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Biomedical subjects

N Horn

Publications and source records attributed to N Horn.

At least 19 recordsLinked to original sources
PubMed2005

Impaired energy metabolism and abnormal muscle histology in mut- methylmalonic aciduria.

The authors report a 27-year-old man with B12-responsive mut- methylmalonic aciduria associated with pure muscle symptoms. Two mutations were found in the methylmalonyl-CoA mutase gene. An exercise test showed a reduced maximal workload and reduced oxygen uptake, and a muscle biopsy showed subsarcolemmal accumulation of mitochondria and normal respiratory chain enzyme activities. These findings may be caused by inhibition of mitochondrial energy metabolism by methylmalonate or its metabolites.

Adult↗
PubMed2005

Congenital cataract, muscular hypotonia, developmental delay and sensorineural hearing loss associated with a defect in copper metabolism.

Deficiencies of different proteins involved in copper metabolism have been reported to cause human diseases. Well-known syndromes, for example, are Menkes and Wilson diseases. Here we report a patient presenting with congenital cataract, severe muscular hypotonia, developmental delay, sensorineural hearing loss and cytochrome-c oxidase deficiency with repeatedly low copper and ceruloplasmin levels. These findings were suggestive of a copper metabolism disorder. In support of this, the patient's fibroblasts showed an increased copper uptake with normal retention. Detailed follow-up examinations were performed. Immunoblotting for several proteins including ATP7A (MNK or Menkes protein), ATP7B (Wilson protein) and SOD1 showed normal results, implying a copper metabolism defect other than Wilson or Menkes disease. Sequence analysis of ATOX1 and genes coding for proteins that are known to play a role in the mitochondrial copper metabolism (COI-III, SCO1, SCO2, COX11, COX17, COX19) revealed no mutations. Additional disease genes that have been associated with cytochrome-c oxidase deficiency were negative for mutations as well. As beneficial effects of copper histidinate supplementation have been reported in selected disorders of copper metabolism presenting with low serum copper and ceruloplasmin levels, we initiated a copper histidinate supplementation. Remarkable improvement of clinical symptoms was observed, with complete restoration of cytochrome-c oxidase activity in skeletal muscle.

Adenosine Triphosphatases↗
PubMed2004

Minimum alveolar concentration (MAC) of xenon in intubated swine.

BACKGROUND: The minimum alveolar concentration (MAC) is a traditional index of the hypnotic potency of an inhalational anaesthetic. To investigate the anaesthetic as well as the unwanted effects of xenon (Xe) in a swine model, it is useful to know MAC(Xe). METHODS: The study was performed using ten swine (weight 27.8-35.4 kg) anaesthetized with halothane and Xe 0, 15, 30, 40, 50 and 65% in oxygen. With each Xe concentration, various concentrations of halothane were administered in a step-by-step design. For each combination, a supramaximal pain stimulus (claw clamp) was applied and the appearance of a withdrawal reaction was recorded. The MAC(Xe) with halothane was calculated using a logistic regression model. RESULTS: During stable ventilation, haemodynamics and temperature, MAC(Xe) value was determined as 119 vol. % (95% confidence limits 103-135). CONCLUSION: MAC(Xe) in swine was calculated by extrapolation of a logistic regression model. Its theoretical value is 119 vol. %.

Anesthesia, Closed-Circuit↗
PubMed2004

Xenon, a modern anaesthesia gas.

Xenon is an interesting anesthetic as it appears to lack negative inotropicy and vasodilatation, giving great advantages to both patients with limited cardiovascular reserve or those who require hemodynamic stability. It has low toxicity and is not teratogenic. Xenon gives rapid induction and recovery, due to its low blood/gas partition coefficient (0.15), and has a MAC of 63%. Several vitro studies showed that Xenon may protect neural cells against ischaemic injury. Its low blood solubility can take to diffusion hypoxia if Xenon is not substituted by 100% oxygen at the end of anesthesia. It has been shown that, compared to other anesthetic regimens, Xenon anesthesia produces the highest regional blood flow in the brain, liver, kidney and intestine. In conclusion, the most important positive effects of Xenon are cardiovascular stability, cerebral protection and favourable pharmacokinetics. Negative points are high cost and the limited number of ventilators supplying Xenon.

Anesthetics, Inhalation↗
PubMed2003

Heterologous production of bacteriocins by lactic acid bacteria.

Over the last two decades, bacteriocins produced by lactic acid bacteria (LAB) have been the subject of considerable research and industrial interest due to their potential as food biopreservatives. The development of heterologous expression systems for such antimicrobial compounds may offer a number of advantages over native systems, such as facilitating the control of bacteriocin gene expression or achieving higher production levels. In addition, the heterologous production by food-grade LAB offers an attractive method for overcoming some of the adverse situations that may affect the effectiveness of some bacteriocins in food systems. Construction of multibacteriocinogenic strains or acquisition of antimicrobial properties by industrial strains are further objectives that can be achieved through the use of heterologous gene expression systems. The development of new biotechnological tools and recent advances in LAB genetics account for the escalating number of studies dealing with heterologous production of bacteriocins by such hosts. This paper reviews the literature published on the subject and compares the different experimental strategies that have been used up to the present for this purpose.

