Insight into the Pathology of a COL1A1 Signal Peptide Heterozygous Mutation Leading to Severe Osteogenesis Imperfecta.

Osteogenesis imperfecta or "brittle bone disease" is a congenital disorder of connective tissue causing the bone to break easily. Around 85-90% of cases are due to autosomal dominant mutations in the genes encoding type I collagen, the major organic component of bone. Genotype-phenotype correlations have shown that quantitative defects of collagen type I lead to mild OI, whereas structural defects show a wide clinical range from mild to perinatal lethal. This may partially be explained by the type of amino acid substitution and the relative location in the domain structure. To fully understand the variability of the clinical manifestation and the underlying pathomechanisms, further investigations are required. Here we provide the first biochemical characterization of a mutation at the signal peptide cleavage site of COL1A1, a domain not yet characterized. By steady-state analysis, we observed reduced production of collagen type I. Furthermore, by pulse-chase analysis we detected delayed secretion and partial intracellular retention of collagen I. In the cellular fraction, the electrophoretic migration was abnormal; however, secreted type I collagen showed a normal migration pattern. The intracellular retention of collagen I was confirmed by immunofluorescent staining. Moreover, transmission electron microscopy of cultured fibroblasts revealed enlargement of ER cisternae. These results further support the hypothesis that mechanisms interfering with ER integrity play an important role in the pathology of severe OI.

Hallazgos bucales en niños con enfermedad renal / Buccal finding in children with renal disease

La frecuencia e incidencia de enfermedades renales en niños ha sido documentada, destacándose las disfunciones tubulares, acidosis tubular, insuficiencia renal crónica, litiasis renal, entre otras. El objetivo de la investigación fue caracterizar los hallazgos bucales en niños de 1 a 13 años con enfermedad renal. Se realizó una investigación de tipo descriptiva con diseño transversal durante enero-abril de 2010, en el Servicio de Nefrología Pediátrica de la Ciudad Hospitalaria "Dr. Enrique Tejera", en Valencia-Estado Carabobo, Venezuela, se evaluaron 50 pacientes con edad promedio 6.85 años, con diversas enfermedades renales, la mayoría con hipercalciuria (58%) y sin otro compromiso sistémico. Los hallazgos bucales encontrados fueron hipoplasias de esmalte 30%, caries dental 36%, retardo en la erupción 16%, maloclusiones 10% y lengua geográfica 6%; ningún paciente presentó cálculo dental. Un dato importante fue que 44% de los pacientes nunca habían asistido a consulta odontológica. Se concluye que los hallazgos bucales con mayor frecuencia encontrados en niños con enfermedad renal fueron hipoplasias del esmalte, caries dental y retardo en la erupción dentaria, destacándose el desinterés por parte de los pacientes a la consulta odontológica

The frequency and incidence of renal disease in children had been documented, highlighting tubular dysfunctions, tubular acidosis, renal insufficiency, renal lithiasis, among others. The aim of this investigation was to characterize the buccal findings in children of 1 to 13 years old with renal disease. The investigation was a descriptive type, in a transversal design during january-april of 2010, in the Pediatric Nephrologic Service of the Hospital City "Dr. Enrique Tejera", in Valencia, Carabobo State, Venezuela, there were 50 patients with a mean age 6.85 years old, with hypercalciuria in 56% with no other systemic disease. The buccal findings were enamel hypoplasia 30%, dental caries 36%, delay eruption 16%, maloclussion 10% and geographic tongue 6%; none of the patients presented dental calculus. An important data was that 44% of the patients never had assisted to a dental appointment. The conclusions of this study are that the buccal findings more seen in children with renal disease are enamel hypoplasias, dental caries and delay eruption, underlying the lack of interest of the patients to the dental consult

Hallazgos bucales en niños con enfermedad bucal / Buccal finding in children with renal disease

