bluuebunny/crossref_metadata_2025 huggingface dataset:
Preparation and characterization of tetracycline‐loaded interpenetrating polymer networks of carboxymethyl cellulose and poly(acrylic acid): water sorption and drug release study
Authors: Bajpai Anil Kumar , Mishra Abhilasha
Published: June 2005

Tetracycline (TC)‐loaded ionic interpenetrating polymer networks (IPNs) of carboxymethyl cellulose (CMC) and crosslinked poly(acrylic acid) (PAA) were prepared and characterized by infrared spectral analysis, differential scanning calorimetry and scanning electron microscopy techniques. The prepared IPNs were evaluated for in vitro blood compatibility by clot formation and hemolysis methods and their water imbibitions capacity was determined. Fractional release dynamics of tetracycline was also investigated from loaded IPNs of CMC and PAA. The entrapped drug was examined for antibacterial activity and structural integrity and effects of various parameters such as percentage loading of the drug, chemical composition of the carrier IPN, pH and temperature of the release medium were investigated on the release profiles of TC. The drug was also released in different simulated biological fluids. Copyright © 2005 Society of Chemical Industry
Structural and end‐group effects on bulk and surface properties of hyperbranched poly(urea urethane)s
Authors: Elrehim Mona Abd , Voit Brigitte , Bruchmann Bernd , Eichhorn Klaus‐Jochen , Grundke Karina , Bellmann C.
Published: June 2005

The hydroxy end groups of aromatic and aliphatic hyperbranched poly‐(urea urethane)s prepared with an AA* + B*B2 one‐pot method were modified with phenylisocyanate, butylisocyanate, and stearylisocyanate. The success of the modification reaction was verified with 1H NMR and IR spectroscopy. Linear model poly‐(urea urethane)s were prepared, too, for comparison. The bulk properties of OH functionalized hyperbranched poly(urea urethane)s, compared with those of linear analogues and modified hyperbranched poly(urea urethane)s, were studied with differential scanning calorimetry, thermogravimetric analysis, and temperature‐dependent Fourier transform infrared measurements. Transparent and smooth thin films could be prepared from all polymer samples and were examined with a light microscope, a microglider, and an atomic force microscope. The properties of the polymer surface were examined by measurements of the contact angle and zeta potential. For all samples, the properties were mainly governed by the strong interactions of the urea and urethane units within the backbone, whereas the influence of the nature of the end groups and of the branched structure was reduced in comparison with other hyperbranched polymer systems. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 3376–3393, 2005
Molecular modeling of cardiac glycoside binding by the human sequence monoclonal antibody 1B3
Authors: Paula Stefan , Monson Nancy , Ball William James
Published: June 2005

The amino acid sequences of the heavy‐ and light‐chain variable regions of the high‐affinity human sequence antidigoxin monoclonal antibody 1B3 (mAb 1B3) were determined, and a structural model for the mAb's variable region was developed by homology modeling techniques. The structural model provided the basis for computationally docking digoxin and eight related cardiac glycosides into the putative binding site of mAb 1B3. Analysis of the consensus binding mode obtained for digoxin showed that the cardenolide moiety of digoxin is deeply embedded in a predominantly hydrophobic, narrow cavity, whereas the terminal, γ‐carbohydrate group is solvent‐exposed. The docking results indicated that the primary driving forces for digoxin binding by mAb 1B3 are hydrophobic interactions with the digoxin steroid ring system and hydrogen bonds with the digitoxose groups. The binding model accounts for the experimentally observed variations in mAb 1B3 binding affinity for various structural analogs of digoxin used previously to develop a 3D structure–activity relationship model of drug binding (Farr CD, Tabet MR, Ball WJ Jr, Fishwild DM, Wang X, Nair AC, Welsh WJ. Three‐dimensional quantitative structure–activity relationship analysis of ligand binding to human sequence antidigoxin monoclonal antibodies using comparative molecular field analysis. J Med Chem 2002;45:3257–3270). In particular, the hydrogen bond pattern is consistent with the unique sensitivity of mAb 1B3's binding affinity to the number of sugar residues present in a cardiac glycoside. The hydrophobic environment about the steroid moiety of digoxin is compatible with the mAb's reduced affinity for ligands that possess hydrophilic hydroxyl and acetyl group modifications in this region. The model also indicated that most of the amino acid residues in contact with the ligand reside in or about the three complementarity determining regions (CDRs) of the heavy chain and the third CDR of the light chain. A comparison of the 1B3 binding model with the crystal structures of two murine antidigoxin mAbs revealed similar binding patterns used by the three mAbs, such as a high frequency of occurrence of aromatic, hydrophobic residues in the CDRs and a dominant role of the heavy chain CDR3 in antigen binding. Proteins 2005. © 2005 Wiley‐Liss, Inc.
Increasing the molecular contacts between maurotoxin and Kv1.2 channel augments ligand affinity
Authors: M'Barek Sarrah , Chagot Benjamin , Andreotti Nicolas , Visan Violeta , Mansuelle Pascal , Grissmer Stephan , Marrakchi Mohamed , El Ayeb Mohamed , Sampieri François , Darbon Hervé , Fajloun Ziad , De Waard Michel , Sabatier Jean‐Marc
Published: June 2005