Bacteriocins↗
PubMed2002

X-linked recessive Menkes disease: carrier detection in the case of a partial gene deletion.

X-linked recessive Menkes disease is a lethal disorder of copper metabolism, caused by defects in the ATP7A gene. About 15% of the mutations causing Menkes disease are partial gene deletions. We have previously demonstrated carrier diagnosis of deletions in heterozygotes by Southern blot analysis. As this technique is very time-consuming alternative methods are obviously of high value. Multiplex polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR) and spanning the deletion on genomic DNA can all be used for detection of partial gene deletions in male patients, but only spanning of the deletion can be applied for carrier detection. Simple multiplex PCR is not applicable for carrier detection because the normal allele of ATP7A will be PCR amplified thus masking the deletion. Here, we demonstrate, in addition to spanning of the deletion on genomic DNA, carrier detection based on the use of a previously unrecognized polymorphism in intron 13 of ATP7A in combination with previously identified intragenic polymorphic markers. We show that these intragenic markers can be used for carrier detection, not only indirectly by determining segregation of the disease related allele but also directly if located within the deleted region. We demonstrate determination of the carrier status of 21 at-risk carriers.

Exons↗
PubMed2002

X-linked recessive Menkes disease: identification of partial gene deletions in affected males.

Menkes disease is an X-linked recessive lethal disorder of copper metabolism, caused by defects in the ATP7A gene. Partial gene deletions comprise about 15% of the mutations causing Menkes disease. We have previously demonstrated identification of partial ATP7A deletions in patients by Southern blot analysis. In the present study, we report the use of three fast and reliable polymerase chain reaction (PCR)-based methods for the identification of partial ATP7A deletions in Menkes disease patients. First we demonstrate the use of multiplex PCR, a fast method for identification and rough localization of partial gene deletions, in which two exons of ATP7A are coamplified. Second, we present PCR amplification of genomic DNA across the deletion junctions, a method enabling identification of the deletion breakpoints and hence the exact size of the deletion. Finally, application of reverse transcription PCR (RT-PCR) for identification and localization of gene deletions at the cDNA level is demonstrated. By studying the mutation at the cDNA level the predicted effect of the mutation on the amino acid sequence and consequently the protein structure and function can be inferred. We demonstrate characterization of partial gene deletions in five patients, and in three of these we were able to determine the breakpoint sequences.

Adenosine Triphosphatases↗
PubMed2001

Cytochrome c oxidase deficiency due to mutations in SCO2, encoding a mitochondrial copper-binding protein, is rescued by copper in human myoblasts.

Mutations in SCO2, a cytochrome c oxidase (COX) assembly gene, have been reported in nine infants with early onset fatal cardioencephalomyopathy and a severe COX deficiency in striated muscle. Studies on a yeast homolog have suggested that human Sco2 acts as a copper chaperone, transporting copper to the Cu(A) site on the Cox II subunit, but the mechanism of action remains unclear. To investigate the molecular basis of pathogenesis of Sco2 defects in humans we performed genetic and biochemical studies on tissues, myoblasts and fibroblasts from affected patients, as well as on a recombinant human C-terminal Sco2 segment (22 kDa), bearing the putative CxxxC metal-binding motif. Recombinant Sco2 was shown to bind copper with a 1:1 stoichiometry and to form homomeric complexes in vitro, independent of the metal-binding motif. Immunohistochemistry using antibodies directed against different COX subunits showed a marked tissue-specific decrease in the Cox II/III subunits that form part of the catalytic core, consistent with the differential tissue involvement, but a more uniform distribution of Cox Vab, a nuclear-encoded subunit. Sco2 was severely reduced in patient fibroblasts and myoblasts by immunoblot analysis. Patient fibroblasts showed increased (64)Cu uptake but normal retention values and, consistent with this, the copper concentration was four times higher in Sco2-deficient myoblasts than in controls. COX activity in patient myoblasts was completely rescued by transduction with a retroviral vector expressing the human SCO2 coding sequence, and more interestingly by addition of copper-histidine (300 microM) to the culture medium. Whether the latter is accomplished by the very low residual levels of Sco2 in the patient cells, direct addition of copper to the Cu(A) site, or by another copper-binding protein remains unknown. Whatever the mechanism, this result suggests a possible therapy for the early treatment of this fatal infantile disease.

