Brain RNA polymerase and nucleolar structure in perinatal asphyxia of the rat.

Ribosomes are integral constitutens of the protein synthesis machinery. Polymerase I (POL I) is located in the nucleolus and transcribes the large ribosomal genes. POL I activity is decreased in ischemia but nothing is known so far on POL I in perinatal asphyxia. We investigated the involvement of POL I in a well-documented model of graded systemic asphyxia at the level of activity, mRNA, protein, and morphology. Caeserean section was performed at the 21st day of gestation. Rat pups still in the uterus horns were immerged in a water bath for asphyctic periods from 5-20 min. Brain was taken for measurement of pH, nuclear POL I activity, and mRNA steady state, and protein levels of RPA40, an essential subunit of POL I and III. Silver staining and transmission electron microscopy with morphometry when appropriate were used to examine the nucleolus. Brain pH and nuclear POL I activity decreased with the length of the asphyctic period while POL-I mRNA and protein levels were unchanged. Accompanying the decrease in brain pH we found significant changes of nucleolar structure in the course of perinatal asphyxia at the light and electron microscopic level. As early as ten min following the asphyctic insult, morphological disintegration of the nucleolus was observed. The changes became more dramatic with longer duration of perinatal asphyxia. We conclude that severe acidosis may be responsible for decreased POL activity and for disintegration of nucleoli in neurons. This condition may lower the ribosome content in neonatal neurons and impair protein synthesis.

Increased steady state mRNA levels of DNA-repair genes XRCC1, ERCC2 and ERCC3 in brain of patients with Down syndrome.

Although deficient DNA-repair was proposed for neurodegenerative disorders including Down Syndrome (DS), repair genes for nucleotide excision repair or X-ray repair have not been studied in brain yet. As one of the hypotheses for the pathogenesis of brain damage in DS is oxidative stress and cells of patients with DS are more susceptible to ionizing irradiation, we decided to study ERCC2, ERCC3 and XRCC1, representatives of repair genes known to be involved in the repair of oxidative DNA-damage. mRNA steady state levels of ERCC2, ERCC3, XRCC1, a transcription activator (TAF-DBP) and an elongation factor (EF1A) were determined and normalized versus the housekeeping gene beta-actin in five individual brain regions of nine controls and nine DS patients. Although different in the individual regions, DNA-repair genes were consistently higher in temporal, parietal and occipital lobes of patients with DS accompanied by comparable changes of TFA-DBP and EF1A. Our results are the first to describe DNA-repair gene patterns in human brain regions providing the basis for further studies in this area. We showed that DNA-repair genes ERCC2 and ERCC3 (excision-repair-cross-complementing-) for nucleotide excision repair and XRCC1 (X-ray-repair-cross-complementing-) for X-ray-repair, were increased at the transcriptional level with the possible biological meaning that this increase may be compatible with permanent (oxidative?) DNA damage.

Apoptosis-associated proteins p53 and APO-1/Fas (CD95) in brains of adult patients with Down syndrome.

In Down syndrome (DS), enhanced apoptosis (programmed cell death) may play a role in the pathogenesis of characteristic mental retardation and precocious dementia of Alzheimer-type. Upregulation of p53 and APO-1/Fas (CD95) precedes apoptosis in many cell types, and a potential role for these molecules has already been demonstrated in Alzheimer's disease (AD) and several other neurodegenerative diseases. We measured p53 and APO-1/Fas (CD95) protein in four different regions of cerebral cortex and cerebellum in nine adult DS patients with Alzheimer-like neuropathologic lesions compared to nine controls. Quantitative ELISA demonstrated higher frontal lobe (mean+/-SD: 0.10+0.035 vs. 0.041+/-0.016 ng/mg protein), temporal lobe (0.062+/-0.021 vs. 0.032+/-0.019 ng/mg protein) and cerebellar levels (0.078+/-0.030 vs. 0.039+/-0.032 ng/mg protein) of p53 protein, and higher temporal lobe (mean+/-SD: 12.3+/-4.3 vs. 5.3+/-2.0 U/mg protein) and cerebellar levels (5.9+/-1.4 vs. 2.9+/-1.1 U/mg protein) of APO-1/Fas (CD95) protein. The results suggest that p53- or APO-1/Fas (CD95)-associated apoptosis may be an important feature of neurodegeneration in DS.

