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Background: Targeted delivery of gene therapy vectors to the mouse respiratory tract is often performed via intranasal or intratracheal administration; however, there can be a great deal of variability between these methods, which could potentially influence experimental results. Improving the accuracy and precision of lung delivery will not only reduce the number of animals required to detect statistically significant differences, but may reduce the variability of studies from different laboratories., Results: Here we evaluated three different methods of adeno-associated virus (AAV) vector administration to the respiratory tract in mice (intranasal, intubation, and intratracheal injection) and discuss the advantages, challenges, and shortcomings of each. We also present a modified-intranasal delivery technique that is superior to passive administration of vector into the nares of anesthetized supine animals. Transgene expression was consistently visible in the nasal cavity, trachea, and proximal to middle aspect of all lung lobes for all four methods, whereas transgene expression was consistently observed in the most distal aspect of lung lobes only with the intubation and intratracheal injection techniques. AAV vector genome copy numbers in the lung were approximately four-fold lower in mice that received vector via intranasal administration in comparison to the other three methods of vector delivery. The modified intranasal, intubation and intratracheal injection methods of vector administration did not yield statistical differences in AAV vector genome copy numbers in the lung. With regard to reproducibility of vector distribution within and between animals, the modified-intranasal technique was superior., Conclusion: Our results show that mode of AAV vector administration to the murine respiratory tract should be selected based on desired target site and skill of the researcher, and that appropriate technique selection may greatly influence experimental outcomes.

Enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) are highly related ovine betaretroviruses that induce nasal and lung tumours in small ruminants, respectively. While the ENTV and JSRV envelope (Env) glycoproteins mediate virus entry using the same cellular receptor, the glycosylphosphatidylinositol-linked protein hyaluronoglucosaminidase, ENTV Env pseudovirions mediate entry into cells from a much more restricted range of species than do JSRV Env pseudovirions. Unlike JSRV Env, ENTV Env does not induce cell fusion at pH 5.0 or above, but rather requires a much lower pH (4.0-4.5) for fusion to occur. The cytoplasmic tail of retroviral envelope proteins is a key modulator of envelope-mediated fusion and pseudotype efficiency, especially in the context of virions composed of heterologous Gag proteins. Here we report that progressive truncation of the ENTV Env cytoplasmic tail improves transduction efficiency of pseudotyped retroviral vectors and that complete truncation of the ENTV Env cytoplasmic tail increases transduction efficiency to wild-type JSRV Env levels by increasing fusogenicity without affecting sensitivity to inhibition by lysosomotropic agents, subcellular localization or efficiency of inclusion into virions. Truncation of the cytoplasmic domain of ENTV Env resulted in a significant advantage in viral entry into all cell types tested, including foetal ovine lung and nasal cells. Taken together, we demonstrate that the cytoplasmic tail modulates the fusion activity of the ENTV Env protein and that truncation of this region enhances Eenv-mediated entry into target cells.

We have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM). The power of the light source needed for illumination in a FLIM system and the signal-to-noise ratio of the detector have led us to a photon "budget." These measures are relevant to many fluorescence microscope users and the results are not restricted to FLIM but applicable to widefield fluorescence microscopy in general. Limitations in photon numbers, however, are more of an issue with FLIM compared to other less quantitative types of imaging. By modeling a typical experimental configuration, examples are given for fluorophores whose absorption peaks span the visible spectrum from Fura-2 to Cy5. We have performed experiments to validate the assumptions and parameters used in our mathematical model. The influence of fluorophore concentration on the intensity of the fluorescence emission light and the Poisson distribution assumption of the detected fluorescence emission light have been validated. The experimental results agree well with the mathematical model. This photon budget is important in order to characterize the constraints involved in current fluorescent microscope systems that are used for lifetime as well as intensity measurements and to design and fabricate new systems.

The double-stranded DNA genome of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) was sequenced and analysed in the context of other group I nucleopolyhedroviruses (NPVs). The genome consists of 129,593 bp with a G + C content of 50.1 mol%. A total of 146 open reading frames (ORFs) of greater than 150 bp, and with no or minimal overlap were identified. In addition, five homologous regions were identified containing 7-10 repeats of a 36 bp imperfect palindromic core. Comparison with other completely sequenced baculovirus genomes revealed that 139 of the CfMNPV ORFs have homologues in at least one other baculovirus and seven ORFs are unique to CfMNPV. Of the 117 CfMNPV ORFs common to all group I NPVs, 12 are exclusive to group I NPVs. Overall, CfMNPV is most similar to Orgyia pseudotsugata MNPV based on gene content, arrangement and overall amino acid identity. Unlike other group I baculoviruses, however, CfMNPV encodes a viral enhancing factor (vef) and has two copies of p26.

A single social defeat by a dominant conspecific induces long-term changes in several physiological and behavioral parameters in rats. These changes may represent an increased vulnerability to subsequent stress and stress-related pathology. Environmental factors, in particular possibilities for social interactions, could modulate these effects. Therefore, we assessed the influence of social environment on susceptibility for the long-term effects of social defeat. Socially housed males of an unselected strain of wild-type rats were equipped with radio-telemetry transmitters that recorded heart rate, temperature and activity. They were individually subjected to defeat and subsequently either housed alone or returned to their group. Behavioral and physiological responses to various novelty stressors were determined during a three-week period after the social defeat. Furthermore, changes in baseline behavior and physiology following defeat were studied in the rat's homecage. The results show a complex interaction between defeat and housing conditions. Depending on the parameters measured, effects were caused by both isolation alone, defeat alone or a combination of both defeat and isolation. Individual housing alone caused a characteristic hyperactive response to novelty stress. Though defeat did not affect behavioral responses, it amplified the physiological response to novelty and social housing did not attenuate this effect. However, social housing did reduce the effects of defeat on heart rate, temperature and activity in the home cage and completely prevented defeat-induced weight loss. Together these results indicate that social housing may indeed positively affect the animal's capacity to cope with stressors.