Scorpion toxins interact with their target ion channels through multiple molecular contacts. Because a “gain of function” approach has never been described to evaluate the importance of the molecular contacts in defining toxin affinity, we experimentally examined whether increasing the molecular contacts between a toxin and an ion channel directly impacts toxin affinity. For this purpose, we focused on two scorpion peptides, the well‐characterized maurotoxin with its variant Pi1‐like disulfide bridging (MTXPi1), used as a molecular template, and butantoxin (BuTX), used as an N‐terminal domain provider. BuTX is found to be 60‐fold less potent than MTXPi1 in blocking Kv1.2 (IC50 values of 165 nM for BuTX versus 2.8 nM for MTXPi1). Removal of its N‐terminal domain (nine residues) further decreases BuTX affinity for Kv1.2 by 5.6‐fold, which is in agreement with docking simulation data showing the importance of this domain in BuTX‐Kv1.2 interaction. Transfer of the BuTX N‐terminal domain to MTXPi1 results in a chimera with five disulfide bridges (BuTX‐MTXPi1) that exhibits 22‐fold greater affinity for Kv1.2 than MTXPi1 itself, in spite of the lower affinity of BuTX as compared to MTXPi1. Docking experiments performed with the 3‐D structure of BuTX‐MTXPi1 in solution, as solved by 1H‐NMR, reveal that the N‐terminal domain of BuTX participates in the increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicate that acting on molecular contacts between a toxin and a channel is an efficient strategy to modulate toxin affinity. Proteins 2005. © 2005 Wiley‐Liss, Inc.
Binding MOAD (Mother Of All Databases)
Authors: Hu Liegi , Benson Mark L. , Smith Richard D. , Lerner Michael G. , Carlson Heather A.
Published: June 2005

Binding MOAD (Mother of All Databases) is the largest collection of high‐quality, protein–ligand complexes available from the Protein Data Bank. At this time, Binding MOAD contains 5331 protein–ligand complexes comprised of 1780 unique protein families and 2630 unique ligands. We have searched the crystallography papers for all 5000+ structures and compiled binding data for 1375 (26%) of the protein–ligand complexes. The binding‐affinity data ranges 13 orders of magnitude. This is the largest collection of binding data reported to date in the literature. We have also addressed the issue of redundancy in the data. To create a nonredundant dataset, one protein from each of the 1780 protein families was chosen as a representative. Representatives were chosen by tightest binding, best resolution, etc. For the 1780 “best” complexes that comprise the nonredundant version of Binding MOAD, 475 (27%) have binding data. This significant collection of protein–ligand complexes will be very useful in elucidating the biophysical patterns of molecular recognition and enzymatic regulation. The complexes with binding‐affinity data will help in the development of improved scoring functions and structure‐based drug discovery techniques. The dataset can be accessed at http://www.BindingMOAD.org. Proteins 2005. © 2005 Wiley‐Liss, Inc.
Phenotype analysis using network motifs derived from changes in regulatory network dynamics
Authors: Cavelier German , Anastassiou Dimitris
Published: June 2005

The intrinsic dynamic response of a transcriptional regulatory network depends directly on molecular interactions in the cellular transcription, translation, and degradation machineries. These interactions can be incorporated into dynamic mathematical models of the biochemical system using the biophysical relationship with the model parameters. Modifications of such interactions bring changes to the biological behavior of the cells, and therefore, many normal and pathological cellular states depend on them. It is important for analysis, prediction, diagnosis, and treatment of cellular function to have an experimentally derived model with parameters that adequately represent the molecular interactions of interest. Finding the model and parameters of a transcriptional regulatory network is a difficult task that has been approached at different levels and with different techniques. We develop here a new analysis method (based on previous work on network inference, modeling, and parameter identification) that finds the most changed parameters from yeast oligonucleotide microarray expression patterns in cases where a phenotype difference exists between two samples. We then relate and examine the changed parameters with their associated genes, corresponding genetic functional categories, and particular subnetworks and connectivities. The biophysical bases for these changes are also identified by studying the relationship of the changed parameters with the transcription, translation, and degradation mechanisms. The method is improved to cases where there are two or more transcription factors influencing transcription, and a statistical analysis is performed to give a measurement of the uniqueness and robustness of the parameter fit. Proteins 2005. © 2005 Wiley‐Liss, Inc.
The role of the liver in metabolic homeostasis: Implications for inborn errors of metabolism
Authors: van den Berghe G.
Published: June 2005

The mechanisms by which the liver maintains a constant supply of oxidizable substrates, which provide energy to the body as a whole, are reviewed. During feeding, the liver builds up energy stores in the form of glycogen and triglyceride, the latter being exported to adipose tissue. During fasting, it releases glucose and ketone bodies. Glucose is formed by degradation of glycogen and by gluconeogenesis from gluconeogenic amino acids provided by muscle. Ketone bodies are produced from fatty acids, released by adipose tissue, and from ketogenic amino acids. The major signals which control the transition between the fed and the fasted state are glucose, insulin and glucagon. These influence directly or indirectly the enzymes which regulate liver carbohydrate and fatty acid metabolism and thereby orient metabolic fluxes towards either energy storage or substrate release. In the fed state, the liver utilizes the energy generated by glucose oxidation to synthesize triglycerides. In the fasted state it utilizes that produced byβ‐oxidation of fatty acids to synthesize glucose. The mechanisms whereby a number of inborn errors of glycogen metabolism, of gluconeogenesis and of ketogenesis cause hypoglycaemia are also briefly overviewed.
Detoxification pathways in the liver
Authors: Grant D. M.
Published: June 2005