Brain vasopressin levels in Down syndrome and Alzheimer's disease.

Performing gene hunting in Down Syndrome fetal brain we detected an overexpressed sequence highly homologous to the human vasopressin gene. As this neuropeptide may be involved in the pathogenetic mechanism and, moreover, was described to play a role in memory and learning, we decided to study the brain gene product level in Down Syndrome (DS), controls and patients with Alzheimer's disease (AD). Subtractive hybridization was used to study the differential expression between steady state mRNA levels in fetal brain of DS and controls at the 23rd week of gestation. A radioimmunological method was used to determine vasopressin (AVP) in five brain regions of each 9 aged DS brains, 9 brains with AD and 9 control individuals, obtained from brain bank. An overexpressed nucleic acid sequence with 91% homology to the vasopressin gene was detected in both fetal brains with DS. AVP levels in controls were of the order cerebellum>occipital>frontal>parietal>temporal lobe and were significantly higher in temporal lobe and lower in cerebellum of patients with DS. AVP levels in brain of AD patients were also significantly increased in temporal lobe but were not reduced in cerebellum. The biological meaning of increased AVP remain unclear but may be linked to the neurodegenerative processes, proposed to be similar in both disorders. Data from gene hunting in fetal DS brain along with our data on aged DS and AD patients suggest the early involvement of AVP in the pathomechanism accompanying cholinergic, monoaminergic and neuropeptidergic deficits described in DS and AD.

N-acetylneuraminic acids (nana): a potential key in renal calculogenesis.

N-Acetylneuraminic acids (NANA) promote binding of calcium ions to macromolecules and cells, increase the intrinsic viscosity of glycoproteins and facilitate gel formation in water. Since these properties are crucial in urinary calculogenesis, we evaluated NANA levels in urine and serum as well as their expression in kidney tissues. Using a modified thiobarbituric acid assay, the evaluation of free and bound NANA in 24-h urine samples revealed a ratio of 1.87 in 33 non-stone-formers but a reversed ratio of 0.84 in 41 recurrent calcium oxalate stone-formers. Time kinetics revealed a gradual rise in NANA expression until 48 h of culture and a significantly higher release into supernatants of papillary renal epithelial cells (REC) when compared with cortical REC. To examine NANA distribution in kidney tissues, paraffin-embedded biopsies from five normal and six stone-forming kidneys were labeled with the biotinylated NANA-specific lectins Maackia amurensis (MAA) and Sambucus nigra (SNA). Immunohistochemistry revealed intense luminal MAA reactivity of distal tubular REC and collecting ducts in 96.7% and 91.5% of normal and stone-forming kidneys respectively. By contrast, there was a marked difference between normal and stone-forming kidneys for SNA reactivity (17.7% vs 95%) at the same locations. Finally, the glycocalyx of recurrent stone-formers showed altered sialylglycoside linkages [alpha(2,6) instead of alpha(2,3)] that may indicate an altered REC function. Given the calcium-binding potential of NANA, their increased local concentration within the glycocalyx layer in the distal nephron may either initiate stone formation or facilitate attachment of microcrystals to REC.

Transcription and activity of antioxidant enzymes after ionizing irradiation in radiation-resistant and radiation-sensitive mice.

