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K Harzer

Publications and source records attributed to K Harzer.

At least 37 records · Page 2Linked to original sources
PubMed1997

Acidic sphingomyelinase mediates entry of N. gonorrhoeae into nonphagocytic cells.

Invasion of human mucosal cells by N. gonorrhoeae via the binding to heparansulfate proteoglycan receptors is considered a crucial event of the infection. Using different human epithelial cells and primary fibroblasts, we show here an activation of the phosphatidylcholine-specific phospholipase C (PC-PLC) and acidic sphingomyelinase (ASM) by N. gonorrhoeae, resulting in the release of diacylglycerol and ceramide. Genetic and/or pharmacological blockade of ASM and PC-PLC cause inhibition of cellular invasion by N. gonorrhoeae. Complementation of ASM-deficient fibroblasts from Niemann-Pick disease patients restored N. gonorrhoeae-induced signaling and entry processes. The activation of PC-PLC and ASM, therefore, is an essential requirement for the entry of N. gonorrhoeae into distinct nonphagocytic human cell types including several epithelial cells and primary fibroblasts.

Bacterial Proteins↗
PubMed1997

Saposins (sap) A and C activate the degradation of galactosylceramide in living cells.

In loading tests using galactosylceramide which had been labelled with tritium in the ceramide moiety, living skin fibroblast lines derived from the original prosaposin-deficient patients had a markedly reduced capacity to degrade galactosylceramide. The hydrolysis of galactosylceramide could be partially restored in these cells, up to about half the normal rate, by adding pure saposin A, pure saposin C, or a mixture of these saposins to the culture medium. By contrast, saposins B and D had little effect on galactosylceramide hydrolysis in the prosaposin-deficient cells. Cells from beta-galactocerebrosidase-deficient (Krabbe) patients had a relatively high residual galactosylceramide degradation, which was similar to the rate observed for prosaposin-deficient cells in the presence of saposin A or C. An SV40-transformed fibroblast line from the original saposin C-deficient patient, where saposin A is not affected, showed normal degradation of galactosylceramide. The findings support the hypothesis, which was deduced originally from in vitro experiments, that saposins A and C are the in vivo activators of galactosylceramide degradation. Although the results with saposin C-deficient fibroblasts suggest that the presence of only saposin A allows galactosylceramide breakdown to proceed at a normal rate in fibroblasts, it remains to be determined whether saposins A and C can substitute for each other with respect to their effects on galactosylceramide metabolism in the whole organism.

Amidohydrolases↗
PubMed1997

Leukodystrophy incidence in Germany.

Through a survey of all departments of pediatrics, neurology and neuropathology in Germany, we calculated the incidence of all major forms of leukodystrophy. Only diagnoses based on specific biochemical tests in association with typical findings and/or neuroradiologically proven white matter involvement were accepted. In accordance with these strict criteria, 617 cases of leukodystrophy were found (incidence of all forms: app. 2.0/100,000). Minimal incidence was estimated at 0.8/100,000 for adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN), 0.6/100,000 for metachromatic leukodystrophy (MLD), and 0.6/100,000 for Krabbe disease. Thus ALD/AMN is apparently underdiagnosed in Germany. A considerable proportion of leukodystrophies could not be classified in spite of adequate diagnostic procedures in experienced centers.

Adolescent↗
PubMed1997

Model SV40-transformed fibroblast lines for metabolic studies of human prosaposin and acid ceramidase deficiencies.