The liver plays an important rôle in protecting the organism from potentially toxic chemical insults through its capacity to convert lipophiles into more water‐soluble metabolites which can be efficiently eliminated from the body via the urine. This protective ability of the liver stems from the expression of a wide variety of xenobiotic biotransforming enzymes whose common underlying feature is their ability to catalyse the oxidation, reduction and hydrolysis (Phase I) and/or conjugation (Phase II) of functional groups on drug and chemical molecules. The broad substrate specificity, isoenzyme multiplicity and inducibility of many of these enzyme systems make them particularly well adapted to handling the vast array of different chemical structures in the environment to which we are exposed daily. However, some chemicals may also be converted to more toxic metabolites by certain of these enzymes, implying that variations in the latter may be important predisposing factors for toxicity. Pharmacogenetic defects of xenobiotic biotransformation enzymes, a subclass of inborn errors of metabolism which are manifested only upon drug challenge, introduce marked variation into human populations for the pharmacokinetics and pharmacodynamics of therapeutic and toxic agents, and thus may have important clinical consequences for drug efficacy and toxicity.
Inborn errors of bile acid metabolism
Authors: Clayton P. T.
Published: June 2005

Cholesterol is converted to cholic acid and chenodeoxycholic acid by a series of reactions involving modifications to the steroid nucleus and oxidation of the side chain. These reactions can be affected by a number of inborn errors of metabolism. When this happens unusual bile acids or bile alcohols are synthesized; these can be identified using gas chromatography‐mass spectrometry and fast atom bombardment mass spectrometry techniques. Two defects affecting the modifications to the steroid nucleus have been described; both present with cholestatic liver disease of neonatal onset. The better characterized of the two — 3β‐hydroxy‐Δ5‐C27‐steroid dehydrogenase deficiency — leads to excretion of 3β‐7α‐dihydroxy‐5‐cholenoic acid and 3β,7α,12α‐trihydroxy‐5‐cholenoic acid in the urine. The liver disease improves dramatically on treatment with chenodeoxycholic acid. Deficient activity of 3‐oxo‐Δ4‐steroid 5β‐reductase is thought to be the cause of familial liver disease in some infants who excrete 7α‐hydroxy‐3‐oxo‐4‐cholenoic acid and 7α,12α‐dihydroxy‐3‐oxo‐4‐cholenoic acid in the urine. However, diagnosis of this disorder is problematical; a similar pattern of metabolite excretion can occur as a result of liver damage caused by viruses or inborn errors of pathways unrelated to bile acid synthesis. Defective side chain oxidation in patients with cerebrotendinous xanthomatosis (CTX) leads to synthesis of bile alcohols such as 5β‐cholestane‐3α,7α,12α,25‐tetrol and 5β‐cholestane‐3α,7α,12α,23,25‐pentol. Patients with CTX do not have cholestatic liver disease. Their major problems (neurological disease, atherosclerosis and xanthomata) are caused by accumulation of cholestanol and cholesterol in the tissues. Bile acid precursors are probably diverted into synthesis of cholestanol. Chenodeoxycholic acid suppresses the production of abnormal metabolites from cholesterol (by inhibition of cholesterol 7α‐hydroxylase) and leads to improvement in the neurological disease. Defective side chain oxidation also occurs in peroxisomal disorders but this time it leads to accumulation of C27 bile acids such as 3α,7α,12α‐trihydroxy‐5β‐cholestanoic acid (trihydroxycoprostanic acid, THCA). This compound is readily detected in the bile and plasma of patients with defects of peroxisome biogenesis. In patients with defects of a single peroxisomalβ‐oxidation enzyme (the 3‐hydroxyacyl‐CoA component of the bifunctional protein or the thiolase), the major C27 bile acid in bile may be 3α,7α,12α,24‐tetrahydroxy‐5β‐cholestanoic acid (varanic acid). In addition to the above inborn errors, others which are less well characterized undoubtedly exist, as do defects of bile acid transport across membranes.
Clinical presentation of metabolic liver disease
Authors: Odievre M.
Published: June 2005

Some clinical clues should alert paediatricians to the possibility of metabolic liver diseases. They can be classified into three categories:

Manifestations due to hepatocellular necrosis, acute or subacute, which can reveal galactosaemia, hereditary fructose intolerance, tyrosinaemia type I, Wilson disease andα1‐antitrypsin deficiency. Symptoms and signs suggestive of Reye syndrome should lead to a study of fatty acid oxidation and urea cycle enzymes. All these manifestations may necessitate a rapid diagnosis and treatment when liver dysfunction is severe.

Cholestatic jaundice can revealα1‐antitrypsin deficiency, Byler's disease, cystic fibrosis, Niemann‐Pick disease and some disorders of peroxisome biogenesis.

Hepatomegaly can reveal disorders with liver damage but also storage diseases such as glycogen storage diseases, cholesteryl ester storage disease and, when associated with splenomegaly, lysosomal storage diseases.

Appropriate investigations for recognizing all these entities are proposed.
Investigation of paediatric liver disease
Authors: Kelly D. , Green A.
Published: June 2005

The investigation of children with liver disease falls into two categories: the investigation of the cholestatic baby and the investigation of the older child (over 2 years) with hepatomegaly. The approach to investigation is directed by the clinical features and employs many different investigational methods including biochemistry, haematology, radiology, electrophysiology and histology. As the clinical presentation of many diseases is similar, it is appropriate to perform a variety of first‐line tests, proceeding to more complex investigations only as indicated.
The role of histochemical investigations in metabolic disorders affecting the liver
Authors: Lake B. D.
Published: June 2005

The application of histochemical techniques to the study of metabolic disorders affecting the liver can yield considerable information, provided the methods used are sound and the interpretation is not over‐enthusiastic. The appropriate methods can give insight into liver function and can identify and localize a wide variety of carbohydrates, lipids, proteins and enzyme activities. It is often thought that tissue taken for histochemical analysis cannot be used for morphology, but properly prepared tissue will provide the architectural and cytological detail necessary for histological assessment. There are several advantages to the histochemical approach, the main ones being economy of use of the valuable tissue sample (in theory about 100 sections and tests can be done on a 1 mm depth of tissue) and that the results of the tests can be assessed in relation to the structure of the liver.