The involvement of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase in radiobiological processes has been described at the enzyme activity level. We irradiated radiation-resistant (RR) and radiation-sensitive (RS) mice and studied antioxidant enzymes at the transcriptional and activity level. In addition, aromatic hydroxylation and lipid peroxidation parameters were determined to study radiation resistance at the oxidation level. RS BALB/c/J Him mice and RR C3H He/Him mice were whole-body-irradiated with x-rays at 2, 4, and 6 Gy and killed 5, 15, and 30 min after irradiation. mRNA was isolated from liver and hybridized with probes for antioxidant enzymes and beta-actin as a housekeeping gene control. Antioxidant enzyme activities were determined by standard assays. Parameters for aromatic hydroxylation (o-tyrosine) and lipid peroxidation (malondialdehyde) were determined by HPLC methods. Antioxidant transcription was unchanged in contrast to antioxidant activities; SOD and CAT activities were elevated within 15 min in RR animals but not in RS mice, at all doses studied. Glutathione peroxidase activity was not different between RR and RS mice and was only moderately elevated after irradiation. No significant differences were found between RR and RS animals at the oxidation level, although a radiation dose-dependent increase of oxidation products was detected in both groups. We found that ionizing irradiation led to increased antioxidant activity only minutes after irradiation in the absence of increased transcription of these antioxidant enzymes. RR animals show higher antioxidant enzyme activities than do RS mice, but oxidation products are comparable in RS and RR mice. As unchanged transcription of antioxidant enzymes could not have been responsible for the increased antioxidant enzyme activities, preformed antioxidant enzymes should have been released by the irradiation process. This would be in agreement with previous studies of preformed, stored SOD. The finding of higher SOD and CAT activities in RR than in RS animals could point to a role for these antioxidant enzymes for the process of radiation sensitivity.

Decrease of brain protein kinase C, protein kinase A, and cyclin-dependent kinase correlating with pH precedes neuronal death in neonatal asphyxia.

BACKGROUND: Acidosis, energy depletion, overstimulation by excitatory amino acids, and free radical-mediated reactions are the major, current concepts for the explanation of damage and death resulting from asphyxia. Impaired protein phosphorylation by protein kinase C represents another mechanism incriminated in cell death. METHODS: We used a nonsophisticated perinatal asphyxia model to study brain (frontal cortex) pH, ATP, protein kinases PKC, PKA, and cyclin-dependent kinase. We used o-tyrosine, a marker for hydroxyl radical attack, and LPO 586, a spectrophotometric assay, to study lipid peroxidation products. The antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase were used in the frontal cortex. In addition, a cell death ELISA and histology to evaluate cell death were performed. RESULTS: Brain pH and protein kinases were decreasing with the length of the asphyctic periods, and energy depletion was shown by a drop of ATP levels, whereas no evidence for the involvement of free radical-mediated mechanisms was obtained. Cell death was shown by the cell death ELISA as early as 10 minutes after the asphyctic period, and histologically, cell death could be revealed but not before day 8 after asphyxia. CONCLUSION: Acidosis and/or impaired protein kinases, but not free radical mechanisms, may play a role in the pathobiochemistry of cell death in neonatal asphyxia of the rat.

Fucosidosis: genetic and biochemical analysis of eight cases.

The molecular basis of the deficiency of alpha-L-fucosidase has been investigated in eight patients who had been diagnosed clinically and enzymatically as suffering from the autosomal recessive lysosomal storage disease fucosidosis. None of the patients had a deletion or gross alteration of the alpha-L-fucosidase gene (FUCA1). Single strand conformation polymorphism (SSCP) analysis followed by direct sequencing of amplified exons and flanking regions identified putative disease causing mutations in six of the patients, who had severe forms of the disease and very low residual alpha-L-fucosidase activity and protein. They were a 10 bp deletion in exon 1 (E113fs), a 1 bp deletion at position -2 of intron 2 (S216fs), a g-->a transition at IVS5+1, point mutations W183X and N329Y in exons 3 and 6, respectively, and a compound allele consisting of a point mutation in the signal peptide in exon 1, P5R, and a 1 bp insertion in exon 6 (Y330fs). One patient in whom an SSCP change was not detected had residual alpha-L-fucosidase activity and cross reacting protein in the heterozygous range and normal metabolism of metabolites containing fucose in his fibroblasts, consistent with the low activity polymorphism. The eighth patient, who had a partial deficiency of alpha-L-fucosidase in her fibroblasts and leucocytes at a young age but normal alpha-L-fucosidase activity and protein at a later age, was homozygous for the common Q281R polymorphism in exon 5. She had no other sequence changes and Kivlin (Peters plus) syndrome has subsequently been diagnosed. The basis of her transient deficiency of alpha-L-fucosidase is not known. The detection of five novel mutations in six severely affected patients confirms the genetic heterogeneity in fucosidosis.