Repeated, intermittent exposure to psychostimulants or stressors results in long-lasting, progressive sensitization of the behavioral effects of a subsequent amphetamine (AMPH) challenge. Although behavioral sensitization has also been observed following a single drug pretreatment, the sensitizing potential of a single exposure to stress is not clear. Both drug- and stress-induced sensitization depend on an enhanced dopaminergic neurotransmission in the mesolimbic DA system. Apart from responding to rewards, this system is also involved in responding towards aversive social stimuli. Therefore, social stressors may be particularly effective in inducing cross-sensitization to stimulant drugs. We examined the time course of sensitization to the locomotor effects of the stimulant, AMPH, following a single social stressor: a social defeat. Wistar rats were exposed in a resident-intruder paradigm to an unfamiliar dominant male conspecific (Wild-Type Groningen), resulting in defeat. The locomotor effects of a subsequent AMPH challenge (0.25 or 1.0 mg/kg) were evaluated 3, 14, and 21 days later by scoring horizontal movement in an open field. AMPH had significantly larger locomotor-activating effects in animals that had been defeated 3 days earlier compared to nondefeated controls. However, this sensitized response was no longer present 14 or 21 days after defeat. Therefore, we conclude that social defeat induces short-lasting cross-sensitization to the locomotor effects of AMPH in rats, but is not sufficient for long-term sensitization. The transient enhancement of responses to dopaminergic drugs may be indicative of a temporary role of dopamine in the cascade of physiological and behavioral changes following social defeat.

The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 3'-5' exonucleases. The CfMNPV v-trex ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 3'-5' exonuclease that cleaves oligonucleotides from the 3' end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 5'-digoxigenin- or 5'-(32)P-labelled oligo(dT)(30) substrate was observed at increasing incubation times or increased amounts of V-TREX. The 3'-excision activity of V-TREX was maximal at alkaline pH (9.5) in the presence of 5 mM MgCl(2), 2 mM dithiothreitol and 0.1 mg BSA ml(-1).

As a result of the decreased incidence of immunological hydrops fetalis and increased insight, the role of inborn errors of metabolism (IEM) as a cause of hydrops fetalis has acquired increased significance. This growing awareness of the manifestation of IEM in pregnancy has revealed that some 20 of these disorders may cause hydrops fetalis, accounting for a few percent of all cases. These IEM are, for the most part, lysosomal storage diseases. We recommend that standard metabolites and enzymes reflecting lysosomal storage diseases be measured in the amniotic fluid and the amniocytes already withdrawn for karyotyping. The value of the diagnosis of lysosomal storage diseases lies in the opportunity for risk evaluation, genetic counselling and targeted prenatal diagnostics in case of subsequent pregnancies. Obtaining insight into the possible therapestic interventions during the pregnancy in which the hydrops is observed is not a goal of this protocol since the necessary investigations are too time-consuming.

Background: There is no comprehensive analytical technique to analyze N-acetylated metabolites in urine. Many of these compounds are involved in inborn errors of metabolism. In the present study, we examined the potential of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy as a tool to identify and quantify N-acetylated metabolites in urine of patients with various inborn errors of metabolism., Methods: We performed (1)H-NMR spectroscopy on a 500 MHz spectrometer. Using a combination of one- and two-dimensional correlation spectroscopy (COSY) (1)H-NMR spectra, we were able to assign and quantify resonances of characteristic N-acetylated compounds products in urine of patients with 13 inborn errors of metabolism., Results: The disease-specific N-acetylated metabolites were excreted at concentrations >100 micromol/mmol of creatinine in the patients' urine. In control urine samples, the concentration of individual N-acetyl-containing compounds was <40 micromol/mmol of creatinine. The combination of one- and two-dimensional COSY NMR spectroscopy led to the correct diagnosis of nine different inborn errors of metabolism. No abnormalities were observed in the spectra of urine from patients with G(M1)- or G(M2)-gangliosidosis. We also determined the (1)H-NMR characteristics of N-acetylated metabolites that may be relevant to human metabolism., Conclusion: (1)H-NMR spectroscopy may be used to identify and quantify N-acetylated metabolites of diagnostic importance for the field of inborn errors of metabolism.

Sanfilippo A syndrome, mucopolysaccharidosis type IIIA, is caused by a deficiency of heparan sulphamidase activity, and usually presents in childhood with neurodegeneration leading to death in teenage years. Visceral symptoms are limited to coarsening and diarrhea. We now describe an adult patient who presented with cardiomyopathy. At age 45 years she had hypertension, and the next year she developed a progressively worsening cardiomyopathy with prominent apical hypertrophy and atrial fibrillation. At age 53, she had severe concentric hypertrophic nonobstructive cardiomyopathy in both ventricles. There was no coarsening of features. Neurologic function, skeleton, cornea, liver, and spleen were normal. Percutaneous endomyocardial biopsy showed ballooned cardiomyocytes with storage vacuoles, containing acid mucopolysaccharides. Leucocytes, uterus, and brain biopsy did not show this storage material. There was a slight increase in total urine mucopolysaccharides, with an increased proportion of heparan sulfates. Heparan sulphamidase activity was deficient in leukocytes and heparan sulphamidase protein and activity were reduced in cultured fibroblasts. No mutations were identified after sequencing of the heparan sulphamidase gene at the cDNA and the genomic level. This new clinical presentation expands the clinical spectrum of Sanfilippo A syndrome to include a primary visceral presentation of cardiomyopathy without neurologic symptoms in the adult. The late onset may be related to the residual heparan sulphamidase activity. The genetic basis of this new variant is still unclear. Physicians evaluating adults must remain aware of possible new adult presentations of storage conditions., (Copyright 2003 Wiley-Liss, Inc.)

In a patient with microcephaly, feeding problems and restlessness, moderately increased serum and urine citrulline concentrations were observed. Protein and allopurinol loading did not result in additional indications for a urea cycle defect. The diagnosis of citrullinaemia was made at both the enzyme and DNA level, resulting from a novel mutation in the argininosuccinate synthetase gene. The fact that the patient has not suffered from severe deterioration, and that there were only minor abnormalities in metabolite concentrations, suggests that the argininosuccinate synthetase capacity was less affected in vivo than in vitro. In vitro nuclear magnetic resonance investigation suggested an active acetylation mechanism for citrulline. This case illustrates the importance of performing extensive biochemical and molecular investigations in order to reach a definitive diagnosis, particularly in instances of moderate citrullinaemia.