There are two areas in which histochemical investigations are used: firstly, to detect cellular constituents, structures and cells not otherwise visible by routine methods. In this mode, histochemistry is an extension of the histological approach and constitutes a ‘super haematoxylin and eosin’ stain. Secondly, it is possible to assess enzyme activities and their localization, and in some well‐defined instances to offer reliable indications of whether there is deficient activity, normal activity or enhanced activity.

Although there is a body of opinion which believes that quantitative enzyme histochemistry is possible and reliable, the author has not found the data, in particular on lysosomal enzymes, to be reliable and remains unconvinced that this technique has a place in the study of pathological tissue.

This review outlines a simple approach to the study of metabolic liver disease and illustrates the findings in a range of disorders of glycogen and lipid metabolism.
Niemann‐Pick disease type C: An update
Authors: Vanier M. T. , Pentchev P. , Rodriguez‐Lafrasse C. , Rousson R.
Published: June 2005

The concept of Niemann‐Pick disease type C as a secondary sphingomyelin storage disorder (in contrast to the sphingomyelinase‐deficient types A and B) has become more and more prevalent, in view of the complex lipid storage pattern and variable sphingomyelinase activities. Although the primary lesion is still unknown, studies conducted over the past six years have led to a breakthrough by showing that this disorder is characterized by unique abnormalities of intracellular translocation of exogenous cholesterol. In cultured fibroblasts of patients, this block leads to a delayed induction of the homeostatic responses to exogenous cholesterol, in particular cholesteryl ester formation, and to the accumulation of unesterified cholesterol in a vesicular, essentially lysosomal, compartment. The transport of endogenous cholesterol is apparently unaffected. The spectrum of phenotypic heterogeneity in relation to abnormal LDL‐processing has been defined in a large patient population. Clinical presentation of the disease is also reviewed and biochemical correlations are discussed. This discovery has had immediate medical applications, by providing the first strategy for reliable prenatal diagnosis of the disorder and easy diagnosis of patients. To date, the exact implication of the cholesterol transport defect in the pathogenesis of Niemann‐Pick type C is not known; recent observations have opened up new possible approaches for the understanding of this lesion. Although final classification of Niemann‐Pick disease type C must await elucidation of the primary defect(s), present knowledge already establishes that the disease is a nosological entity distinct from Niemann‐Pick disease type A and B, and suggests that it might be the model for a new molecular concept of neurolipidosis — and even of inherited metabolic disease.
Screening and economics
Authors: Stilwell J. A.
Published: June 2005

Screening for disease involves expenditure now in order to reap benefits in the future. It is important to understand why future benefits should bediscounted in the cost‐benefit calculation. The reasons are derived from a societal preference for consumption now over consumption tomorrow, combined with the productivity of capital, which enables goods today to be transformed into more goods tomorrow.

It is also necessary to put costs and values on human lives. This is simplified at present in the UK because a highly restrictive immigration policy implies that the net value of the average additional citizen is zero (or even negative).

The cost‐benefit calculations that are presented must be carried out highly systematically in order to avoid double counting or omission. A computerized spreadsheet is ideal for this purpose.
Economic evaluation of cost‐benefit ratio of neonatal screening procedure for phenylketonuria and hypothyroidism
Authors: Dhondt J. ‐L. , Farriaux J. ‐P. , Sailly J. ‐C. , Lebrun T.
Published: June 2005

A comparison between the cost of identification and care of patients with phenylketonuria (PKU) and congenital hypothyroidism (CH) and the expenditure for the care of untreated retarded patients has been established on the basis of the activity of the Nord‐Pas‐de‐Calais regional screening centre and of interviews with patients' families. The analysis yields a benefit‐cost ratio of 6.6 for PKU and 13.8 for CH prophylaxis. However, cost‐benefit varies depending on the economic partner, i.e. the patient's family, Social Security or Administration.
Studies on pyruvate carboxylase from cultured human fibroblasts and amniotic fluid cells
Authors: Hansen Torben L. , Christensen Ernst
Published: June 2005

The properties of pyruvate carboxylase in cultured human fibroblasts were investigated. A pH optimum around pH 7.6 was found in Tris buffer at 37°C. The apparentKm for pyruvate and bicarbonate were 0.22 mmol/l and 2.1 mmol/l respectively. The activity of the crude homogenate was most stable at room temperature. The major end product was identified as citric acid during the assay conditions used. During growth the specific activity increased from 0.5 to 2 nmol/min per mg protein.