[The Austrian Metabolic Register]. / Osterreichisches Stoffwechselregister.

The "Austrian Register for Metabolic Disorders" was founded in 1995 on the initiative of the "Work Group for Congenital Metabolic Disorders" within the Austrian Society for Pediatrics. It is designed as a clearinghouse for reporting and recording congenital metabolic disorders and aims to determine the frequency and regional distribution of these diseases. Another objective is to have patient data handy if and when new therapeutic options or new means of diagnostic verification, including carrier status or prenatal diagnosis, become available. Currently more than 400 patients are on record with disorders of amino acid, organic acid, lipid or carbohydrate metabolism, as well as disorders of the mitochondria, peroxisomes and lysosomes; furthermore, dyschromia, porphyria and diseases of connective tissue and disturbances in the metabolism of purines, pyrimidines, metals or vitamins. Lysosomal enzyme defects, mitochondrial and peroxisomal disorders account for the majority of cases. Patients have to be reported on a continuous basis as prerequisite for this initiative to be successful. It is, therefore, planned to incorporate reporting of metabolic disorders to the "Register" on a national scale as integral part of the diagnosis of these conditions.

Comparison of sialic acids excretion in spot urines and 24-hour-urines of children and adults.

Sialic acids comprise all N- and O-acyl derivatives of neuraminic acid and are components of glycoproteins and glycolipids. Their concentrations vary physiologically with age but also in diseases such as inflammation, neoplastic tumours or in inborn genetic disorders causing abnormal sialic acid metabolism. Determination of free and bound sialic acids in urine using the thiobarbituric acid method according to Warren (J Biol Chem 1959; 234:1971-5) was shown to be useful for the diagnosis of diseases that involve sialic acid metabolic disorders. This test-also used for the diagnosis of inborn errors of metabolic diseases, such as sialidosis, infantile sialic acid storage disease, Salla's disease, neuraminidase deficiency and others-should be included in the selective screening for storage diseases. With the reported number of mild, juvenile and adult forms of genetic disorders increasing, this diagnosis will also be useful for teenagers and adults. We therefore considered it important not to confine our investigation to children and compared the diagnostic value of 24-hour and spot urines. As shown in 24-hour urines (n = 242, 128 males, 114 females) the average excretion of sialic acids increases constantly during life, from 67.6 mumol to 444.0 mumol per day, as does the free (27.5 mumol to 217.1 mumol) and bound fraction (40.1 mumol to 226.9 mumol). The relative proportion of free and bound sialic acid shows only slight lifetime variations, the free fraction increases from about 40 percent the first few years to about 53 percent of total in the fifth decade. In the spot urines, the mean ratio of total free sialic acids and urinary creatinine (mmol/mol) decreases constantly during the first few decades, with a sharp drop during the first years of life (from 3 months-2 years: from 203.9 to 94.2 and 82.1 to 42.3 respectively; with 10 years: 52.3 and 22.4 respectively; in the sixth decade: 44.8 and 21.9). Similar findings could also be observed in the investigated 24-hour urines (correlation coefficient of ratios, R = +0.981). The comparison of 24-hour urines and spot urines confirms the reliability of results for spot urines, however, the urine collection over an extended period yields additional information.

The effects of acid glycosaminoglycans on neonatal calvarian cultures--a role of keratan sulfate in Morquio syndrome?