Animal studies suggest that overactivity of the hexosamine pathway, resulting in increased UDP-hexosamines [UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc)] is an important mechanism by which hyperglycemia causes insulin resistance. This study was performed to test this hypothesis in patients with type 2 diabetes mellitus (DM). Eight obese patients with uncontrolled DM type 2 and severe insulin resistance were treated with iv insulin for 28 +/- 6 d aimed at euglycemia. Before and after iv insulin treatment, insulin sensitivity was measured using a hyperinsulinemic euglycemic clamp, and a muscle biopsy was taken for measurement of UDP-GlcNAc, UDP-GalNAc, UDP-glucose, and UDP-galactose levels. Also, isoelectric focusing patterns of serum transferrin and the urinary excretion of glycosaminoglycans as measures of final products of the hexosamine pathway were examined. After euglycemia, insulin resistance improved, as demonstrated by an increase in the glucose infusion rate during the clamp from 12.7 +/- 5.6 to 22.4 +/- 8.8 micro mol/kg.min (P < 0.0005) and a decrease in insulin requirement from 1.7 +/- 0.9 to 1.1 +/- 0.6 U/kg.d (P < 0.005), whereas metabolic control improved. Surprisingly, both UDP-GlcNAc, from 8.81 +/- 1.21 to 12.31 +/- 2.52 nmol/g tissue (P < 0.005), and UDP-GalNAc concentrations, from 4.49 +/- 0.85 to 5.89 +/- 1.55 nmol/g tissue (P < 0.05) increased. Isoelectric focusing patterns of serum transferrin and excretion of glycosaminoglycans were similar before and after euglycemia. In conclusion, after amelioration of hyperglycemia- induced insulin resistance, UDP-hexosamines increased in skeletal muscle of patients with type 2 DM. These results do not support the hypothesis that accumulation of products of the hexosamine pathway plays a major role in hyperglycemia-induced insulin resistance.

Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNAc (alpha 1-->3)[Fuc alpha 1-->2]Gal(beta1-->4)GlcNAc(beta 1-->3)Gal(beta 1--> 4)Glc (beta 1-->1')Cer, IV(2)-alpha-fucosyl-IV(3)-alpha-N-acetylgalactosaminylneolactotetraosylceramide), B-6-2 (Gal(alpha 1-->3)[Fuc alpha 1--> 2] Gal (beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc(beta 1-->1')Cer, IV(2)- alpha-fucosyl-IV(3)-alpha-galactosylneolactotetraosylceramide), and globoside (GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc(beta 1-->1') Cer, globotetraosylceramide) were tritium labeled in their ceramide moiety and used as natural substrates. The degradation rate of glycolipid A-6-2 was very low in fibroblasts of all the alpha-NAGA-deficient patients (less than 7% of controls), despite very heterogeneous clinical pictures, ruling out different residual enzyme activities as an explanation for the clinical heterogeneity. Strongly elevated urinary excretion of blood group A glycolipids was detected in one patient with blood group A, secretor status (five times higher than upper limit of controls), in support of the notion that blood group A-active glycolipids may contribute as storage compounds in blood group A patients. When glycolipid B-6-2 was fed to alpha-galactosidase A-deficient cells, the degradation rate was surprisingly high (50% of controls), while that of globotriaosylceramide was reduced to less than 15% of control average, presumably reflecting differences in the lysosomal enzymology of polar glycolipids versus less-polar ones. Relatively high-degree degradation of substrates with alpha-D-Galactosyl moieties hints at a possible contribution of other enzymes.

Individual differences in aggressive behaviour have been linked to variability in central serotonergic activity, both in humans and animals. A previous experiment in mice, selectively bred for high or low levels of aggression, showed an up-regulation of postsynaptic serotonin-1A (5-HT(1A)) receptors, both in receptor binding and in mRNA levels, in the aggressive line [Brain Res 736 (1996) 338]. The aim of this experiment was to study whether similar differences in 5-HT(1A) receptors exist in individuals from a random-bred rat strain, varying in aggressiveness. In addition, because little is known about the functional consequences of these receptor differences, a response mediated via postsynaptic 5-HT(1A) receptors (i.e., hypothermia) was studied both in the selection lines of mice and in the randomly bred rats. The difference in receptor binding, as demonstrated in mice previously, could not be shown in rats. However, both in rats and mice, the hypothermic response to the 5-HT(1A) agonist alnespirone was larger in aggressive individuals. So, in the rat strain as well as in the mouse lines, there is, to a greater or lesser extent, an enhanced sensitivity of postsynaptic 5-HT(1A) receptors in aggressive individuals. This could be a compensatory up-regulation induced by a lower basal 5-HT neurotransmission, which is in agreement with the serotonin deficiency hypothesis of aggression.

Sjögren-Larsson syndrome (SLS) is an autosomal recessively inherited neurocutaneous disorder caused by a deficiency of the microsomal enzyme fatty aldehyde dehydrogenase (FALDH). We report the clinical characteristics and the results of molecular studies in 19 SLS patients. Patients 1-17 show the classical triad of severe clinical abnormalities including ichthyosis, mental retardation and spasticity. Most patients were born preterm, and all patients exhibit ocular abnormalities and pruritus. Electro-encephalography shows a slow background activity, without other abnormalities. MRI of the brain shows an arrest of myelination, periventricular signal abnormalities of white matter and mild ventricular enlargement. Cerebral (1)H-MR spectroscopy reveals a characteristic, abnormal lipid peak. The degree of white matter abnormality in the MRIs and the height of the lipid peak in (1)H-MR spectra do not correlate with the severity of the neurological signs. The clinical presentation and the clinical course is strikingly similar in these patients. Patient 18 shows a mild phenotype that essentially contains the same, but less severe, clinical features. Patient 19 exhibits the typical, but very mild, dermatological and ocular abnormalities, without any clinical neurological involvement. The diagnosis of SLS was confirmed by demonstration of the enzyme defect in cultured skin fibroblasts. Furthermore, as might be predicted from the essential role of FALDH in leucotriene B(4) (LTB(4)) metabolism, elevated urinary concentrations of LTB(4) and 20-OH-LTB(4) were found in all patients studied. Molecular studies of the FALDH gene revealed eight different mutations, including three new ones: a large 26-base pair deletion (21-46del), a missense mutation (80C-->T) and an insertion mutation (487-488insA). The vast majority of SLS patients seem to be severely affected independent of their genotype.