The activity of pyruvate carboxylase in the crude homogenate from cultured human fibroblasts was 0.76±0.12 nmol/min per mg protein, while the activity in cultured amniotic fluid cells was 0.66±0.17 nmol/min per mg protein, suggesting the possibility of prenatal diagnosis of pyruvate carboxylase deficiency.
Biochemical studies in the cerebro‐hepato‐renal syndrome of Zellweger: A disturbance in the metabolism of pipecolic acid
Authors: Trijbels J. M. F. , Monnens L. A. H. , Bakkeren J. A. J. M. , Van Raay‐Selten A. H. J. , Corstiaensen J. M. B.
Published: June 2005

The metabolism of pipecolic acid has been studied in three patients suffering from the cerebro‐hepato‐renal syndrome of Zellweger. A marked pipecolic aciduria was observed in these patients and serum levels of pipecolic acid were also elevated. Fromin vivo studies evidence was obtained that a disturbance in the catabolic pathway of pipecolic acid was present in all patients. This conclusion was based on the delayed return of the serum pipecolic acid concentration to the fasting concentration after oral loading of the patients withdl‐pipecolic acid. Moreover, no increase in the excretion of α‐amino adipic acid was observed in the patients after loading, in contrast with the control subjects, who showed a marked increase in the excretion of this metabolite of pipecolic acid. Further evidence for the presence of a metabolic defect in the catabolism of pipecolic acid was obtained from the observation that patients excreted significantly higher amounts of pipecolic acid during the loading experiment.
Review: Preimplantation diagnosis of inherited disease
Authors: Lissens W. , Sermon K. , Staessen C. , Van Assche E. , Janssenswillen C. , Joris H. , Van Steirteghem A. , Liebaers I.
Published: June 2005

Preimplantation diagnosis of inherited diseases has become possible with the techniques ofin vitro fertilization, blastomere biopsy of the 6‐ to 10‐cell embryo and DNA analysis of the single blastomeres. Disease‐free embryos are selected for transfer to the uterus, thereby avoiding the need for termination of a fetus diagnosed as affected in prenatal diagnosis in the first or early‐second trimester of pregnancy. The genetic indications for preimplantation diagnosis are theoretically the same as for prenatal diagnosis, but the defects must be detectable by the polymerase chain reaction. For X‐linked recessive diseases, fluorescencein situ hybridization can be used as an alternative for the selection of female embryos. So far almost 40 healthy children have been born worldwide after preimplantation diagnosis for genetic disease. The possibilities and limitations of preimplantation diagnosis, especially in prevention of inherited disease, are discussed in this review.
Plasma total odd‐chain fatty acids in the monitoring of disorders of propionate, methylmalonate and biotin metabolism
Authors: Çoker M. , de Klerk J. B. C. , Poll‐The B. T. , Huijmans J. G. M. , Duran M.
Published: June 2005

Total plasma odd‐numbered long‐chain fatty acids were analysed in patients with methylmalonic acidaemia (vitamin B12‐responsive and unresponsive), combined methylmalonic acidaemia/homocystinuria (CblC), propionic acidaemia (both neonatal‐onset and late‐onset), biotinidase deficiency and holocarboxylase synthase deficiency, as well as in hospital controls. Total odd‐numbered long‐chain fatty acids (C15:0, C17:1 and C17:0) were expressed as a percentage of total C12–C20 fatty acids. Control values were 0.72%±0.31% (n=12). Normalization of the percentage of odd‐chain fatty acids occurred in all vitamin‐responsive patients, following the institution of vitamin treatment. In general the neonatal‐onset propionic acidaemia and B12‐unresponsive methylmalonic acidaemia patients had the highest plasma odd‐chain fatty acid concentrations, which correlated with the clinical condition but not with the urinary excretion of methylcitrate or methylmalonate. Plasma odd‐chain fatty acid concentrations and methylmalonate excretions in CblC patients reacted very well to vitamin B12 treatment, but with no clinical response. Measurement of plasma odd‐chain fatty acids is of no value for the monitoring of defects of biotin metabolism.
Ataxia associated with increased plasma concentrations of pristanic acid, phytanic acid and C27 bile acids but normal fibroblast branched‐chain fatty acid oxidation
Authors: Clayton P. T. , Johnson A. W. , Mills K. A. , Lynes G. W. , Wilson J. , Casteels M. , Mannaerts G.
Published: June 2005

Investigations of peroxisomal function were undertaken in an 8‐year‐old girl who developed motor difficulties at the age of 3.5 years and went on to develop a progressive ataxia and dysarthria. There were no other neurological abnormalities and she was of normal intelligence. Analysis of plasma very long‐chain fatty acids revealed a normal C26 concentration and normal C24/C22 and C26/C22 ratios. Analysis of branched‐chain fatty acids showed an elevated plasma phytanic acid concentration of 60 µmol/L (normal<15) and a considerably elevated pristanic acid concentration of 50 µmol/L (normal<2). Plasma concentrations of the C27 bile acids 3α,7α‐dihydroxycholestanoic acid (DHCA) and 3α,7α,12α‐trihydroxycholestanoic acid (THCA) and of the C29‐dicarboxylic acid were also increased. We postulated that these results might be due to deficiency of the peroxisomal branched‐chain acyl‐CoA oxidase, but when oxidation of branched‐chain fatty acids was studied in cultured skin fibroblasts it was found to be normal. Alternative explanations for the accumulation of branched‐chain substrates for peroxisomalβ‐oxidation are discussed. Treatment with a low‐phytanic acid diet arrested the progression of the ataxia and led to a slight improvement.
Co‐cultivation of Niemann‐Pick disease type C fibroblasts belonging to complementation groupsα andβ stimulates LDL‐derived cholesterol esterification
Authors: Steinberg S. J. , Mondal D. , Fensom A. H.
Published: June 2005

Niemann‐Pick disease type C (NPC) is a neurovisceral storage disorder with an unknown primary deficiency. Somatic cell hybridization experiments using human cultured fibroblasts have shown that two complementation groups (NPC‐α and NPC‐β) are associated with the biochemical and clinical phenotypes comprising NPC. We identified the rarer complementation group NPC‐β originally using the technique of filipin staining as a marker for complementation. In this study we show that the esterification of cholesterol derived from the LDL pathway can be used as an isotopic assay. However, multinuclear hybrids exhibit a delayed induction in this pathway. Furthermore, we discovered that, in the presence of an LDL source, co‐cultivation of fibroblasts belonging to NPC‐α and NPC‐β stimulated cholesterol esterification.
Cytochrome c oxidase deficiency in subacute necrotizing encephalopathy (Leigh syndrome)
Authors: Hayasaka K. , Brown G. K. , Danks D. M. , Droste M. , Kadenbach B.
Published: June 2005

Tissues and cultured fibroblasts from two patients with Leigh syndrome (subacute necrotizing encephalopathy) were examined. A systemic defect in cytochrome oxidase was identified by enzyme assay and estimation of cytochrome concentrations. Immunochemical analysis showed a reduction of most subunits of the cytochrome oxidase complex.