La frecuencia e incidencia de enfermedades renales en niños ha sido documentada, destacándose las disfunciones tubulares, acidosis tubular, insuficiencia renal crónica, litiasis renal, entre otras. El objetivo de la investigación fue caracterizar los hallazgos bucales en niños de 1 a 13 años con enfermedad renal. Se realizó una investigación de tipo descriptiva con diseño transversal durante enero-abril de 2010, en el Servicio de Nefrología Pediátrica de la Ciudad Hospitalaria "Dr. Enrique Tejera", en Valencia-Estado Carabobo, Venezuela, se evaluaron 50 pacientes con edad promedio 6.85 años, con diversas enfermedades renales, la mayoría con hipercalciuria (58%) y sin otro compromiso sistémico. Los hallazgos bucales encontrados fueron hipoplasias de esmalte 30%, caries dental 36%, retardo en la erupción 16%, maloclusiones 10% y lengua geográfica 6%; ningún paciente presentó cálculo dental. Un dato importante fue que 44% de los pacientes nunca habían asistido a consulta odontológica. Se concluye que los hallazgos bucales con mayor frecuencia encontrados en niños con enfermedad renal fueron hipoplasias del esmalte, caries dental y retardo en la erupción dentaria, destacándose el desinterés por parte de los pacientes a la consulta odontológica

The frequency and incidence of renal disease in children had been documented, highlighting tubular dysfunctions, tubular acidosis, renal insufficiency, renal lithiasis, among others. The aim of this investigation was to characterize the buccal findings in children of 1 to 13 years old with renal disease. The investigation was a descriptive type, in a transversal design during january-april of 2010, in the Pediatric Nephrologic Service of the Hospital City "Dr. Enrique Tejera", in Valencia, Carabobo State, Venezuela, there were 50 patients with a mean age 6.85 years old, with hypercalciuria in 56% with no other systemic disease. The buccal findings were enamel hypoplasia 30%, dental caries 36%, delay eruption 16%, maloclussion 10% and geographic tongue 6%; none of the patients presented dental calculus. An important data was that 44% of the patients never had assisted to a dental appointment. The conclusions of this study are that the buccal findings more seen in children with renal disease are enamel hypoplasias, dental caries and delay eruption, underlying the lack of interest of the patients to the dental consult

Mare's colostrum globules stimulate fibroblast growth in vitro: a biochemical study.

The wound repair function of mare's milk and colostrum was investigated. Mare's colostrum improved wound healing in vivo; thus fibroblast growth activation by mare's milk and colostrum was examined. As expected, colostrum was more effective than milk. To establish the biochemical nature of the bioactive molecules involved, colostrum was fractionated into whey, casein, and fat globules, and the efficacy of these fractions on fibroblast proliferation was studied. The fat globule fraction provided the strongest stimulation; its composition was studied and compared with the less-active milk fat globule fraction. The lipid pattern highlighted several differences between mare's colostrum and milk; in particular, total lipid, linoleic acid, linolenic acid, ganglioside, and glycolipid contents were higher in colostrum. A proteomic investigation revealed some differences between the protein composition of colostrum and milk fat globules. Adipophylin and lactadherin were significantly overexpressed in colostrum fat globules. The role of specific lipids on skin wound repair and that of the epidermal growth factor-like domain, embedded within the lactadherin molecule and probably released in conditions stimulating proteolysis, are discussed.

Influence of ethanol, malate and arginine on histamine production of Lactobacillus hilgardii isolated from an Italian red wine.

Wine, like other fermented foods, may contain biogenic amines produced by lactic acid bacteria via amino acids decarboxylation. The most relevant amines from the toxicological standpoint are histamine and tyramine. The complexity of fermented substrates makes it difficult to suggest a priori how variables can modulate amine production. Lactobacillus hilgardii ISE 5211 was isolated from an Italian red wine. Besides producing lactate from malate, this strain is also able to convert arginine to ornithine and histidine to histamine. In the present investigation we studied the influence of malate, arginine and ethanol on histamine accumulation by L. hilgardii ISE 5211. Ethanol concentrations above 13% inhibit both histamine accumulation and bacterial growth; concentrations below 9% affect neither growth nor histamine production. However, an ethanol concentration of 11% allows a low but continuous accumulation of histamine to occur. Arginine also delays histamine accumulation, while malate appears to have no effect on histidine-histamine conversion.

A cluster of autosomal recessive spondylocostal dysostosis caused by three newly identified DLL3 mutations segregating in a small village.