Skin fibroblasts from patients with Farber disease (acid ceramidase deficiency) and from two siblings of the only known family affected with prosaposin deficiency were transformed by transfection with a plasmid carrying the SV40 large T antigen. The prosaposin-deficient transformed cell lines conserved their original metabolic defects, and in particular they were free of detectable immunoreactivity when using anti-saposin B and anti-saposin C antisera. Ultrastructurally, the cells contained heterogeneous lysosomal storage products. As found for their parental cell lines, the SV40-transformed fibroblasts exhibited deficient in vitro activities of lysosomal ceramidase and beta-galactosylceramidase, but a normal activity of acid sphingomyelinase. As observed for SV40-transformed fibroblasts from Farber disease, degradation of radioactive glucosylceramide or low density lipoprotein-associated radiolabelled sphingomyelin by the prosaposin-deficient cells in situ showed a clear impairment in the turnover of lysosomal ceramide. Ceramide storage in prosaposin-deficient cells was also demonstrated by ceramide mass determination. In contrast to acid ceramidase deficient cells, both the accumulation of ceramide and the reduced in vitro activity of acid ceramidase in cells from prosaposin deficiency could be corrected by addition of purified saposin D. The data confirm that prosaposin is required for lysosomal ceramide degradation, but not for sphingomyelin turnover. The SV40-transformed fibroblasts will be useful for pathophysiological studies on human prosaposin deficiency.

Acid Ceramidase↗
PubMed1997

Hydrops fetalis: manifestation in lysosomal storage diseases including Farber disease.

UNLABELLED: The authors describe a case of disseminated lipogranulomatosis (Farber disease) presenting as nonimmune hydrops fetalis. This is the tenth lysosomal storage disease which can show this clinical manifestation. The literature is reviewed for all hydrops cases associated with lysosomal storage diseases. CONCLUSION: Although rare, the lysosomal storage diseases collectively are significant causes of nonimmune hydrops and appropriate investigations are required in all cases of unexplained hydrops fetalis.

Humans↗
PubMed1997

Progressive cerebellar ataxia, proximal neurogenic weakness and ocular motor disturbances: hexosaminidase A deficiency with late clinical onset in four siblings.

Tay-Sachs disease is a genetically determined neurodegenerative disorder, resulting from mutations of the hexosaminidase (Hex) A gene coding for the alpha-subunit of beta-D-N-acetyl-hexosaminidase. Clinically, there is severe encephalomyelopathy leading to death within the first few years of life. Hex A activity is usually absent in tissue and body fluids of these patients. Juvenile and adult Hex A deficiencies are less severe but rare variants with some residual Hex A activity. All these variants are most prevalent among Ashkenazi Jews. We describe a non-Jewish family in which four adult brothers and sisters had markedly reduced Hex A activities and onset of symptoms in the second decade of life. The phenotypical expression was remarkably homogeneous, consisting in a combination of slowly progressive motor neuron disease, ataxia and ocular motor disturbances. None of the patients were demented at this stage of their illness. Magnetic resonance studies showed severe cerebellar atrophy, but were otherwise normal. Hex A deficiency was established by biochemical measurements in the serum and skin fibroblasts using the fluorogenic substrates 4-MUG and 4-MUGS as well as by gel electrophoresis. Molecular genetic studies revealed that the patients are compound heterozygotes for the 'adult' mutation Gly269 --> Ser and the 'infantile' 4-base insertion in exon 11 of the Hex A gene.

Adult↗
PubMed1996

Neuraminidase assay in cultured human fibroblasts: in situ versus in vitro procedures.

Further investigations have been carried out to characterize a published procedure of neuraminidase assay, in which the activity is measured directly on the cell culture layer. The pH optimum was 4.0. A Vmax value of 130 nmol/mg/h and a K(m) of 0.3 mmol/l were found. During incubation in the acid buffer, arylsulphatase activity was released into the medium, whereas neuraminidase activity remained attached to the cells. The in situ method allowed an unequivocal diagnosis of primary and secondary neuraminidase deficiencies. Because of its simplicity and reliability, the method appears useful as a routine method in clinical laboratories.

Adult↗
PubMed1996

Methylamine accumulation in cultured cells as a measure of the aqueous storage compartment in the laboratory diagnosis of genetic lysosomal diseases.