Morquio syndrome (mucopolysaccharidosis IV) presents with multiple bone dysplasia and is characterized by the inability to degrade keratan sulfate due to deficient N-acetylgalactosamine-6-sulfate sulfatase in Morquio A syndrome and deficient beta-D-galactosidase in Morquio B syndrome. The aim of our study was to investigate into the pathogenetic mechanism as it is not clear whether the accumulation of keratan sulfate is toxic for osteoblasts or inhibits osteoblast activity as e.g. bone resorption. The glycosaminoglycans keratan sulfate, heparan sulfate, dermatan sulfate, chondroitin-4,6-sulfate and hyaluronic acid were tested in rat neonatal calvarian cultures for their effects on bone resorption, osteoblast activity and toxicity. Bone resorption was evaluated by calcium release into the medium, osteoblast activity by the determination of alkaline phosphatase and toxicity by measuring lactate dehydrogenase in the culture media. Keratan sulfate had no effect on bone resorption but inhibited osteoblast activity at the low, nontoxic concentration of 10 ng per ml organ culture supernatant significantly (p<0.05). At a concentration of 100 ng per ml keratan sulfate revealed toxic effects as reflected by significantly (p<0.05) elevated lactate dehydrogenase activity. None of the other glycosaminoglycans inhibited osteoblast activities. Heparan sulfate showed at toxic levels (10 microg per ml supernatant) significantly increased bone resorption (p<0.05) accompanied by increased alkaline phosphatase activity. The specific keratan sulfate effects of inhibiting osteoblast activity and toxicity towards bone, which were never tested before, suggest a role for this glycosaminoglycan in the pathogenesis of bone dysplasia in Morquio syndrome.

Juvenile hyaline fibromatosis: impaired collagen metabolism in human skin fibroblasts.

Juvenile hyaline fibromatosis (JHF) is inherited as a fatal autosomal recessive disorder characterised by multiple tumorous mucocutaneous proliferations. In this paper a 14 month old girl with JHF is described. For this condition, a malfunction of collagen synthesis is considered as the pathogenetic cause. Recently published data have revealed an absent band for type III collagen (TIIIC) chain in western blot studies of clinically unaffected JHF skin. Therefore supernatants of skin fibroblast cell cultures, obtained from normal human skin, were analysed for type 1 collagen (TIC) and TIIIC metabolites by radioimmunoassays. Besides the typical morphological connective tissue changes in the skin lesions, TIC synthesis and degradation were found increased in JHF fibroblasts compared with control fibroblasts. In contrast, TIIIC overall metabolism was significantly reduced by 36% compared with controls.

Homocysteine increases cyclin-dependent kinase in aortic rat tissue.

BACKGROUND: Hyperhomocyst(e)inemia is strongly associated with occlusive arterial disease. A direct effect of homocysteine on the proliferation of smooth muscle cells was proposed recently. This observation led us to examine the effect of homocysteine on cyclin-dependent kinase, the starter of mitosis and reflecting proliferation. METHODS AND RESULTS: Seventy Him:OFA rats were divided into seven groups. For 12 weeks, 10 rats were fed homocysteine 25 mg/kg body weight per day, 10 were fed 50 mg/kg body wt per day, and 10 were fed 100 mg/kg body weight per day; 10 were given homocysteic acid 100 mg/kg body weight per day, 10 were administered cysteine 100 mg/kg body weight per day, and 10 were given ascorbic acid 270 mg/kg body weight per day. Ten remained untreated and served as controls. Aortic cyclin-dependent kinase was determined at the transcriptional (mRNA) and protein levels. Phosphokinase C and aortic homocyst(e)ine also were evaluated in aortic tissue. Aortic cyclin-dependent kinase protein was significantly (P = .0001) elevated in the three homocysteine-treated groups, and mRNA cyclin-dependent kinase levels were significantly elevated in the rats given the 50 and 100 mg/kg body weight per day protocol. Endothelial damage was shown at higher homocysteine doses as reflected by circulating ACE and von Willebrand factor changes. Proliferation of cells of the aortic wall by bromodeoxyuridine incorporation could be shown in the high-dose homocysteine group only. CONCLUSIONS: Our findings indicate that homocysteine specifically stimulates aortic cyclin-dependent kinase at the transcriptional level, with the possible consequence of proliferation of aortic cells as revealed by incorporation of bromodeoxyuridine in the aortic wall.

Evidence for McKusick's hypothesis of deficient collagen cross-linking in patients with homocystinuria.