In 1982, one of us reported a cluster of eight individuals affected by spondylocostal dysostosis (SD, MIM 277300) in four nuclear families indigenous to a village from eastern Switzerland. We tested the hypothesis that the molecular basis for this cluster was segregation of a single mutation in the DLL3 gene, recently linked to SD. Marker haplotypes around the DLL3 locus contradicted this hypothesis as three different haplotypes were seen in affected individuals, but sequence analysis showed that three unreported DLL3 mutations were segregating: a duplication of 17 bp in exon 8 (c.1285-1301dup), a single-nucleotide deletion in exon 5 (c.615delC), and a R238X nonsense mutation in exon 6. Contrary to our initial assumption of a single allele segregating in this small community, three different pathogenic alleles were observed, with a putative founder mutation occurring at the homozygous state but also compounding with, and thus revealing, two other independent mutations. As all three mutations predict truncation of the DLL3 protein and loss of the membrane-attaching domain, the results confirm that autosomal recessive spondylocostal dysostosis represents the null phenotype of DLL3, with remarkable phenotypic consistency across families.

Homozygous Gly530Ser substitution in COL5A1 causes mild classical Ehlers-Danlos syndrome.

Skin hyperelasticity, tissue fragility with atrophic scars, and joint hypermobility are characteristic for the classical type of Ehlers-Danlos syndrome (EDS). The disease is usually inherited as an autosomal dominant trait; however, recessive mode of inheritance has been documented in tenascin-X-deficient EDS patients. Mutations in the genes coding for collagen alpha1(V) chain (COL5A1), collagen alpha2(V) chain (COL5A2), tenascin-X (TNX), and collagen alpha1(I) chain (COL1A1) have been characterized in patients with classical EDS, thus confirming the suspected genetic heterogeneity. Recently, we described a patient with severe classical EDS due to a Gly1489Glu substitution in the alpha1(V) triple-helical domain who was, in addition, heterozygous for a disease-modifying Gly530Ser substitution in the alpha1(V) NH(2)-terminal domain [Giunta and Steinmann, 2000: Am. J. Med. Genet. 90:72-79; Steinmann and Giunta, 2000: Am. J. Med. Genet. 93:342]. Here, we report on a 4-year-old boy with mild classical EDS, born to healthy consanguineous Turkish parents; the mother presented a soft skin, while the father had a normal thick skin. Ultrastructural analysis of the dermis revealed in the patient the typical "cauliflower" collagen fibrils, while in both parents variable moderate aberrations were seen. Mutation revealed the presence of a homozygous Gly530Ser substitution in the alpha1(V) collagen chains in the patient, while both parents were heterozygous for the same substitution. An additional mutation in either the COL5A1 and COL5A2 genes was excluded. Furthermore, haplotype analysis with polymorphic microsatellite markers excluded linkage to the genes coding for alpha3(V) collagen (COL5A3), tenascin-X (TNX), thrombospondin-2 (THBS2), and decorin (DCN). These new findings support further our previous hypothesis that the heterozygous Gly530Ser substitution is disease modifying and now suggest that in the homozygous state it is disease causing.

The catechol 1,2 dioxygenase system of Acinetobacter radioresistens: isoenzymes, inductors and gene localisation.

Two different isozymes (Iso A and Iso B) of catechol 1,2 dioxygenase (C1,2O) were isolated from cultures of A. radioresistens grown in two different media, containing phenol and benzoate respectively. In the phenol medium the bacteria expressed about 90% of Iso A, whereas in the benzoate medium the Iso A/Iso B ratio was 40:60. The two proteins have different molecular masses, isoelectric points and N-terminal sequences that are not consistent with simple post-translational modifications. Furthermore, their behaviour differs at high temperatures (42 degrees C-47 degrees C) and at moderately acidic pH (pH 6.0): Iso A proved to be the more stable under conditions of environmental stress. Hybridisation analysis with an A. calcoaceticus catA-derived probe revealed that A. radioresistens C1,2O proteins are encoded by two chromosomally located genes. Bidimensional electrophoresis (2DE) maps of crude extracts of cells grown in different carbon sources (phenol, benzoate and acetate) clearly demonstrated a differential induction pattern for the two proteins. The hypothesis of a double set of genes, one for benzoate catabolism and the other for phenol catabolism, is discussed, and analogies are drawn with other known C1,2Os.