The rate of synthesis of cytochrome oxidase subunits, determined by labelling experiments in cultured fibroblasts, was the same in the patients and normal controls. The reduced cytochrome oxidase content of the patients' tissues must therefore result from abnormal turnover of the protein subunits.
Deficit of uridine diphosphate galactose in galactosaemia
Authors: Ng W. G. , Xu Y. K. , Kaufman F. R. , Donnell G. N.
Published: June 2005

The levels of uridine diphosphate galactose (UDPGal) and uridine diphosphate glucose (UDPGlc) have been determined in liver autopsy samples, erythrocytes and cultured skin fibroblasts from galactosaemic patients and compared to non‐galactosaemic controls. In patients with undetectable erythrocyte galactose‐1‐phosphate uridyltransferase (transferase) activity, the levels of UDPGal were substantially lower than in controls. In patients with detectable transferase activity, even though in less than 1% of normal values, both UDPGal and UDPGlc levels were in the normal range. Incubation of erythrocytes from both galactosaemic patients and normal individuals with 10 mmol/L uridine increased UDPGal and UDPGlc levels several‐fold, both in the presence or absence of galactose in the incubation medium. We hypothesize that a deficit of UDPGal is responsible for the late onset clinical manifestations in galactosaemia which include ovarian failure, speech defect and neurological abnormalities. We suggest that uridine administration may be of therapeutic value in raising the intracellular concentrations of UDPGal. We conclude that the transferase reaction, however small in activity, is essential for optimal UDPGal formation.
A fluorimetric assay of steroid sulphatase in leukocytes: Evidence for two genetically different enzymes with arylsulphatase C activity
Authors: van Diggelen O. P. , Konstantinidou A. E. , Bousema M. T. , Boer M. , Bakx Th. , Jöbsis A. C.
Published: June 2005

Arylsulphatase C (ASC) activity in leukocytes and fibroblasts measured with 4‐methylumbelliferylsulphate, is caused by at least two genetically different sulphatases. One of these is steroid sulphatase (STS). Depending on the substrate concentration, about 10–50% of the ASC activity in leukocytes can be attributed to sulphatases other than STS. Steroid sulphatase can be measured specifically with 4‐methylumbelliferylsulphate as the fraction of total ASC activity which is inhibitable by dehydroepiandrosterone sulphate. Using this assay, the adjusted ASC activity in leukocytes from patients with X‐linked ichthyosis was 2% of normal. Obligate heterozygotes showed reduced activity.
Use of placental enzyme analysis in assessment of the newborn at risk for nonketotic hyperglycinaemia (NKH)
Authors: Toone J. R. , Applegarth D. A.
Published: June 2005

The incidence of non‐ketotic hyperglycinaemia (NKH), McKusick 23830, in the population of British Columbia, Canada was found to be the same as that reported for Finland. We present the plasma glycine levels in ten non‐ketotic hyperglycinaemia patients and outline difficulties in interpreting plasma glycine levels in their newborn siblings. We propose the use of a placental glycine cleavage enzyme (EC 2.1.1.10) assay to rule out a diagnosis of non‐ketotic hyperglycinaemia in at‐risk infants during the first few days of life.
Prenatal diagnosis of 3‐Hydroxy‐3‐methylglutaric aciduria by GC‐MS and enzymology on cultured amniocytes and chorionic villi
Authors: Chalmers R. A. , Tracey B. M. , Mistry J. , Stacey T. E. , McFadyen I. R.
Published: June 2005

This paper reports the prenatal diagnosis of HMG CoA lyase deficiency at 16 weeks' gestation by direct chemical analysis of cell‐free amniotic fluid and by measurement of HMG CoA lyase activity in cultured amniocytes. Termination of an affected fetus allowed study of chorionic villus tissue, the results providing the basis for future first trimester prenatal diagnoses of this condition. An abstract report of this work has appeared elsewhere (Chalmerset al., 1985).
Normal growth and development with unrestricted protein intake after severe infantile propionic acidaemia
Authors: Luder A. S. , Yannicelli S. , Green C. L.
Published: June 2005

A child with propionic acidaemia, after a stormy infantile course complicated by microcephaly, has shown normal subsequent growth and development without dietary protein restriction.
Recent advances in the molecular genetics of the neuronal ceroid lipofuscinoses
Authors: Mole S. E.
Published: June 2005