Amino Acid Motifs↗
PubMed2001

Homozygosity (E140K) in SCO2 causes delayed infantile onset of cardiomyopathy and neuropathy.

OBJECTIVE: To report three unrelated infants with a distinctive phenotype of Leigh-like syndrome, neurogenic muscular atrophy, and hypertrophic obstructive cardiomyopathy. The patients all had a homozygous missense mutation in SCO2. BACKGROUND: SCO2 encodes a mitochondrial inner membrane protein, thought to function as a copper transporter to cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. Mutations in SCO2 have been described in patients with severe COX deficiency and early onset fatal infantile hypertrophic cardioencephalomyopathy. All patients so far reported are compound heterozygotes for a missense mutation (E140K) near the predicted CxxxC metal binding motif; however, recent functional studies of the homologous mutation in yeast failed to demonstrate an effect on respiration. METHODS: Here we present clinical, biochemical, morphologic, functional, MRI, and MRS data in two infants, and a short report in an additional patient, all carrying a homozygous G1541A transition (E140K). RESULTS: The disease onset and symptoms differed significantly from those in compound heterozygotes. MRI and muscle morphology demonstrated an age-dependent progression of disease with predominant involvement of white matter, late appearance of basal ganglia lesions, and neurogenic muscular atrophy in addition to the relatively late onset of hypertrophic cardiomyopathy. The copper uptake of cultured fibroblasts was significantly increased. CONCLUSIONS: The clinical spectrum of SCO2 deficiency includes the delayed development of hypertrophic obstructive cardiomyopathy and severe neurogenic muscular atrophy. There is increased copper uptake in patients' fibroblasts indicating that the G1541A mutation effects cellular copper metabolism.

Age of Onset↗
PubMed2001

Multisystem disorder associated with a missense mutation in the mitochondrial cytochrome b gene.

Mitochondrial cytochrome b mutations have been reported to have a homogenous phenotype of pure exercise intolerance. We describe a novel mutation in the cytochrome b gene of mitochondrial DNA (A15579G) associated with a selective decrease of muscle complex III activity in a patient who, besides severe exercise intolerance, also has multisystem manifestations (deafness, mental retardation, retinitis pigmentosa, cataract, growth retardation, epilepsy). The point mutation is heteroplasmic in muscle (88%) and leukocytes (15%), and changes a highly conserved tyrosine to cysteine at amino acid position 278.

Adult↗
PubMed2001

Cosmix-plexing: a novel recombinatorial approach for evolutionary selection from combinatorial libraries.

The efficiency of existing combinatorial biological library methods has been moderate in terms of the success rates, the affinities of the ligands selected and the time and effort involved in trying to optimize the initial leads. Although mimicking natural evolution, existing strategies take little notice of the importance of recombination within a selected population to generate increased diversity. We present an overview of our recent progress which has resulted in the successful development of such a strategy, which we designate cosmix-plexing. We incorporate recombination as a central feature in obtaining high success rates and high affinities, even for short monomer peptides, in a very short time. The method uses type II restriction enzymes to re-assort small hypervariable DNA cassettes from an intermediate pre-selected population (e.g. from a phagemid display library), while maintaining the original open-reading frame. Since, in the naive library, each cassette contains all possible combinations of the polypeptide sequences it encodes, much longer regions can be optimized than was possible with methods which depend on a simple selection from the naive library. Short peptides can now be rapidly selected, which exhibit the same, or higher, specificity and affinity for a defined target molecule, than (say) an antibody or even the natural ligand.

Amino Acid Sequence↗
PubMed2001

An mtDNA mutation, 14453G-->A, in the NADH dehydrogenase subunit 6 associated with severe MELAS syndrome.

We report a novel point mutation in the gene for the mitochondrially encoded ND6 subunit of the NADH:ubiquinone oxidoreductase (complex I of the respiratory chain) in a patient with MELAS syndrome. The mutation causes a change from alanine to valine in the most conserved region of the ND6 subunit. The patient was heteroplasmic for the mutation in both muscle and blood, but the mutation was not detected in the patient's mother. A marked reduction of complex I activity was found in the patient's muscular tissue. This is the first report of a mutation in the ND6 subunit causing MELAS. Our data confirm the genetic heterogeneity in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome, and confirms that MELAS can be caused by mutation in polypeptide-coding mtDNA genes.

Base Sequence↗
PubMed2001

Disturbed copper transport in humans. Part 1: mutations of the ATP7A gene lead to Menkes disease and occipital horn syndrome.