Media containing aromatic compounds induce peculiar proteins in Acinetobacter radioresistens, as revealed by proteome analysis.

An Acinetobacter radioresistens strain able to grow on phenol or benzoate as sole carbon and energy source through the beta-ketoadipate pathway was isolated in our laboratories. In previous research, we found a different expression of catechol-1,2-dioxygenase isoenzymes (C-1,2-O) depending on the growth substrate (phenol or benzoate). In the present study, we used proteome techniques to extend our investigation to other enzymes involved in the aromatic degradation pathway. Since the first nontoxic metabolite in this route is cis,cis-muconic acid, we focused our attention on the enzymes leading to this compound, chiefly phenol hydroxylase (PH), benzoate dioxygenase (BD), cis-1,2-dihydroxycyclohexa-3,5-diene-1-carboxylate dehydrogenase (D) and C-1,2-O. In particular, the A. radioresistens proteome was monitored under different growth substrate conditions, using acetate, benzoate, or phenol as sole carbon source. We compared the protein maps by software image analysis and detected marked differences, suggesting the inducibility of most enzymes. This research also sought to evaluate the conditions allowing the best expression of enzymes to be used in immobilized systems suitable for bioremediation. The experimental data indicate that benzoate is the best carbon source to gain the highest amount of C-1,2-O and D, while phenol is the best growth substrate to obtain PH.

Human proteome enhancement: high-recovery method and improved two-dimensional map of colostral fat globule membrane proteins.

The human milk fat globule membrane protein composition is still largely unknown, although it counts for 2-4% of the total milk protein content and contains several important biologically active components. The aim of this work was to create a two-dimensional electrophoresis (2-DE) map of the human milk fat globule membrane proteins, both integral and membrane-associated, and to identify and characterize as many protein components as possible. A new protocol for the solubilization and extraction of the human milk fat globule membrane proteins with a double extraction procedure is presented, and the results compared with the extraction methods reported in the literature. The proteins were separated, in the first dimension, by isoelectric focusing (IEF) in the pH range 3-10 on strips of 13 cm length and, in the second dimension, by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 11.5% T homogeneous gels. A reproducible 2-DE map of integral and membrane-associated proteins was obtained and the first 23 spots, representing the major components, were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis and/or by amino acid sequencing.

Improved resistance to transition metals of a cobalt-substituted alcohol dehydrogenase 1 from Saccharomyces cerevisiae.

Cobalt-substituted alcohol dehydrogenase 1 was purified from a yeast culture of Saccharomyces cerevisiae. Its reactivity towards different transition metals was tested and compared with the native zinc enzyme. The cobalt enzyme displayed a catalytic efficiency 100-fold higher than that of the zinc enzyme. Copper, nickel and cadmium exerted a mixed-type inhibition, with a scale of inhibition efficiency: Cu(2+)>Ni(2+)>Cd(2+). In general, a higher resistance of the modified protein to the inhibitory action of transition metals was observed, with two orders of magnitude for copper I(50). The presence of nickel in the complexes enzyme-coenzyme-inhibitor-substrate resulted in a decrease of the ampholytic nature of the catalytic site. On the contrary, cadmium and copper exerted an enhancement of this parameter. Electrostatic or other types of interactions may be involved in conferring a good resistance in the basic pH range, making cobalt enzyme very suitable for biotechnological processes.

Characterization of 11 new mutations in COL3A1 of individuals with Ehlers-Danlos syndrome type IV: preliminary comparison of RNase cleavage, EMC and DHPLC assays.