The Sjögren-Larsson Syndrome (SLS) is a neurocutaneous disorder, caused by deficient activity of the microsomal enzyme fatty aldehyde dehydrogenase (FALDH). FALDH catalyzes the oxidation of medium- and long-chain fatty aldehydes to their corresponding carboxylic acids. SLS is diagnosed by demonstrating the enzyme deficiency or by mutation analysis of the FALDH gene, while laboratory investigations of plasma, urine, and cerebrospinal fluid do not reveal any diagnostic abnormality. Leukotriene (LT) B4 is a pro-inflammatory mediator synthesized from arachidonic acid. LTB4 is inactivated by microsomal omega-oxidation, successively yielding 20-OH-LTB4, 20-CHO-LTB4 and 20-COOH-LTB4. Since FALDH is involved in LTB4 degradation, we have analyzed LTB4 and its metabolites in urine and cerebrospinal fluid as well as the degradation capacity for LTB4 in fresh polymorphonuclear leukocytes (PMN) of SLS patients. The urinary concentrations of LTB4, 20-OH-LTB4 and 20-COOH-LTB4 are below the detection limit in healthy controls. The urine of all SLS patients (n=13) exhibited highly elevated concentrations of LTB4 and 20-OH-LTB4, while 20-COOH-LTB4 was absent. Cerebrospinal fluid levels of LTB4, 20-OH-LTB4 and 20-COOH-LTB4 were found to be normal (n=7). PMN isolated from four patients were shown to be unable to convert 20-OH-LTB4 to 20-COOH-LTB4. Our findings provide unambiguous evidence for defective LTB4 degradation in SLS patients, and offer new and non-invasive diagnostic tools. Moreover, they open new pathophysiological considerations, with the prospect of rational treatment strategies.

Most ovarian tumors are cystic structures containing variable amounts of fluid. Several studies of ovarian cyst fluid focus on one specific metabolite using conventional assay systems. We examined the potential of (1)H-nuclear magnetic resonance spectroscopy in evaluation of the overall metabolic composition of cyst fluid from different ovarian tumors. Ovarian cyst fluid samples obtained from 40 patients with a primary ovarian tumor (12 malignant and 28 benign) were examined. After deproteinization and pD standardization, we performed (1)H-NMR spectroscopy on a 600 MHz instrument. With (1)H-NMR spectroscopy we found detectable concentrations of 36 metabolites with high intersample variation. A number of unassigned resonances as well as unexpected metabolites were found. We introduce an overall inventory of the low-molecular-weight metabolites in ovarian cyst fluid with corresponding resonances. Significant differences in concentration (p < 0.01) were found for several metabolites (including an unknown metabolite) between malignant and benign ovarian cysts. Furthermore, higher concentrations in malignant- and lower in benign fluids were found compared to normal serum values, indicating local cyst wall metabolic processes in case of malignant transformation. We conclude that (1)H-nuclear magnetic resonance spectroscopy can give an overview of low-molecular-weight proton-containing metabolities present in ovarian cyst fluid samples. The metabolic composition of cyst fluid differs significantly between benign and malignant ovarian tumors. Furthermore, differences between benign subgroups possibly related to histopathological behaviour can be detected. The presence of N-acetyl aspartic acid and 5-oxoproline exclusively in serous cystadenoma samples is remarkable. Future studies will concentrate on these findings and explore the possibilities of extrapolating information from the in vitro studies to in vivo practice, in which metabolic differences between malignant and benign subtypes can be of great importance in a pre-operative phase., (Copyright 2000 John Wiley & Sons, Ltd.)

In 6 patients with Sjögren-Larsson syndrome (SLS), the urinary excretion of leukotriene B(4) (LTB(4)) and omega-hydroxy-LTB(4) was found to be highly elevated, whereas omega-carboxy-LTB(4) was absent. This abnormal pattern of urinary excretion of LTB(4) and its metabolites appears to be unique to patients with SLS and offers a new approach to the diagnosis of this disorder. Moreover, defective inactivation of LTB(4) might be of pathophysiologic significance in the disease.

As carbohydrate-deficient glycoprotein syndromes (CDGS) are multisystemic disorders with impaired central nervous function in nearly all cases, we tested isoforms of beta-trace protein (beta TP), a 'brain-type' glycosylated protein in cerebrospinal fluid (CSF) of nine patients with the characteristic CDGS type I pattern of serum transferrin. Whereas the serum transferrin pattern did not discriminate between the various subtypes of CDGS type I (CDGS type Ia, type Ic, and patients with unknown defect), beta TP isoforms of CDGS type Ia patients differed from that of the other CDGS type I patients. The percentage of abnormal beta TP isoforms correlated with the severity of the neurological symptoms. Furthermore, two patients are described, who illustrate that abnormal protein N-glycosylation can occur restricted to either the 'peripheral' serum or the central nervous system compartment. This is the first report presenting evidence for an N-glycosylation defect restricted to the brain. Testing beta TP isoforms is a useful tool to detect protein N-glycosylation disorders in the central nervous system.

We have calculated the relative frequency and the birth prevalence of lysosomal storage diseases (LSDs) in The Netherlands based on all 963 enzymatically confirmed cases diagnosed during the period 1970-1996. The combined birth prevalence for all LSDs is 14 per 100,000 live births. Glycogenosis type II is the most frequent LSD with a birth prevalence of 2.0 per 100,000 live births, representing 17% of all diagnosed cases. Within the group of lipidoses, metachromatic leukodystrophy (MLD) is the most frequent LSD. MLD was diagnosed in 24% of lipidoses and the calculated birth prevalence was 1.42 per 100,000 for all types combined. Krabbe disease, diagnosed in 17% of cases, also belongs to the more frequent lipid storage diseases in The Netherlands with a birth prevalence of 1.35 per 100,000. The birth prevalence of Gaucher disease, commonly regarded as the most frequent lipid storage disease is 1.16 per 100,000 for all types combined. The combined birth prevalence for all lipid storage diseases is 6.2 per 100,000 live births. Within the group of mucopolysaccharidoses (MPSs), MPS I has the highest calculated birth prevalence of 1.19 per 100,000 (25% of all cases of MPS diagnosed), which is slightly more frequent than MPS IIIA with an estimated birth prevalence of 1.16 per 100,000. As a group, MPS III comprises 47% of all MPS cases diagnosed and the combined birth prevalence is 1.89 per 100,000 live births. The birth prevalence of MPS II is 0.67 per 100,000 (1.30 per 100,000 male live births). All other MPSs are rare. The combined birth prevalence for all MPSs is 4.5 per 100,000 live births. Mucolipidoses and oligosaccharidoses are very rare with birth prevalences between 0.04 and 0.20 for individual diseases. Only 49 cases were diagnosed between 1970 and 1996. Their combined birth prevalence is 1.0 per 100,000 live births.

Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS; MIM 260920) is a rare, apparently monogenic, autosomal recessive disorder characterized by recurrent episodes of fever accompanied with lymphadenopathy, abdominal distress, joint involvement and skin lesions. All patients have high serum IgD values (>100 U/ml) and HIDS 'attacks' are associated with an intense acute phase reaction whose exact pathophysiology remains obscure. Two other hereditary febrile disorders have been described. Familial Mediterranean fever (MIM 249100) is an autosomal recessive disorder affecting mostly populations from the Mediterranean basin and is caused by mutations in the gene MEFV (refs 5,6). Familial Hibernian fever (MIM 142680), also known as autosomal dominant familial recurrent fever, is caused by missense mutations in the gene encoding type I tumour necrosis factor receptor. Here we perform a genome-wide search to map the HIDS gene. Haplotype analysis placed the gene at 12q24 between D12S330 and D12S79. We identified the gene MVK, encoding mevalonate kinase (MK, ATP:mevalonate 5-phosphotransferase; EC 2.7.1.36), as a candidate gene. We characterized 3 missense mutations, a 92-bp loss stemming from a deletion or from exon skipping, and the absence of expression of one allele. Functional analysis demonstrated diminished MK activity in fibroblasts from HIDS patients. Our data establish MVK as the gene responsible for HIDS.

Objective: To determine the spectrum of clinical and MRI/1H MRS features of patients with fatty aldehyde dehydrogenase (FALDH) deficiency., Background: The Sjogren-Larsson syndrome (SLS) was originally defined as a clinical triad consisting of ichthyosis, spastic di- or tetralegia, and mental retardation, with autosomal recessive inheritance. By now, both the deficiency of the enzyme FALDH, and the genetic mutations on chromosome 17 responsible for this deficiency, have been identified. SLS, defined by fibroblast FALDH deficiency, seems to be a much broader syndrome., Methods: The clinical findings of 11 FALDH-deficient patients of different ages and one patient with the characteristic SLS-like ichthyosis, but without FALDH deficiency, were evaluated in relation to their cerebral MRI, and to 1H MRS in six patients., Results: The severity of neurologic symptoms showed considerable variation. Fundoscopic perifoveal glistening dots and the characteristic SLS-like ichthyosis were present in all patients. Serial MRI findings showed evidence of retarded myelination and a variable degree of dysmyelination. 1H MRS showed an accumulation of free lipids in the periventricular white matter, even before the stage of visible dysmyelination., Conclusions: The neurologic consequences of FALDH deficiency show considerable variation. The characteristic pattern of ichthyosis and retinal degeneration are seen consistently, yet they are not pathognomonic. MRI and 1H MRS findings suggest an accumulation of long-chain fatty alcohol intermediates, resulting in retarded myelination and dysmyelination.

Background: A38-year-old man presented with a history of fish odor (since age 5) and unusual muscle fatigue with increased serum creatine kinase. Our aim was to identify the metabolic error in this new condition., Methods: We used 1H NMR spectroscopy to study serum and urine from the patient., Results: The concentration of N, N-dimethylglycine (DMG) was increased approximately 100-fold in the serum and approximately 20-fold in the urine. The presence of DMG as a storage product was confirmed by use of 13C NMR spectroscopy and gas chromatography-mass spectrometry. The high concentration of DMG was caused by a deficiency of the enzyme dimethylglycine dehydrogenase (DMGDH). A homozygous missense mutation was found in the DMGDH gene of the patient., Conclusions: DMGDH deficiency must be added to the differential diagnosis of patients complaining of a fish odor. This deficiency is the first inborn error of metabolism discovered by use of in vitro 1H NMR spectroscopy of body fluids., (Copyright 1999 American Association for Clinical Chemistry)

Background: The diagnosis of inborn errors of purine and pyrimidine metabolism is often difficult. We examined the potential of 1H-NMR as a tool in evaluation of patients with these disorders., Methods: We performed 1H-NMR spectroscopy on 500 and 600 MHz instruments with a standardized sample volume of 500 microL. We studied body fluids from 25 patients with nine inborn errors of purine and pyrimidine metabolism., Results: Characteristic abnormalities could be demonstrated in the 1H-NMR spectra of urine samples of all patients with diseases in the pyrimidine metabolism. In most urine samples from patients with defects in the purine metabolism, the 1H-NMR spectrum pointed to the specific diagnosis in a straightforward manner. The only exception was a urine from a case of adenine phosphoribosyl transferase deficiency in which the accumulating metabolite, 2,8-dihydroxyadenine, was not seen under the operating conditions used. Similarly, uric acid was not measured. We provide the 1H-NMR spectral characteristics of many intermediates in purine and pyrimidine metabolism that may be relevant for future studies in this field., Conclusion: The overview of metabolism that is provided by 1H-NMR spectroscopy makes the technique a valuable screening tool in the detection of inborn errors of purine and pyrimidine metabolism., (Copyright 1999 American Association for Clinical Chemistry)

The nucleotide excision repair (NER) pathway is able to remove a wide variety of structurally unrelated lesions from DNA. NER operates throughout the genome, but the efficiencies of lesion removal are not the same for different genomic regions. Even within a single gene or DNA strand repair rates vary, and this intragenic heterogeneity is of considerable interest with respect to the mutagenic potential of carcinogens. In this study, we have analyzed the removal of the two major types of genotoxic DNA adducts induced by UV light, i.e., the pyrimidine (6-4)-pyrimidone photoproduct (6-4PP) and the cyclobutane pyrimidine dimer (CPD), from the Saccharomyces cerevisiae URA3 gene at nucleotide resolution. In contrast to the fast and uniform removal of CPDs from the transcribed strand, removal of lesions from the nontranscribed strand is generally less efficient and is modulated by the chromatin environment of the damage. Removal of 6-4PPs from nontranscribed sequences is also profoundly influenced by positioned nucleosomes, but this type of lesion is repaired at a much higher rate. Still, the transcribed strand is repaired preferentially, indicating that, as in the removal of CPDs, transcription-coupled repair predominates in the removal of 6-4PPs from transcribed DNA. The hypothesis that transcription machinery operates as the rate-determining damage recognition entity in transcription-coupled repair is supported by the observation that this pathway removes both types of UV photoproducts at equal rates without being profoundly influenced by the sequence or chromatin context.

