid–activated receptor , and (RXR , , ), 5–AMP activated protein kinase (AMPK) 1 and 2, adiponectin receptor 1 and 2 (AR 1 and AR 2) were measured in heart tissue of neonatal 0–day, 7–day and 21– day old rats. Results mRNA expression of all three members of the PPAR family were upregulated significantly from day 0 to day 21 ( +117%, +133%, +203%). In addition, m–RNA expression of all RXR isoforms increased from day 0 to day 7 ( +125%, +69%; +41%). AR 2 exhibited a small but significant increase in mRNA expression (+ 46%). Conclusions We were able to demonstrate for the first time that in addition to PPAR, also PPAR and , as well as all RXR isoforms and AR 2 are upregulated in the heart during neonatal development.Drs. Steinmetz and Quentin contributed equally to this publication     

A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease)

4-Methylumbelliferyl--iduronate 2-sulphate was synthesized and shown to be a specific substrate for the lysosomal iduronate-2-sulphate sulphatase (IDS). Fibroblasts (n = 17), leukocytes (n = 3) and plasmas (n = 9) from different MPS II patients showed <5% of mean normal IDS activity. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl--iduronate 2-sulphate requires the sequential action of IDS and -iduronidase. A normal level of -iduronidase activity was insufficient to complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl--iduronide formed by IDS. A second incubation step in the presence of excess purified -iduronidase is needed to avoid underestimation of the IDS activity.     

Diagnosis of the mucopolysaccharidoses using cultured skin fibroblasts and amniotic fluid cells

Assay of-L-iduronidase, heparin sulphamidase,N-acetyl--D-glucosaminidase, arylsulphatase B,-L-fucosidase,-glucuronidase,-galactosidase and-D-mannosidase in cultured cells is described. Activities in deficient fibroblast strains are compared to control fibroblast strains. The first case of Sanfilippo B in the United Kingdom is reported. A comparison of enzyme activities in cultured fibroblasts and amniotic fluid cells is made.     

Expression of growth factors and their receptors in human esophageal carcinomas: regulation of expression by epidermal growth factor and transforming growth factor α

The expression of mRNAs for epidermal growth factor (EGF), transforming growth factor (TGF), EGFR, platet-derived growth factor (PDGF) A and B chain, PDGF receptor (PDGFR), transforming growth factor (TGF),erbB-2 and estrogen receptor (ER) genes was first examined in 6 human esophageal carcinoma cell lines, 6 xenoplanted and 15 surgically resected esophageal carcinomas. Secondly, the effect of EGF and TGF on the expression of these genes by the TE-1 esophageal carcinoma cell line was investigated. The expression of EGF mRNA was detected in 8 (29.6%) of 27 tumors including the cell lines, whereas the TGF and EGFR genes were expressed in 21 (77.8%) and 24 (88.9%) tumors respectively. PDGF B chain and PDGFR were detected in 18 (66.7%) and 20 (74.1%), respectively, and ER mRNA was observed in 16 (59.3%) tumors. Genes for PDGF A chain and TGF and theerbB-2 gene were commonly expressed. On the other hand, exogenous EGF and TGF stimulated the expressions offos andmyc genes by TE-1 cells. The expression of mRNAs for TGF, PDGF A and B chain and theerbB-2 genes was also increased after treatment with EGF. TGF increased the accumulation of mRNAs for EGF, Moreover, the expression of mRNAs for interstitial collagenase, stromelysin and type IV collagenase was increased after EGF or TGF treatment. These results indicate that EGF and TGF may regulate the multi-growth-factor receptor expression and may play a central role for tumor invasion and metastasis as autocrine modulators for human esophageal carcinoma.Abbreviations EGF epidermal growth factor - TGF transforming growth factor - PDGF platelet-derived growth factor - ER estrogen receptor - R receptor     

Heterophilic interactions between cell adhesion molecule L1 and α<Subscript>v</Subscript> β<Subscript>3</Subscript>-integrin induce HUVEC process extension <Emphasis Type="Italic">in vitro</Emphasis> and angiogenesis <Emphasis Type="Italic">in vivo</Emphasis>

Cell adhesion molecule L1 was implicated in angiogenic processes, tumor formation and metastasis. Here, we provide evidence that the sixth Ig-like domain of L1 (L1Ig6) interacts with _{v 3} to induce process extension of human umbilical vein endothelial cells (HUVECs) in vitro and angiogenesis in vivo. HUVECs formed network-like structures on full-length L1 or L1Ig6 substrates comparable to structures found on matrigel. In the presence of mab _{v 3} or cyclic RGD, apoptosis was induced. In fibrin matrices where L1Ig6 was covalently incorporated, HUVECs formed multicellular and hollow processes through interactions between cell-surface _{v 3} and RGD-sites of matrix-immobilized L1Ig6. No such processes were induced by L1Ig6 having non-functional RDG-sites, or in the presence of mab _{v 3} or cyclic RGD. In those matrices, increased apoptosis was found. Co-immunoprecipitation of L1 or L1Ig6 with _{v 3} suggests close interactions. Furthermore, L1Ig6 stimulated HUVECs showed increased tyrosine phosphorylation of _{v 3} and phosphorylation of MAP kinases (ERK1 and ERK2) but not AKT indicating specific activation of _v and _{v 3} followed by activation of downstream kinases. Application of L1Ig6-modified fibrin matrices on CAMs induced 50–60% increased _v and _v3 protein expression and in vivo angiogenesis indicated by ~50% increased mean vascular length density. The results demonstrate angiogenic potential of L1Ig6 involving ligation and activation of _v3     