Osteoporosis occurs commonly in homocystinuria. The underlying pathobiochemical mechanism remains unclear; disturbed cross-linking of collagen has been suggested but this hypothesis has not been fully tested, nor have studies on collagen synthesis been performed. We therefore used recently available noninvasive tests for collagen synthesis and cross-linking to examine 10 patients with homocystinuria. Synthesis of collagen type I and type III was not different from age-matched healthy controls as reflected by comparable plasma levels of carboxyterminal propeptide of type I procollagen (PICP) and of plasma levels of N-terminal propeptide of procollagen type III (PIIINP). Collagen type I cross-links expressed by serum carboxyterminal telopeptide of collagen type I (ICTP) were 1.14 +/- 0.24 micrograms/l in the patient group versus 3.29 +/- 0.32 micrograms/l in the control group. This significant reduction of cross-links in the group with homocystinuria did not correlate with serum homocysteine or homocysteic acid concentrations. Our data clearly indicate that the disturbed cross-linking hypothesis still holds and that the bone manifestations of homocystinuria are not due to deficient collagen synthesis.

[Mucopolysaccharidoses from the view point of mucopolysaccharidoses families--10 years' experiences of the "Austrian Society for Mucopolysaccharidoses"]. / Die Mukopolysaccharidosen aus der Sicht der MPS-Familien--10 Jahre Erfahrungen der "Osterreichischen Gesellschaft für Mukopolysaccharidosen".

The mucopolysaccharidoses (MPS) are rare inborn errors of metabolism. They are caused by defects in enzymes which are necessary for the degradation of mucopolysaccharides. An effective causal treatment is not available as yet. Nevertheless, it is the duty of the doctor, both from the medical and human aspect to assist MPS patients and their families physically and psychologically over many years. This task needs much empathy, working morale and knowledge of these diseases on the part of the medical adviser involved, but also demonstrates the limitations of active medical treatment. Since 10 years the "Austrian Society for Mucopolysaccharidoses" has tried to fill the gap between the MPS families' need for help and the still discouraging current medical treatment prospectives.

Skin collagen defects in a patient with juvenile hyaline fibromatosis.

Juvenile hyaline fibromatosis is a rare disorder characterised by multiple subcutaneous tumours, gum hypertrophy, muscle weakness, and flexion contractures of the large joints. Histology shows an abundance of a homogenous, amorphous, acidophilic extracellular matrix in which spindle shaped cells are embedded forming minute streaks. It has been previously suggested that collagen abnormalities may be involved. A 14 month old girl with this syndrome is described in whom postmortem western blot studies were performed. These studies revealed an absent pro-alpha 2(I) chain and an absent collagen type III chain in skin but not in the other organs examined.

Histology and electron microscopy of fucosidosis of the skin. Subtle clues to diagnosis by electron microscopy.

Fucosidosis is an autosomal recessive inborn error of metabolism in which fucose-containing glycolipids, glycoproteins, and oligo- and polysaccharides accumulate in tissues as a consequence of alpha-L-fucosidase deficiency. Since the detection of this entity in 1966 several cases have been described, but until now investigations of clinically uninvolved skin have not been performed. In this study we have investigated clinically normal skin obtained from a patient with fucosidosis and his healthy sister, by light and electron microscopy, to determine whether normal skin in this condition yields clues that may have prognostic relevance. We found "empty"- appearing storage vesicles in melanocytes, endothelial cells, sweat glands, and fibroblasts in the skin.

[Mucopolysaccharidoses--current aspects of diagnosis and therapy]. / Die Mukopolysaccharidosen--aktuelle Aspekte über Diagnose und Therapie.

The mucopolysaccharidoses, first described at the beginning of this century, are still subject of research. The accumulation of pathological metabolites and the underlying enzyme defects are now correlated to specific gene mutations. A comparison of genotype and phenotype of the individual forms of the mucopolysaccharidoses is the subject of ongoing studies. In many cases, symptomatic treatment was not able to increase the quality of life of patients suffering from mucopolysaccharidosis to a satisfactory degree. International working groups are, thus, currently trying to improve and standardize symptomatic therapies. A causal therapeutic approach was attempted by implanting different cells and tissues that are able to produce the missing enzymes. Bone-marrow transplantations were also performed, but both treatment approaches were not very effective and in some cases even proved fatal for the patients. An intensive international research effort focuses on enzyme-replacement therapy and gene therapy. Mucopolysaccharidoses are rare diseases, affecting only about one hundred patients in Austria. Nevertheless, Austria plays an active role in researching these metabolic disorders.