Intracellular accumulation of the lysosomotropic compound [14C]methylamine was used to estimate the size of the lysosomal compartment in fibroblasts cultured from patients with a variety of lysosomal storage diseases. In previous work from our laboratory, it was shown that methylamine accumulation was significantly increased in diseases with infantile or juvenile onset and storage of predominantly water-soluble material such as in the mucopolysaccharidoses, mucolipidoses, and oligosaccharidoses. In the present study, methylamine incorporation was abnormally increased in cells from patients with glycogenosis type II and with Niemann-Pick type C disease, whereas it was normal in other sphingolipidoses and in the late-infantile and juvenile forms of neuronal ceroid lipofuscinoses. The methylamine test was also checked regarding its potential use for prenatal diagnostic testing. In model systems with cultured amniotic or chorionic villus cells, lysosomal storage was experimentally induced by the cathepsin inhibitor leupeptin and was readily detected when compared to untreated controls. Cultured amniotic cells from a fetus with mucopolysaccharidosis II were found to incorporate significantly higher amounts of [14C]methylamine than the normal controls. The results indicate that the methylamine accumulation method is an additional tool in the diagnosis and prenatal diagnosis of lysosomal diseases with abnormal storage of water-soluble material.

Amnion↗
PubMed1996

A simple method for screening for Farber disease on cultured skin fibroblasts.

Farber disease is an inborn lysosomal storage disorder characterized by accumulation of ceramide in the patient's tissues due to the deficient activity of acid ceramidase. Currently, confirmation of the diagnosis is performed in an extremely limited number of laboratories. We therefore developed a procedure which does not require any particular sphingolipid substrate and is based on the quantitation of ceramide levels in cultured skin fibroblasts. In the method we devised, the ceramide present in cellular lipid extracts subjected to mild alkaline hydrolysis was quantified using the commercially available diacylglycerol kinase kit. We show that both primary cultures of skin fibroblasts and SV40-transformed fibroblasts derived from a series of patients with Farber disease exhibit ceramide excess as compared to their normal counterparts (2345-17 153 pmol/mg cell protein in Farber cells vs. 432-1298 pmol/mg cell protein in controls). Use of this simple method should greatly facilitate the biochemical diagnosis of Farber disease.

Acid Ceramidase↗
PubMed1996

Ouchterlony double immunodiffusion method demonstrates absence of ferritin immunoreactivity in visceral organs from nine patients with Niemann-Pick disease type C.

Ouchterlony double immunodiffusion clearly demonstrated absence of ferritin, the principal iron storage protein, in spleen and/or liver extracts from nine patients with Niemann-Pick disease type C (NPC). The patients died from different clinical forms of this disease of still unknown etiology. The absence of ferritin immunoreactivity was shown using two different antisera raised in rabbits against ferritin from human spleen or liver, organs which predominantly contain light chain subunits (L-ferritin). A diagnostic double immunodiffusion assay of ferritin is, therefore, feasible with small amounts of NPC liver tissue, e.g., needle biopsy specimens. Furthermore, SDS-polyacrylamide gel electrophoresis after Coomassie blue staining revealed an almost complete absence of the L-ferritin protein band in crude spleen heat extracts from two NPC patients. The absence of visceral ferritin in all nine patients studied is suggestive of a biochemical abnormality that is as characteristic as the known impairment of cellular trafficking of LDL-derived cholesterol in this complex lysosomal storage disorder. According to recent data a relationship exists between ferritin-dependent lipid peroxidation and oxidative modification of LDL. We suggest that deficiency of the antioxidant ferritin-whatever the nature of this deficiency might be-could lead to uncontrolled LDL oxidation with subsequent multisubstrate lipidosis in NPC disease.

Adolescent↗
PubMed1996

[Disseminated lipogranulomatosis (Farber disease) with hydrops fetalis].