Mutations of the ATP7A gene (OMIM 300011) lead to the Menkes disease (MD, OMIM 309400) involving impaired brain development, neurological degeneration, connective tissue abnormalities, and high lethality in early infancy. Occipital horn syndrome (OHS, OMIM 304150), a milder phenotype, is also caused by ATP7A gene mutations. In MD patients, an early copper-histidine treatment may prevent the neurological impairment and prolong survival leading to an OHS phenotype. To demonstrate the genotype/phenotype correlation, two male patients are reported with different ATP7A gene mutations and several phenotypes. In the first patient with the MD phenotype, a mutation within the exon 20 (Gln1288Ter) was found producing a stop codon just prior to the highly conserved ATP binding domain. The OHS phenotype of the second patient was caused by a splice site mutation involving the position +6 of intron 6 within a copper binding domain. Small amounts of correctly spliced ATP7A transcript were sufficient to develop the milder OHS phenotype in this patient (OMIM 30001.0006). In conclusion, mutations of the copper transporting P-type ATPase ATP7A gene cause distinct human diseases showing some genotype/phenotype correlation and implications for treatment.

Adenosine Triphosphatases↗
PubMed2001

Disturbed copper transport in humans. Part 2: mutations of the ATP7B gene lead to Wilson disease (WD).

Mutations in the Wilson disease gene ATP7B, a P-type ATPase, are responsible for copper accumulation in the liver and other organs leading to Wilson disease (WD, OMIM 277900). Clinical manifestations of Wilson disease (WD) include chronic liver disease, acute hepatic failure or neuropsychiatric diseases. Since potent medical treatments are available to prevent disabling residual symptoms, early diagnosis is crucial. To demonstrate the clinical course and genetic findings, a male patient with a novel mutation in the ATP7B gene, a 10 base pair insertion in exon 6 (1927ins 10), and a second missense mutation in exon 13 (P992L) is reported. The patient presented with signs of chronic liver disease at the age of 10 years. Clinical findings included hepatomegaly, elevated liver enzymes and coagulopathy. A combination treatment with the copper chelating agent D-penicillamine and zinc acetate was started leading to normalization of liver function and no appearance of neurological signs or Kayser-Fleischer ring after 7 years follow-up. Truncating mutations of the ATP7B gene (insertions, deletions, nonsense mutations) leading to gross loss of C-terminal parts of the protein, thereby probably completely destroying the protein function, may correlate with a hepatic phenotype and early onset as seen in the patient presented.

Adenosine Triphosphatases↗
PubMed2000

Characterization of the hCTR1 gene: genomic organization, functional expression, and identification of a highly homologous processed gene.

The human hCTR1 gene was originally identified by its ability to complement a yeast mutant deficient in high-affinity copper uptake (Zhou, B., Gitschier, J., 1997. A human gene for copper uptake identified by complementation in yeast. Proc. Natl. Acad. Sci. USA 94, 7481-7486). Here, we have determined the DNA sequence of the exon-intron borders of the hCTR1 structural gene and report that the coding sequence is disrupted by three introns, all of which comply with the GT/AG rule. Furthermore, human fibroblasts, transfected with hCTR1 cDNA, were shown to have a dramatically increased capacity for (64)Cu uptake, indicating that the hCtr1 protein is functional in copper uptake in human cells. In contrast, no evidence was found for involvement of the hCTR2 gene product in copper uptake. Finally, we have identified a highly homologous processed pseudogene, hCTR1psi, which was localized to chromosome 3q25/26. The processed gene was found to be transcribed, but due to a frame shift mutation, it only had the potential to encode a truncated protein of 95 amino acid residues, and cells transfected with hCTR1psi DNA showed no increase of (64)Cu uptake.

Amino Acid Sequence↗
PubMed2000

Similar splice-site mutations of the ATP7A gene lead to different phenotypes: classical Menkes disease or occipital horn syndrome.

More than 150 point mutations have now been identified in the ATP7A gene. Most of these mutations lead to the classic form of Menkes disease (MD), and a few lead to the milder occipital horn syndrome (OHS). To get a better understanding of molecular changes leading to classic MD and OHS, we took advantage of the unique finding of three patients with similar mutations but different phenotypes. Although all three patients had mutations located in the splice-donor site of intron 6, only two of the patients had the MD phenotype; the third had the OHS phenotype. Fibroblast cultures from the three patients were analyzed by reverse transcriptase (RT)-PCR to try to find an explanation of the different phenotypes. In all three patients, exon 6 was deleted in the majority of the ATP7A transcripts. However, by RT-PCR amplification with an exon 6-specific primer, we were able to amplify exon 6-containing mRNA products from all three patients, even though they were in low abundance. Sequencing of these products indicated that only the patient with OHS had correctly spliced exon 6-containing transcripts. We used two different methods of quantitative RT-PCR analysis and found that the level of correctly spliced mRNA in this patient was 2%-5% of the level found in unaffected individuals. These findings indicate that the presence of barely detectable amounts of correctly spliced ATP7A transcript is sufficient to permit the development of the milder OHS phenotype, as opposed to classic MD.

Adenosine Triphosphatases↗