Major advances in the molecular genetic analysis of the neuronal ceroid lipofuscinoses (NCL) have recently been made: the genes for two major types have been identified and the chromosomal location for a third defined.CLN1, the gene for infantile NCL (Santavuori‐Haltia disease) encodes palmitoyl protein thioesterase (PPT). Most patients (75% of disease chromosomes) have the same point mutation. In contrast,CLN3, the gene for juvenile NCL (Batten or Spielmeyer‐Vogt‐Sjögren disease) is not a previously known gene, nor does its product display homology to any previously described proteins. The same 1 kb genomic deletion is present in the majority of patients (81% of disease chromosomes).CLN5, the gene for Finnish variant late infantile NCL, has been mapped to 13q and should be identified in the near future. The gene for late‐infantile NCL (Jansky‐Bielschowsky disease) has not yet been localized to a chromosome despite intensive research. It is likely that this type of NCL is caused by mutations in more than one gene each resulting in the same phenotype.
A fluorimetric enzyme assay for the diagnosis of sanfilippo disease type A (MPS IIIA)
Authors: Karpova E. A. , Voznyi Ya. V. , Keulemans J. L. M. , Hoogeveen A. T. , Winchester B. , Tsvetkova I. V. , van Diggelen O. P.
Published: June 2005

4‐Methylumbelliferyl‐α‐d‐N‐sulphoglucosaminide (MU‐α‐GlcNS) was synthesized and shown to be a substrate for the lysosomal heparin sulphamidase. Sanfilippo A patients' fibroblasts (n=42) and lymphocytes (n=1) showed 0–3% of mean normal heparin sulphamidase activity; in total leukocytes from patients (n=8) sulphamidase activity was clearly deficient. In fibroblasts from obligate heterozygotes for Sanfilippo A, the sulphamidase activity was reduced in 9 out of 10 cases. Heparin sulphamidase desulphates MU‐αGlcNS to MU‐αGlcNH2 and further hydrolysis during a second incubation is required to liberate 4‐methylumbelliferone, which can be measured. Yeastα‐glucosidase, which has low but sufficientα‐glucosaminidase activity, was used to hydrolyse the reaction intermediate MU‐αGlcNH2 to release 4‐methylumbelliferone and free glucosamine.
Clinical and molecular heterogeneity of cytochromec oxidase deficiency in the newborn
Authors: Lombes A. , Romero N. B. , Touati G. , Frachon P. , Cheval M. A. , Giraud M. , Simon D. , Ogier de Baulny H.
Published: June 2005

We report 8 cases of severe cytochromec oxidase deficiency with onset in the neonatal period. Clinical symptoms were heterogeneous: antenatal cerebral malformations, neurological distress with ketoacidosis, severe myopathy, or isolated respiratory control failure. Lactic acid was elevated in blood and/or CSF in 7 cases. Muscle biopsy (7 patients), liver biopsy (4 patients), and cultured skin fibroblasts (7 patients) were used to assess the cytochromec oxidase deficiency. Among the patients, the enzymatic defect differed in the level of residual activity, expression in different tissues and subunit composition in muscle (as analysed by immunohistochemistry). Southern blot analysis of the mitochondrial DNA was normal in 7 patients. The heterogeneity of cytochromec oxidase deficiency was therefore demonstrated by these clinical presentations and by the biochemical assessment of the enzyme defect. This reflects, most probably, the diverse nature of the causal mutations.
Two siblings with episodic ketoacidosis and decreased activity of succinyl‐CoA:3‐ketoacid CoA‐transferase in cultured fibroblasts
Authors: Pretorius C. J. , Loy Son G. G. , Bonnici F. , Harley E. H.
Published: June 2005

Succinyl‐CoA:3‐ketoacid CoA‐transferase deficiency leads to a severe ketoacidosis presenting in infancy. We describe two siblings of African ancestry who presented with repeated episodes of ketoacidosis. Both had a positive test for salicylate in the absence of salicylate ingestion. Analysis of urine for organic acids revealed the presence of acetoacetate and 3‐hydroxybutyrate. Succinyl‐CoA:3‐ketoacid CoA‐transferase activities in cultured fibroblasts were 11% and 18% of control values.
Metabolic abnormalities in feline Niemann‐Pick type C heterozygotes
Authors: Brown D. E. , Thrall M. A. , Walkley S. U. , Wurzelmann S. , Wenger D. A. , Allison R. W. , Just C. A.
Published: June 2005

Niemann‐Pick disease type C (NPC) is an autosomal recessive neurovisceral lysosomal storage disorder in which cholesterol lipidosis results from defective intracellular transport of unesterified cholesterol. The primary molecular defect of NPC is unknown; regulatory mechanisms of cholesterol metabolism are impaired, resulting in retarded esterification of exogenous cholesterol with accumulation of unesterified cholesterol in lysosomes and secondary storage of glycolipids and sphingomyelin. In obligate heterozygotes from a feline NPC model, cultured skin fibroblasts challenged with exogenously derived cholesterol exhibited intermediate rates of cholesterol esterification and accumulation of unesterified cholesterol. Liver lipid analyses of obligate heterozygote cats demonstrated intermediate cholesterol and sphingomyelin concentrations. Vacuolated skin fibroblasts were found in 2 of 3 heterozygote cats, and occasional cortical neurons exhibited intracellular inclusions immunoreactive for GM2‐ganglioside. Ultrastructural studies provided evidence of storage in liver and brain. We believe these morphological and biochemical findings are the first example of manifestations of CNS abnormalities in a genetic carrier for a neuronal storage disease.
Marked increase of methylumbelliferyl‐tetra‐N‐acetylchitotetraoside hydrolase activity in plasma from gaucher disease patients
Authors: den Tandt W. R. , van Hoof F.
Published: June 2005

Methylumbelliferyl‐tetra‐N‐acetylchitotetraoside hydrolase activity was increased 53‐ to 484‐fold in plasma from Gaucher disease patients and no activator could be found. High activity was also measured in other lysosomal storage diseases including Krabbe disease, Wolman disease, GM1‐gangliosidosis and to a lesser extent Niemann‐Pick disease type B, but the activities were lower than the lowest values in Gaucher patients.