We report on a female preterm infant of 29 weeks' gestation with severe hydrops fetalis who died 3 days post natum as a result of disseminated intravascular coagulation. Autopsy findings included anasarca, bilateral pleural effusions, ascites and hepatosplenomegaly as well as multiple, up to pinhead sized, white granulomas on the surface of liver, spleen and lungs. Microscopy revealed storage macrophages of the reticuloendothelial system, especially in liver, spleen and bone marrow, the lymphatic organs, the salivary glands, the thyroid gland and the suprarenal medulla. Cerebrum, heart, kidneys, intestines and placenta were not afflicted. Atrophy of the lymphatic compartments in the spleen, lymph nodes and thymus, as well as disorder of the liver texture, are presumably a secondary result. The diagnosis of Farber's disease was established biochemically by the demonstration of ceramide depositions in the spleen, and in fibroblast cultures in situ by the accumulation of ceramide released from loaded radioactive glucosylceramide. Ultrastructurally, corresponding storage lysosomes were found in macrophages. To our knowledge this is the first account of Farber's disease in a preterm infant with hydrops fetalis.

Amidohydrolases↗
PubMed1996

Differentiation of rare leukodystrophies by post-mortem morphological and biochemical studies: female adrenoleukodystrophy-like disease and late-onset Krabbe disease.

Two 6-year-old patients with clinical signs of leukodystrophy had no nosological diagnoses in vivo. Neuropathological studies revealed scavenger cells to be clustered in perivascular regions of the demyelinated brains. Histochemical and ultrastructural details of the non-metachromatic storage macrophages suggested lipid storage and prompted a biochemical analysis of cerebral tissue. The detection of increased amounts of very long chain fatty acids in the cholesterol ester fraction from formalin tissue in one patient was consistent with a diagnosis of an adrenoleukodystrophy-like condition, while the marked reduction in beta-galactocerebrosidase activity in a frozen brain sample of the second patient indicate Krabbe disease. The diagnostic potential of post-mortem studies in rare leukodystrophies is addressed.

Adrenoleukodystrophy↗
PubMed1995

Significance of lipopigments with fingerprint profiles in eccrine sweat gland epithelial cells.

Lipopigments with fingerprint profiles in eccrine sweat gland epithelial cells are regular findings in childhood NCL. They have also been described in adult NCL (ANCL) a few times, but not consistently. However, they have been considered nonspecific when not matched by similar abnormal profiles in noneccrine sweat gland epithelial cells. These conflicting reports may pose a diagnostic dilemma as outlined in the following 2 examples. Patient 1 is a 20-year-old man who developed severe tetraparesis and dementia over 2 years. Electroencephalogram was abnormal with epileptiform discharges. The patient died at age 21 years without autopsy; no other relatives are known to have a similar disease. Patient 2, a 49-year-old woman, developed ataxia and gait abnormalities when 44 years old, and, later, psychosis and dementia. The patient is still alive; no other family members are similarly affected. Both patient lacked evidence of a retinopathy clinically, funduscopically, and by electroretinography. Both patients showed lipopigments within secretory eccrine sweat gland epithelial cells which harbored unequivocal fingerprint profiles, but not within noneccrine sweat gland cells. Regular lipofuscin was observed in other cells, including skeletal muscle fibers of patient 2.

Adult↗
PubMed1995

Sulfatide and sphingomyelin loading of living cells as tools for the study of ceramide turnover by lysosomal ceramidase--implications for the diagnosis of Farber disease.

The ceramide turnover by lysosomal ceramidase in intact, living cells was investigated by loading radiolabeled sulfatide or sphingomyelin in situ on skin fibroblasts and lymphoid cells. The cells originated from normal individuals and from patients with acid ceramidase deficiency (Farber disease). While fibroblasts from individuals with Farber disease exhibited some impairment in the degradation of the ceramide produced by sulfatide hydrolysis, lymphoid cells from individuals with Farber disease metabolized the ceramide as readily as did normal cells, suggesting the existence in lymphoid cells of a non-lysosomal degradation pathway for the sulfatide-derived ceramide. In contrast, sphingomyelin loading in the presence of serum showed a considerably decreased turnover of ceramide in both fibroblasts and lymphoid cells from individuals with Farber disease. Further methodologic variation led to the use of LDL-associated radioactive sphingomyelin; LDL-association promoted the targeting of exogenous sphingomyelin to lysosomes. As a result, an almost complete deficiency of ceramide degradation was found in cells from severely affected patients with Farber disease. Our data with this novel method show that sphingomyelin loading of intact living cells is a simple, alternative means for determining ceramide degradation by lysosomal ceramidase and for diagnosing Farber disease.