The essential Saccharomyces cerevisiae KIN28 gene encodes a subunit of general transcription factor TFIIH, a multiprotein complex required for RNA polymerase II transcription initiation and nucleotide excision repair (NER). Kin28 is implicated in the transition from transcription initiation to transcription elongation by phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of the RNA polymerase II complex. Here, we explore the possibility that Kin28 like the other subunits of TFIIH is involved in NER in vivo, using yeast cells carrying either a wildtype or a thermosensitive KIN28 allele. The removal of UV induced cyclobutane pyrimidine dimers (CPDs) was monitored at base resolution from both strands of the RNA polymerase II transcribed genes RPB2 and URA3. Cells carrying the thermosensitive KIN28 allele display a transcription-coupled repair (TCR) defect at the non-permissive temperature, which was most pronounced directly downstream of transcription initiation, probably as an indirect result of a general decrease in the level of RNA polymerase II transcription. The fact that CPD removal in non-transcribed DNA is completely unaffected in these cells indicates that Kin28 is not essential for general NER in vivo, providing the first example of a TFIIH subunit that is required for TCR but not for NER in general.

The flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and its metabolite 2-bromoacrolein (2BA) are very potent bacterial mutagens in Salmonella typhimurium (S. typhimurium) TA 100. In this study, we showed that 2BA and Tris-BP are also mutagenic in S. typhimurium TA 104, which detects mutations at AT base pairs, while TA 100 detects mutations at CG basepairs. We also studied the mutagenicity of 2BA in mammalian cells in vitro and in the rat in vivo. Firstly, 2BA was tested in the human lymphoblastoid cell line TK6. The results showed that there was no increase in mutation frequency at the hprt locus, whereas there was a large decrease in cell survival. Secondly, a shuttle vector system was used to study the induction of mutations by 2BA:DNA adducts. The vector was modified by insertion of a single-stranded oligonucleotide containing on average one 2BA:DNA adduct. No increase in mutation frequency above background was detected after replication of this vector in SV40 transformed normal human fibroblasts. Because the liver is a major site for bioactivation of Tris-BP to 2BA in vivo, we tested the initiating capacity of Tris-BP in the rat liver in a modified Solt & Farber initiation and promotion system. Administration of Tris-BP resulted in a small increase in the number of preneoplastic gamma-glutamyl-transpeptidase positive (GGT+) foci in the liver compared to control animals (only significant in the lowest size class). Modification of the experimental protocol by performing partial hepatectomy 24 h after the administration of Tris-BP, did not increase the number of GGT+ or glutathione S-transferase-P (GST-P+) positive foci above the control level. Taken together, these results indicate that, in spite of a high mutagenicity in S. typhimurium, 2BA and Tris-BP have low or negligible mutagenic effects in mammalian systems. The lack of mutagenic activity may explain why Tris-BP is not a carcinogen in the rat liver.

Objective: To determine whether cerebrospinal fluid (CSF) methylmalonic acid (MMA) is increased in neurological patients with low serum cobalamin (Cbl, vitamin B12) concentrations as opposed to neurological patients with normal serum Cbl concentrations., Material and Methods: We measured MMA concentrations in serum and CSF of neurological patients with low serum cobalamin concentrations, but without overt cobalamin related manifestations such as anemia or combined disease of the cord, and neurological patients with normal serum cobalamin concentrations (controls)., Results: Serum and CSF MMA concentrations were significantly higher in patients than in controls. Serum MMA was elevated in 4 patients of whom 3 had clearly elevated CSF MMA concentrations., Conclusion: Strong indications for cobalamin deficiency can be found not only in serum but also in CSF of patients with seemingly asymptomatic low serum cobalamin concentrations.

Gaucher disease is a lysosomal storage disease characterized by storage of glucocerebroside due to lysosomal glucocerebrosidase deficiency. Increased urinary excretion of sialyloligosaccharides and mannosylglycoasparagines has been described for two patients with the infantile form of the disease, probably as a consequence of obstruction of lysosomal functioning due to the glycolipid accumulation in lysosomes. By thin-layer chromatography, we found increased urinary oligosaccharide excretion in a series of adult non-neuronopathic patients. Oligosaccharide patterns were comparable between patients and also with the pattern observed in infantile Gaucher disease. Composition was analysed by methanolysis and gas chromatography. Mannose and N-acetylglucosamine are the main carbohydrates in all oligosaccharide bands. A statistically significant correlation was found between oligosaccharide excretion and the severity of the disease expressed as severity score index. Patients treated with enzyme replacement therapy showed a reduction up to 65% of the original oligosaccharide excretion after 1 year of treatment, comparable with the reduction in spleen volume.

The molecular mechanism of transcription-coupled nucleotide excision repair in eukaryotes is poorly understood. The identification of the dual role of basal transcription factor TFIIH in DNA repair and transcription provided a plausible link between both processes. However, TFIIH is not part of the elongating transcription complex, suggesting that additional components are required to recruit TFIIH when RNA polymerase II (RNAPII) stalls at the site of DNA damage. Previously, we have shown that the yeast Rad26 protein is involved in transcription-coupled DNA repair. This paper describes the differential contribution of the Rad26 protein to efficient removal of UV-induced cyclobutane pyrimidine dimers (CPDs) from transcribed DNA. Two distinct regions within the transcribed strand of RNAPII-transcribed genes are identified that differ in their requirement for the RAD26 gene product. Using high-resolution repair analysis, we determined the in vivo repair kinetics of cyclobutane pyrimidine dimers positioned around the transcription initiation site of RNAPII-transcribed genes RPB2 and URA3. Although transcription-coupled repair is severely reduced in rad26 mutants, lesions positioned in a small region immediately downstream of transcription initiation are efficiently removed in the absence of Rad26. The observed transition in repair characteristics is abrupt and in excellent agreement with the region where TFIIH dissociates from RNAPII in vitro, strongly suggesting an inverse correlation between TFIIH association and Rad26 requirement. These data suggest that a transcription repair coupling factor (Rad26/CSB) is required for efficient repair only during the elongating stages of RNAPII transcription.