Kinetic properties of the high activity in Gaucher plasma were similar to those of controls. It is not known whether the increased activity represents intrinsic enzyme activity or increased enzyme concentration. It is possible that this enzyme may help in the detection of Gaucher disease or in the assessment of enzyme therapy withβ‐d‐glucosidase (Ceredase®).
Chorionic villus sampling: Diagnostic uses and limitations of enzyme assays
Authors: Fowler B. , Giles L. , Cooper A. , Sardharwalla I. B.
Published: June 2005

Control ranges for enzymes in uncultured chorionic villi were established, based on: (1) 21 of 22 enzymes (mainly lysosomal) in villi had similar properties to the enzyme in cultured fibroblasts; (2) isoenzyme patterns in villi were similar to those in fibroblasts for five lysosomal enzymes but different for aryl sulphatases; (3) control ranges were determined for 12 enzymes in abortion villi and for 21 enzymes in biopsy villi, values tending to be higher in the latter for those enzymes studied in both types of sample; (4) storage of samples under various conditions revealed no major changes in activity of seven lysosomal enzymes.

A number of potential pitfalls in the use of chorionic villus samples for diagnosis of metabolic disorders by enzyme assay are described: (1) the presence of aryl sulphatase C in chorionic villi, an isoenzyme which may interfere in assays of aryl sulphatase A; (2) the presence of maternal enzyme in chorionic villus material illustrated by the detection of the A isoenzyme of B‐hexosaminidase in chorionic villus from a pregnancy affected with Sandhoff's disease; (3) the finding of falsely normal levels of α‐iduronidase in chorionic villus samples from a pregnancy affected with Hurler's disease, probably due to contamination with maternal tissue which has relatively high levels of this enzyme compared with fetal chorionic material: (4) the inadequacy of indirect assays of incorporation of radiolabel into macromolecules using chorionic villi, for example [14C]propionate incorporation for prenatal diagnosis of methylmalonic aciduria.

Provided that such pitfalls are recognized and great care is taken in selection of villus samples and interpretation of results, chorionic villus sampling allows reliable prenatal diagnosis of a large number of disorders using enzyme asays.
Prenatal diagnosis and prevention of inherited abnormalities of collagen
Authors: Pope F. M. , Daw S. C. M. , Narcisi P. , Richards A. R. , Nicholls A. C.
Published: June 2005

There is now strong evidence for the implication of collagen α1(I), α2(I) and α1(III) mutations in many forms of osteogenesis imperfecta and inherited arterial aneurysms (Ehlers Danlos syndrome type IV). A sizeable proportion of these disorders have detectable abnormalities by conventional protein chemistry, immunofluorescence, or more sophisticated DNA analysis. Everyone of them with specific defects or with linkage to appropriate gene markers is therefore amenable to prevention using conventional prenatal diagnosis by chorionic villus biopsy (with fibroblast culture), fetoscopic biopsy (with fibroblast culture), ultrasound diagnosis of the severely deformed fetus, or gene linkage studies by chorionic villus biopsy or amniocentesis.

Already many collagen α1(I), α2(I) and α1(III) mutations have been characterized including point mutations, small and large deletions and regulatory mutations. Many others are likely to be rapidly studied by exploiting recent advances in DNA technology, and other strong candidate genes include collagen II (some chondrodystrophies), collagen VI (certain arterial and cardiovascular diseases) and collagen VII (dystrophic epidermolysis bullosa). Other important common diseases are likely to include osteoporosis, osteoarthritis and cerebral aneurysms.

A detailed review is provided of collagen interstitial genes and proteins, together with a description of the various forms of osteogenesis imperfecta and Ehlers Danlos syndrome in which either collagen α1(I), α2(I) or α1(III) mutations have been identified. Appropriate restriction length polymorphisms (RFLPs) useful in identifying carriers of these mutant genes are also described.
Prenatal diagnosis of Duchenne muscular dystrophy: A three‐year experience in a rapidly evolving field
Authors: Bakker E. , Bonten E. J. , Veenema H. , den Dunnen J. T. , Grootscholten P. M. , van Ommen G. J. B. , Pearson P. L.
Published: June 2005

Application of molecular genetic techniques has greatly increased diagnostic possibilities of hereditary disorders. In 1983 the first linkage of Duchenne muscular dystrophy with flanking DNA probes was described, which made carrier detection possible in a limited number of cases. The first published prenatal diagnosis for Duchenne muscular dystrophy dates from 1985. DNA‐analysis for Duchenne muscular dystrophy and Becker muscular dystrophy has become increasingly informative, firstly by the development of more flanking markers, followed by intragenic probes detecting deletions and, more recently, by the use of cDNA probes detecting a deletion or duplication mutation in over 60% of the Duchenne and Becker muscular dystrophy patients. Although these developments allow a highly reliable (>99%) carrier detection and prenatal diagnosis in over 90% of cases, the continuing introduction of new probes and/or technologies has necessitated constant reappraisal of many families to derive maximum information. During the past 3 years we applied prenatal diagnosis for Duchenne and Becker muscular dystrophies with DNA‐analysis on 53 male fetuses in 47 families. Twenty‐two healthy male babies were born, after being diagnosed to have a low Duchenne muscular dystrophy risk. Two pregnancies also diagnosed as low risk have not yet come to term. In the other cases a high risk for Duchenne muscular dystrophy was found and the parents chose abortion. Our studies also revealed a number of important diagnostic pitfalls, such as non‐paternity, karyotypic anomalies and gonadal mosaicism.