Acid Ceramidase↗
PubMed1995

Deficient ferritin immunoreactivity in visceral organs from four patients with Niemann-Pick disease type C.

Ferritin, the major iron storage protein, was found to be undetectable on immunoblot analysis of spleen and liver extracts from four patients with Niemann-Pick disease type C (NPC). The patients had died from different clinical forms of this storage disease of still unknown etiology. The absence of ferritin immunoreactivity was shown using two different antisera, raised in rabbits, against ferritin from human spleen containing predominantly light-chain subunits (L-ferritin). Further evidence of absent L-ferritin in visceral tissues was provided by immunohistochemical studies performed in one of the four NPC patients. However, heavy-chain and light-chain ferritin mRNAs could be identified in cultured fibroblasts from this patient. The finding of deficient ferritin immunoreactivity is suggestive of an additional biochemical abnormality that is as marked as the known impairment of the transport of exogenously derived cholesterol in this complex lysosomal storage disorder.

Animals↗
PubMed1995

A family with combined Farber and Sandhoff, isolated Sandhoff and isolated fetal Farber disease: postnatal exclusion and prenatal diagnosis of Farber disease using lipid loading tests on intact cultured cells.

UNLABELLED: An earlier described patient with combined sphingolipidoses, Farber and Sandhoff disease, had two healthy older brothers and two further sibs, one with Sandhoff disease and one (a fetus) with Farber disease, showing segregation of the respective genes. The prenatal diagnosis in the latter was performed using lipid (sphingomyelin and glucosylceramide) loading tests on the cultured amniotic fluid cells. After 1-3 days of incubation the cells' lipid extract revealed radioactive ceramide to be released and highly accumulated. The deficiency in acid ceramidase was known from the patient with the combined diseases. Confirmation of the prenatal Farber diagnosis was done by similar loading tests on the fetal fibroblasts and by analysis of liver lipids of the less than 18-week-old fetus. CONCLUSION: This is the first report on the use of lipid loading tests on intact cultured cells for prenatal diagnosis of Farber disease. The postnatal diagnosis of Farber disease can also be readily made using those tests, as was shown in four further cases.

Abortion, Eugenic↗
PubMed1995

Neurodegenerative course in ceramidase deficiency (Farber disease) correlates with the residual lysosomal ceramide turnover in cultured living patient cells.

Farber's lipogranulomatosis is an inborn lipid storage disease characterized by tissue accumulation of ceramide due to deficient activity of lysosomal ceramidase. Symptoms include painful swelling of joints, subcutaneous nodules, a hoarse cry, hepatosplenomegaly and nervous system dysfunction of markedly variable degree. In most cases the neural dysfunction rather than the general dystrophy, seems to limit the duration of Farber disease. We examined whether the severity can be shown as a function of ceramide turnover by lysosomal ceramidase. The lysosomal degradation of sphingomyelin-derived ceramide was studied in situ in patient skin fibroblasts and lymphoid cells loaded with LDL-associated radioactive sphingomyelin. We could show for the first time a significant correlation between the ceramide accumulated in situ and the severity of Farber disease. Our method provides an alternative means for determining ceramide degradation by lysosomal ceramidase, but in intact cells. The relatively simple method is at least of the same diagnostic use for Farber disease as the in vitro assay of acid ceramidase using cell homogenates and may also have some prognostic use.

Acid Ceramidase↗