Serum cytokines in patients with rheumatoid arthritis

Serum cytokines such as interleukin 1 (IL-1), interferon (IFN-), and tumor necrosis factor (TNF) were measured in 40 patients with rheumatoid arthritis (RA). In the 40 patients studied, serum IL-1 was detected in 5 patients, IFN- in 10 patients, and TNF in 20 patients. The IL-1-positive group showed increased values of activity indices compared to the IL-1-negative group. Values of serum IFN- correlated well with the number of peripheral blood lymphocytes and CD3⁺ cells and with the percentage of CD3⁺ CD26⁺ cells. Values of serum TNF correlated positively with the number of peripheral blood monocytes and the percentage of CD3⁺ HLA-DR⁺ and CD3⁺ CD25⁺ cells. These results indicated that serum IL-1 in RA patients reflects the activity of RA, while the serum IFN- and TNF in RA patients may be related to circulating activated lymphocytes and monocytes, respectively.     

Increased serum levels of transforming growth factor-α in patients with colorectal cancer

PURPOSE: This study was conducted to investigate the serum levels of transforming growth factor- in patients with colorectal cancer and to investigate the clinical significance of these levels in association with tumor stage and histologic differentiation. Also, serum levels of transforming growth factor- were measured after curative surgical resection. METHODS: Serum levels of transforming growth factor- were measured in 42 consecutive patients with colorectal cancer before surgery, in 21 patients after surgical resection (part of the 42 preoperative patients), and in 20 healthy volunteers. We used TGF- Assay. RESULTS: Serum levels of transforming growth factor- in patients with colorectal cancer were significantly higher than in the healthy control group (P=0.001). Significant elevations in serum levels of transforming growth factor- were found in 50 percent (21/42) of patients with colorectal cancer when the mean + 2 standard deviations (80.4 pg/ml) of the control group were used as the upper limit of the normal range. Serum levels of transforming growth factor- tended to decrease with increasing tumor size (n=31;r=–0.52;P=0.002). Serum levels of transforming growth factor- before surgery (89.7±44.4 pg/ml; n=21) significantly decreased to 60.3±19.8 pg/ml after surgical resections of tumors (P=0.017). Serum levels of transforming growth factor- completely decreased to the same serum levels of the control group after surgical resections in all patients who had serum levels of transforming growth factor- greater than mean + 2 standard deviations (80.4 pg/ml) of the control group preoperatively (n=11;P=0.002). CONCLUSIONS: Levels of preoperative transforming growth factor- in patients with colorectal cancer appeared to be higher than levels measured in control subjects. Serum levels of transforming growth factor- before surgery significantly decreased after surgical resections of tumors. Additional studies are warranted to determine if serum levels of transforming growth factor- may be useful as a potential biomarker in the management of patients with colorectal cancer.Supported by a grant from Ewha Womans University Mokdong Hospital, Seoul, Korea.Read at the meeting of The American Society of Colon and Rectal Surgeons, Philadelphia, Pennsylvania, June 22 to 26, 1997.     

Na,K-ATPase isoform gene expression in normal and hypertrophied dog heart

Objectives The catalytic subunit of the sodium-potassium ATPase, the target of digitalis glycosides, has three isoforms is tissuespecific and developmentally regulated. While the effect of pressure overload on Na, K-ATPase isoform expression has been studied in rodent heart, there are no systematic data on this question in hearts of larger animals, which differ from those of rodents both in isoform composition and in glycoside sensitivity. Thus, we investigated the expression of Na, K-ATPase isoforms in normal dog heart; we also examined the effect of experimental left ventricular hypertrophy on isoform expression.Methods hypertrophy was produced by aortic banding. Expression was assessed by quantitative Northern and Western blotting, immuno-fluorescence, and³H-ouabain binding.Results RNA blotting indicated that the 3 isoform represented 11% of Na, K-ATPase mRNA in normal dog LV. Normal dog LV expressed 1 and 3 protein, but no detectable 2; immunoreactive 1 and 3 protein were also present in Purkinje fibers. There was a statistically significant decrease in total expression of all isoform mRNA's in hypertrophied dog LV, resulting in a greater proportion of 1. The expression level of the 3 isoform mRNA and protein was lower in hypertrophied hearts.Conclusions These results indicate a greater proportion of 1 isoform pumps in experimental canine hypertrophy. Thus, shifts in Na, K-ATPase isoforms occur in pressure-overloaded heart in large animals as well as rodents.     