We report on 12 patients with EDS IV in whom clinical diagnosis was confirmed by biochemical analysis of collagen type III, and further proven by mutation analysis of the COL3A1 gene. Four overlapping RT-PCR products covering the coding sequence for the triple-helical domain of type III collagen were analyzed by direct sequencing. So far, we have identified, 4 base changes at donor splice junctions, and 1 base change at an acceptor splice site, which all affect mRNA splicing; 1 genomic deletion, which removes exon 45; and 6 nucleotide changes, which cause substitutions of glycine residues within the triple helix. Eleven of the 12 identified mutations are newly recognized. Furthermore, we report a preliminary comparison of RNase cleavage, EMC and DHPLC assays in mutation detection in the COL3A1 gene.

COL5A1 haploinsufficiency is a common molecular mechanism underlying the classical form of EDS.

We have identified haploinsufficiency of the COL5A1 gene that encodes the proalpha1(V) chain of type V collagen in the classical form of the Ehlers-Danlos syndrome (EDS), a heritable connective-tissue disorder that severely alters the collagen-fibrillar structure of the dermis, joints, eyes, and blood vessels. Eight of 28 probands with classical EDS who were heterozygous for expressed polymorphisms in COL5A1 showed complete or nearly complete loss of expression of one COL5A1 allele. Reduced levels of proalpha1(V) mRNA relative to the levels of another type V collagen mRNA, proalpha2(V), were also observed in the cultured fibroblasts from EDS probands. Products of the two COL5A1 alleles were approximately equal after the addition of cycloheximide to the fibroblast cultures. After harvesting of mRNAs from cycloheximide-treated cultured fibroblasts, heteroduplex analysis of overlapping reverse transcriptase-PCR segments spanning the complete proalpha1(V) cDNA showed anomalies in four of the eight probands that led to identification of causative mutations, and, in the remaining four probands, targeting of CGA-->TGA mutations in genomic DNA revealed a premature stop at codon in one of them. We estimate that approximately one-third of individuals with classical EDS have mutations of COL5A1 that result in haploinsufficiency. These findings indicate that the normal formation of the heterotypic collagen fibrils that contain types I, III, and V collagen requires the expression of both COL5A1 alleles.

Purification and catalytic properties of two catechol 1,2-dioxygenase isozymes from benzoate-grown cells of Acinetobacter radioresistens.

Two catechol 1,2-dioxygenase (C1,2O) isozymes (IsoA and IsoB) have been purified to homogeneity from a strain of Acinetobacter radioresistens grown on benzoate as the sole carbon and energy source. IsoA and IsoB are both homodimers composed of a single type of subunit with molecular mass of 38,600 and 37,700, Da respectively. In conditions of low ionic strength, IsoA can aggregate as a trimer, in contrast to IsoB, which maintains the dimeric structure, as also supported by the kinetic parameters (Hill numbers). IsoA is identical to the enzyme previously purified from the same bacterium grown on phenol, whereas the IsoB is selectively expressed using benzoate as carbon source. This is the first evidence of the presence of differently expressed C1,2O isozymes in A. radioresistens or more generally of multiple C1,2O isozymes in benzoate-grown Acinetobacter cells. Purified IsoA and IsoB contain approximately 1 iron(III) ion per subunit and both show electronic absorbance and EPR features typical of Fe(III) intradiol dioxygenases. The kinetic properties of the two enzymes such as the specificities toward substituted catechols, the main catalytic parameters, and their behavior in the presence of different kind of inhibitors are, unexpectedly, very similar, in contrast to most of the previously known dioxygenase isozymes.

Compound heterozygosity for a disease-causing G1489E [corrected] and disease-modifying G530S substitution in COL5A1 of a patient with the classical type of Ehlers-Danlos syndrome: an explanation of intrafamilial variability?