Correct quantitative results for plasma cholesterol, 7-dehydrocholesterol (7-DHC), and 8-dehydrocholesterol (8-DHC) are invaluable for making the correct diagnosis in patients with the Smith-Lemli-Opitz syndrome (SLO) and for biochemical monitoring of these patients during therapy. The enzymatic method for cholesterol measurement based on cholesterol oxidase gives falsely high values for plasma cholesterol in samples from patients with SLO. Both 7-DHC and 8-DHC contribute substantially to the test result, given that they are accepted substrates of cholesterol oxidase. All cholesterol methods making use of this enzyme are expected to give unreliable results with plasma samples from SLO patients. Cholesterol values found with these methods may be low-normal in individual cases with SLO. Therefore, other techniques for measuring cholesterol, 7-DHC, and 8-DHC, e.g., gas chromatography, should be used for diagnosing these patients and for follow-up during therapy. However, a normal value for plasma cholesterol, as obtained by gas chromatography, does not exclude SLO. The diagnosis should always be confirmed or excluded by testing for the presence of high concentrations of 7-DHC and 8-DHC in plasma. We found that one patient with a severe form of the disease had a plasma cholesterol concentration of 20 micromol/L-to our knowledge, the lowest value ever recorded in a human being.

Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was examined at single nucleotide resolution in the yeast Saccharomyces cerevisiae, using an improved protocol for genomic end-labelling. To obtain the sensitivity required for adduct detection in yeast, an oligonucleotide-directed enrichment step was introduced into the current methodology developed for adduct detection in Escherichia coli. With this method, heterogeneous repair of CPDs within the RPB2 locus is observed. Individual CPDs positioned in the transcribed strand are removed very efficiently with identical kinetics. This fast repair starts within 23 bases downstream of the transcription initiation site. The non-transcribed strand of the active gene exhibits slow repair without detectable repair variations between individual lesions. In contrast, CPDs positioned in the promoter region show profound repair heterogeneity. Here, CPDs at specific sites are removed very quickly, with comparable rates to CPDs positioned in the transcribed strand, while at other positions lesions are not repaired at all during the period studied. Interestingly, the fast repair in the promoter region is dependent on the RAD7 and RAD16 genes, as are the slowly repaired CPDs in this region and in the non-transcribed strand. This indicates that the global genome repair pathway is not intrinsically slow and at specific positions can be as efficient as the transcription-coupled repair pathway.

Up to now eight patients with alpha-NAGA deficiency have been described. This includes the newly identified patient reported here who died unexpectedly aged 1 1/2 years of hypoxia during convulsions; necropsy was not performed. Three patients have been genotyped previously and here we report the mutations in the other five patients, including two new mutations (S160C and E193X). The newly identified patient is consanguineous with the first patients reported with alpha-NAGA deficiency and neuroaxonal dystrophy and they all had the alpha-NAGA genotype E325K/E325K. Clinical heterogeneity among patients with alpha-NAGA deficiency is extreme. Two affected sibs, homozygotes for E325K, are severely affected and have the signs and symptoms of infantile neuroaxonal dystrophy, but prominent vacuolisation is lacking. The mildly affected patients (two families, three patients) at the opposite end of the clinical spectrum have clear vacuolisation and angiokeratoma but no overt neurological manifestations. Two of them are homozygous for the stop mutation E193X, leading to complete loss of alpha-NAGA protein. These observations are difficult to reconcile with a simple genotype-phenotype correlation and we suggest that factors or genes other than alpha-NAGA contribute to the clinical heterogeneity of the eight patients with alpha-NAGA deficiency. At the metabolic level, the patients with alpha-NAGA deficiency are similar. The major abnormal urinary oligosaccharides are sialylglycopeptides of the O linked type. Our enzymatic studies indicated that these compounds are not the primary lysosomal storage products.

Mutation spectra at the nucleotide sequence level of five hprt cDNA genes integrated in different genomic positions of a HPRT(-) derivative of the human lymphoblastoid TK6 cell line were compared with each other and with the spectrum of mutations confined to the 657 bp coding region of the endogenous hprt gene in the parental TK6 cells. The mutation rates in these genomic positions vary significantly and also the mutation spectra are different. In each genomic position the majority of mutations are basepair substitutions and deletions. the ratios of which vary among the genomic positions. Although it is likely that the different rates of deletion are to a large extent the net result of different rates of misalignment and repair of these errors in the various genomic positions, for the basepair substitutions it is not possible to deduce which mechanisms have caused these mutations and what causes the differences among the genomic positions. Taken together, the differences in mutation rates and spectra cannot be explained by a single mutagenic process.

6-Mercaptopurine, a hypoxanthine antimetabolite, is used in the treatment of acute lymphoblastic leukemia (ALL) in children. Extensively metabolized before it exerts cytotoxic action, it is catabolized into 6-mercapto-2,8-dihydroxypurine (thiouric acid), which is excreted by the kidneys. We describe a metabolite of 6-mercaptopurine, 6-methylmercapto-8-hydroxypurine, whose presence has not been previously reported in plasma. This compound was found in high concentrations in plasma during high-dose 6-mercaptopurine infusions (1300 mg/m2 in 24 h). This previously unknown compound was identified by reversed-phase HPLC with absorbance detection and by gas chromatography-mass spectrometry. The pathways leading to 6-methylmercapto-8-hydroxypurine in vivo are not yet fully understood. In a group of 17 patients treated with four courses of high-dose 6-mercaptopurine infusions according to the ALL-8 treatment protocol of the Dutch Childhood Leukemia Study Group, the steady-state concentrations of 6-methylmercapto-8-hydroxypurine in plasma were one-fifth of the parent drug concentrations, with wide interindividual variation. The formation of high concentrations of 6-methylmercapto-8-hydroxypurine in plasma, especially during the infusion, probably indicates another catabolic pathway of high-dose 6-mercaptopurine, apart from its conversion into thiouric acid.

In this study free kappa light chains in cerebrospinal fluid (CSF) were determined both by an affinity mediated capillary blotting technique after isoelectric focusing (IEF) in agarose gel and by a quantitative enzyme linked immunosorbent assay (ELISA). The free kappa results were compared with the IgG findings in 4 neurological patient groups with a distinct CSF IgG pattern: (1) CSF without oligoclonal IgG bands, (2) CSF with serum derived IgG bands, (3) CSF restricted IgG bands and (4) CSF restricted and serum derived IgG bands. Oligoclonal free kappa bands are nearly absent in CSF of groups 1 + 2, and present in 88% of group 3 and 84% of group 4 patients. We could also establish free kappa indices from specimens in the 4 groups in analogy to IgG indices. Group 1 had a median free kappa index of 1.1, group 2: 1.0 and groups 3 + 4: 10.0. The correspondence between immunoblot and index findings for free kappa is better than for IgG. Free kappa index is more sensitive but somewhat less specific than IgG index for establishing intrathecal immune production.