Interactive influences of peroxisome proliferator-activated receptor α activation and glucocorticoids on pancreatic beta cell compensation in insulin resistance induced by dietary saturated fat in the rat

Aims/hypothesis We sought to elucidate whether excess glucocorticoids and increased dietary lipids act synergistically to impair glucose tolerance and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR) has an adverse or beneficial effect on glucose tolerance.Methods Dexamethasone (100 g kg^–1 body weight day^–1; 5 days) was administered to insulin-resistant rats fed a high-saturated-fat (HF) diet for 4weeks. The PPAR agonist WY14643 was administered (50 mg kg^–1 body weight intraperitoneally) 24 h before sampling. Glucose-stimulated insulin secretion (GSIS) was assessed in vivo after an acute glucose bolus injection, and in vitro using step-up and step-down islet perifusions.Results Although neither PPAR activation nor dexamethasone alone affected fasting glycaemia in the HF group, dexamethasone in combination with PPAR activation elicited marked postabsorptive hyperglycaemia. Dexamethasone treatment of HF rats had little effect on GSIS after an acute glucose challenge in vivo, but induced glucose intolerance. PPAR activation augmented GSIS in dexamethasone-treated HF rats in vivo, restoring glucose tolerance. Contrasting with data obtained in vivo, greatly enhanced peak rates of GSIS were observed ex vivo in perifusions of islets from dexamethasone-treated HF rats compared with those from untreated HF rats, an effect attenuated by antecedent PPAR activation.Conclusions/interpretation The study demonstrates that glucocorticoid excess precipitates the development of glucose intolerance in rats maintained on a high-saturated-fat diet. It does this by interrupting the negative feedback loop between insulin sensitivity and secretion in vivo, such that further enhancement of compensatory insulin secretion is not possible. PPAR activation restores the coupling between insulin secretion and action.     

Triplication (/ααα<Superscript>Anti3.7</Superscript>) or deletion (-α<Superscript>3.7</Superscript>/) association in Argentinian β-thalassemic carriers

Prevalence of alpha gene triplication or deletion in -thalassemia carriers was studied in 109 unrelated individuals in Rosario, Argentina. In different populations -^3.7 allele presents a higher prevalence than ^anti3.7; thus, -thalassemia associated with -thalassemia is more frequently observed. Nevertheless, this event was detected in only one case (0.9%), while the association with alpha triplication was present in two subjects (1.8%).     

A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease type D (MPS IIID)

Summary 4-Methylumbelliferyl--N-acetylglucosamine 6-sulphate was synthesized and shown to be a substrate for the lysosomalN-acetylglucosamine-6-sulphate sulphatase (GlcNAc-6S sulphatase). Fibroblasts and leukocytes from 3 different Sanfilippo D patients showed <1% of mean normal GlcNAc-6S sulphatase activity. The enzymatic liberation of the fluorochrome from 4-methyl-umbelliferyl--N-acetylglucosamine 6-sulphate requires the sequential action of the GlcNAc-6S sulphatase and -N-acetylglucosaminidase. A normal level of -N-acetylglucosaminidase activity was insufficient to complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl--N-acetylgluco-saminide formed by the GlcNAc-6S sulphatase. A second incubation in the presence of excess -N-acetyglucosaminidase is needed to avoid underestimation of the GlcNAc-6S sulphatase activity.     

Uptake and metabolism of radiolabelled GM1-ganglioside in skin fibroblasts from controls and patients with GM1-gangliosidosis

Summary The uptake and metabolism of [3-³H-sphingosine]GM1-ganglioside was measured in cultured skin fibroblasts from controls and patients with infantile, juvenile and adult GM1-gangliosidosis. When dissolved in medium with phosphatidylserine, GM1-ganglioside was efficiently taken up by cultured skin fibroblasts and transferred into lysosomes. A linear increase in GM1-ganglioside endocytosis was shown with phosphatidylserine concentrations of up to 40m/ml. A pulse-chase study revealed that [³H]GM1-ganglioside was metabolized to GM2-ganglioside, GM3-ganglioside, ceramide dihexoside, ceramide monohexoside, ceramide and sphingosine. Sphingosine was recycled to sphingomyelin. In a 20-h pulse study, cell lines from patients with GM1-gangliosidosis of infantile, juvenile and adult types hydrolysed 2–5%, 20–44% and 54–58% of the total endocytosed GM1-ganglioside respectively. These values were lower than in control cells (64.17 ± 5.43% (n=10)). The hydrolysis rates of exogenous [³H]GM1-ganglioside in cultured fibroblasts from patients with various types of GM1-gangliosidosis closely reflected the clinical severity.Abbreviations GM1 Gal13GalNAc14(NeuAc23)Gal14Galc-1-ceramide - GM2 GalNAc14(NeuAc23)Gal14Glc-1-ceramide - GM3 NeuAc23Gal14Glc-1-ceramide - CDH Gal14Glc-1-ceramide     

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.