The classical type of Ehlers-Danlos syndrome (EDS) is an autosomal dominant connective tissue disorder characterized by skin hyperelasticity, tissue fragility, and joint hypermobility. We investigated the molecular defect of EDS in a three-generation family. Cultured dermal fibroblasts from the propositus and his daughter produced abnormal alpha1(V) and alpha2(V) collagen molecules. Mutation analysis by means of RNase cleavage and direct sequencing of reverse transcription-polymerase chain reaction products showed in both the presence of a heterozygous G1489E [correction] mutation in the COL5A1 gene, which represents the first report of a glycine substitution in the main triple-helical region of alpha1(V) collagen. In the propositus, his unaffected daughter, and mother we identified a further newly recognized G530S substitution in the NH2-terminal domain, which did not cosegregate with the EDS phenotype and was found in only one of 51 unrelated control individuals. Because the NH2-terminal domain plays a crucial role in modulating fibril formation, the G530S substitution may alter the structure and function of this region and consequently the formation of collagen fibrils. Indeed, indirect evidence supports our hypothesis: (1) the EDS phenotype in the compound heterozygous propositus is more severe than that of his affected daughter with the G1489E [correction] mutation only; (2) his unaffected daughter and mother with the G530S substitution present with thin skin and delayed wound healing; (3) as does the only control individual with the same substitution. Thus, in the compound heterozygous propositus the EDS phenotype is caused by the G1489E [correction] mutation and possibly aggravated by the G530S substitution, which may explain intrafamilial variability.

Phenol hydroxylase from Acinetobacter radioresistens is a multicomponent enzyme. Purification and characterization of the reductase moiety.

This paper reports the isolation and characterization of phenol hydroxylase (PH) from a strain belonging to the Acinetobacter genus. An Acinetobacter radioresistens culture, grown on phenol as the only carbon and energy source, produced a multicomponent enzyme system, located in the cytoplasm and inducible by the substrate, that is responsible for phenol conversion into catechol. Because of the wide diffusion of phenol as a contaminant, the present work represents an initial step towards the biotechnological treatment of waste waters containing phenol. The reductase component of this PH system has been purified and isolated in large amounts as a single electrophoretic band. The protein contains a flavin cofactor (FAD) and an iron-sulfur cluster of the type [2Fe-2S]. The function of this reductase is to transfer reducing equivalents from NAD(P)H to the oxygenase component. In vitro, the electron acceptors can be cytochrome c as well as other molecules such as 2, 6-dichlorophenolindophenol, potassium ferricyanide, and Nitro Blue tetrazolium. The molecular mass of the reductase was determined to be 41 kDa by SDS/PAGE and 38.8 kDa by gel permeation; its isoelectric point is 5.8. The N-terminal sequence is similar to those of the reductases from A. calcoaceticus NCIB 8250 (10/12 identity) and Pseudomonas CF600 (8/12 identity) PHs, but much less similar (2/12 identity) to that of benzoate dioxygenase reductase from A. calcoaceticus BD413. Similarly, the internal peptide sequence of the A. radioresistens PH reductase displays a good level of identity (9/10) with both A. calcoaceticus NCIB 8250 and Pseudomonas CF600 PH reductase internal peptide sequences but a poorer similarity (3/10) to the internal peptide sequence of benzoate dioxygenase reductase from A. calcoaceticus BD413.

Ehlers-Danlos syndrome type VII: clinical features and molecular defects.

We evaluated the clinical features, molecular defects, and problems associated with the management of two patients who had type-VII Ehlers-Danlos syndrome and reviewed the cases of eighteen patients with this condition who had been reported on previously. The typical clinical features associated with this syndrome include bilateral congenital dislocation of the hip; severe generalized hypermobility of the joints; multiple dislocations of joints other than the hip; muscular hypotonia; and hyperelasticity, fragility, and a doughy texture of the skin. Collagen and DNA analyses demonstrated that both of our patients had type-VIIB Ehlers-Danlos syndrome, which is caused by heterozygous new mutations of the COL1A2 gene that encodes the proalpha2(I) chain of type-I procollagen. The obligatory GT dinucleotide at the splice donor site of intron 6 was altered in both of our patients: one patient (Case 1) had an A substitution of the G nucleotide, and the other patient (Case 2) had a C substitution of the T nucleotide. Abnormal splicing resulted in the loss of the exon 6-encoded N-telopeptide, which includes the N-proteinase cleavage site. Despite multiple operative procedures, one of our patients, who was thirty-seven years old at the time of the most recent follow-up, continued to have persistent subluxation of the right hip and osteoarthritis of the left hip. Closed reduction of the dislocated hips, regardless of the type of immobilization used, was unsuccessful in all twenty patients. The results of open reduction were improved when capsulorrhaphy was combined with iliac or femoral osteotomy, or both.