Sequencing of the arylsulfatase A gene in a late infantile metachromatic leukodystrophy patient showed the presence of a 12-bp deletion in exon 2. This deletion was found in a compound heterozygous state with the previously described 287 C-->T transition.

This study describes a standardized method for recording single-pulse 1H-nuclear magnetic resonance (1H-NMR) spectra from cerebrospinal fluid (CSF). Quantitative data for alanine, valine, threonine, and lactic acid correlated well with data obtained with conventional techniques. The pH of the samples is important for the reproducibility of the chemical shift of resonances, and should be standardized to improve recognition and assignment of resonances. A database of resonances from various metabolites is presented. Fifty compounds could be identified in CSF, 15 of which had not been observed earlier in NMR studies of CSF. We describe for the first time in the literature, to our knowledge, 3-hydroxyisovaleric acid as a regular component of many CSF samples. As examples of the diagnostic power of the technique, spectra are shown of CSF from patients with three different inborn errors of metabolism. We found high concentrations of N-acetylaspartic acid, citric acid, and succinic acid in CSF from a patient with Canavan disease. This is indirect evidence for the existence of a carrier mechanism that is shared by these di- and tricarboxylic acids.

Objective: To determine the clinical symptoms in adult metachromatic leukodystrophy and in adult pseudodeficiency for arylsulfatase A., Design: Case series., Setting: University hospital., Patients: Twenty-five adult patients with very low arylsulfatase A activity., Results: In 13 patients, a diagnosis of adult metachromatic leukodystrophy was made. The main symptoms were dementia, behavioral abnormalities, ataxia, and polyneuropathy. In 12 patients, a diagnosis of arylsulfatase A pseudodeficiency was made. No characteristic clinical syndrome could be detected in these patients., Conclusions: Adult metachromatic leukodystrophy is a progressive metabolic disease with symptoms of demyelination of the central and peripheral nervous systems. Diagnosis must be confirmed by determination of arylsulfatase A activity and accumulation of sulfatides. Pseudodeficiency for arylsulfatase A can be confirmed or excluded by means of DNA analysis.

A spectrum of 100 mutations in the endogenous hprt gene of the human lymphoblastoid TK6 cell line is presented. The majority of the mutations originates in sequences outside the coding region of the gene. Large deletions are a major cause of inactivation of the hprt gene (57% of the mutants). Mutations in the splice sites that result in several forms of aberrantly spliced mRNA are relatively frequently recovered (16%) compared with mutants containing alterations in the coding region of the hprt gene (27%). The majority, but not all, of the splice mutants contain an alteration in the consensus sequences of the splice sites. A spectrum of mutations in the coding region of the hprt gene enlarged to a total of 42 mutants shows that basepair substitutions predominate (71%) and that small deletions and insertions are less frequently recovered. Basepair substitutions arise slightly more frequently at GC basepairs than at AT basepairs.

A procedure to extract and purify gangliosides from small volumes of plasma (0.6 mL), cerebrospinal fluid (1 mL) and fibroblasts is described. Gangliosides were extracted with chloroform/methanol and purified by means of reversed phase chromatography and gel filtration before analysis by thin layer chromatography. The procedure proved to be useful in confirming deficiency of lysosomal enzyme activity affecting ganglioside breakdown. The new procedure also appeared to be useful to monitor ganglioside catabolism in cultured fibroblasts loaded with ganglioside.

The influence of the genomic position of a gene on UV-induced mutations was studied in the endogenous hprt gene in human lymphoblastoid TK6 cells and in cell lines derived from TK6 each containing a single copy of a hamster hprt cDNA gene integrated on a retroviral vector in different positions of the human genome. Previous studies showed that the genomic sequences surrounding the integration site influence spontaneous mutagenesis, resulting in a 10-fold difference in mutation rates among the hprt cDNA genes. Here we demonstrate that the genomic positions of three integrated hprt cDNA genes do not influence UV-induced mutagenesis. The mutability by UV irradiation in these cell lines is approximately the same (16.0 x 10(-6) per J/m2). The nature of the UV-induced mutations determined in two of the cell lines containing the integrated hprt cDNA gene (approximately 30 mutants each) was also found not to be different. The endogenous hprt gene in the parental TK6 cells exhibits a significantly lower mutability (2.1 x 10(-6) per J/m2) than the cDNA genes, but the spectrum is very similar. The spectrum in TK6 shows no influence of strand-specific repair and resembles most closely the spectrum obtained by McGregor et al. after irradiation of human cells synchronized in S-phase. This suggests that mutations arising in cells that are in S-phase at the time of irradiation constitute the majority of the mutants in an asynchronous TK6 cell population. We hypothesize that repair in the endogenous hprt gene in TK6 cells is very efficient, removing virtually all lesions before replication takes place except in cells that were in S-phase at the time of irradiation when there is not enough time for repair. Furthermore we suggest that the higher mutability of the integrated hprt cDNA genes compared with the endogenous gene is caused by a less efficient repair in the cDNA genes.

Analysis of urinary oligosaccharides by thin-layer chromatography (TLC) is used as screening procedure for 10 different lysosomal diseases. We tested the usefulness of HPLC in screening, using a CarboPac PA1 column (Dionex), pulsed amperometric detection (PAD), and post-column derivatization (PCD). Patterns from six types of oligosaccharidoses were compared with normal urinary patterns and with the TLC patterns. PAD appeared to be nonspecific and therefore is applicable only to desalted urine samples. PCD was more specific and applicable to nondesalted urine samples, albeit with a lower resolving power. Peaks in urines from oligosaccharidoses patients were identified on the basis of retention times of commercially available oligosaccharides or TLC bands after isolation and HPLC of the corresponding oligosaccharides. Abnormal oligosaccharide peaks were seen in urines from patients with alpha-mannosidosis, GM1-gangliosidosis (juvenile), GM2-gangliosidosis (Sandhoff disease), Pompe disease, and beta-mannosidosis. HPLC detected no abnormal oligosaccharides in urine from patients with fucosidosis. Although TLC is a simple and reliable screening procedure for detecting classical lysosomal diseases with oligosaccharide excretion, HPLC, by its higher resolution and possibility of quantification, can more generally be used for recognition of abnormal oligosaccharides or detection of increased excretion or content for known oligosaccharides in urine, other body fluids, and cells.