Fernando Segade

Prothymosin α is a nuclear protein

Prothymosin α, a protein first isolated from rat thymus and widely distributed in animal tissues, has an attributed role in the stimulation of the immune system. Its structure contains thymosin α1, a Glu‐rich domain and a putative nuclear location signal. Furthermore, the amount of this protein seems to be associated with the relative size of the nucleus and is inducible during cell growth. We postulate that prothymosin α is located inside the cell nucleus and that its activity might be to organize some protein complexes.

Chronic and Recurrent Otitis Media: A Genome Scan for Susceptibility Loci

Otitis media (OM) is the most common childhood disease. Almost all children experience at least one episode, but morbidity is greatest in children who experience chronic/recurrent OM (COME/ROM). There is mounting evidence that COME/ROM clusters in families and exhibits substantial heritability. Subjects who had tympanostomy tube surgery for COME/ROM (probands) and their families were recruited for the present study, and an ear examination was performed, without knowledge of the subjects history, to determine presence of OM sequelae. In addition, tympanometric testing was performed at three frequencies (226, 630 or 710, and 1,400 Hz) to detect abnormal middle-ear mechanics, and hearing was screened at 20 dB for the speech frequencies. Of these families, 121 had at least two individuals who had received the diagnosis of COME/ROM (364 affected and genotyped individuals), of whom 238 affected and informative relative pairs were used for analyses. Single-point nonparametric linkage analysis provided evidence of linkage of COME/ROM to chromosome 10q at marker D10S212 (LOD 3.78; P=3.0 x 10(-5)) and to chromosome 19q at marker D19S254 (LOD 2.61; P=5.3 x 10(-4)). Analyses conditional on support for linkage at chromosomes 10q and 19q resulted in a significant increase in LOD score support on chromosome 3p (between markers D3S4545 and D3S1259). These results suggest that risk of COME/ROM is determined by interactions between genes that reside in several candidate regions of the genome and are probably modulated by other environmental risk factors.

Deficiency in Microfibril-associated Glycoprotein-1 Leads to Complex Phenotypes in Multiple Organ Systems

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight component of the fibrillin-rich microfibril. Gene-targeted inactivation of MAGP-1 reveals a complex phenotype that includes increased body weight and size due to excess body fat, an altered wound healing response in bone and skin, and a bleeding diathesis. Elastic tissues rich in MAGP-1-containing microfibrils develop normally and show normal function. The penetrance of MAGP-1-null phenotypes is highly variable and mouse strain-dependent, suggesting the influence of modifier genes. MAGP-1 was found to bind active transforming growth factor-β (TGF-β) and BMP-7 with high affinity, suggesting that it may be an important modulator of microfibril-mediated growth factor signaling. Many of the phenotypic traits observed in MAGP-1-deficient mice are consistent with loss of TGF-β function and are generally opposite those associated with mutations in fibrillin-1 that result in enhanced TGF-β signaling. Increased body size and fat deposition in MAGP-1-mutant animals are particularly intriguing given the localization of obesity traits in humans to the region on chromosome 1 containing the MAGP-1 gene.

Differential expression of facilitative glucose transporters in normal and tumour kidney tissues

To investigate the differences in the pattern of glucose transporter (GLUT) gene expression between normal and tumour tissues and among histological subtypes of renal cell carcinomas (RCCs), as malignant cells are characterized by increased glucose uptake and use.

Identification of a Matrix-binding Domain in MAGP1 and MAGP2 and Intracellular Localization of Alternative Splice Forms

MAGP1 is a small molecular mass protein associated with microfibrils in the extracellular matrix (ECM). To identify the molecular basis of its interaction with other microfibrillar proteins, deletion constructs of MAGP1 were expressed in a mammalian cell system that served as a model for microfibril assembly. This study identified a 54-amino acid sequence in the carboxyl-terminal region of the protein that defines a matrix-binding domain that is sufficient to target MAGP1 to the ECM. Site-directed mutagenesis demonstrated that binding activity is dependent on the presence of 7 cysteine residues in this sequence. MAGP2 contains a sequence similar to the matrix-binding domain of MAGP1, but could not associate with the ECM because of a single amino acid change. Two naturally occurring MAGP1 splice variants, MAGP1B (human-specific) and MAGP1D (found in mice), localized intracellularly when expressed as chimeric proteins with green fluorescent protein in rat lung fibroblasts. This suggests a second action site for MAGP1.

Glucose transporter 10 and arterial tortuosity syndrome: the vitamin C connection.

Arterial Tortuosity Syndrome (ATS) is a heritable disease characterized by twisting and lengthening of the major arteries, hypermobility of the joints, and laxity of skin. ATS is caused by mutations in SLC2A10, encoding Glucose Transporter 10 (GLUT10). The current model of ATS holds that loss of GLUT10 at the nuclear periphery induces a glucose‐dependent increase in Transforming Growth Factor‐β (TGFβ) that stimulates vessel wall cell proliferation. Instead, we propose that GLUT10 transports ascorbate, a cofactor for collagen and elastin hydroxylases, into the secretory pathway. In ATS, loss of GLUT10 results in defective collagen and/or elastin. TGFβ activation represents a secondary response to a defective extracellular matrix.

Molecular evolution of the fibulins: Implications on the functionality of the elastic fibulins

The fibulins form a family of secreted proteins associated with the basement membrane, cell adhesive structures, and elastic fibers characterized by the presence of a unique fibulin-like C-terminal domain preceded by a rod-like tandem array of calcium-binding EGF modules. We traced the origin of the fibulin gene family to the base of the metazoans. In invertebrates, Fibulin-1 and Hemicentin comprise the fibulin gene set. Diversification of the fibulins took place in the last common ancestor to the chordates by gene duplication of an ancestral Fibulin-1 gene. Further duplications at the vertebrate stem and in teleost fishes increased the number of fibulin genes to nine, including the novel Fibulin-8. Extensive gene loss has happened repeatedly, including Fibulin-8 in placental mammals and Fibulin-4 in birds. The Fibulin-3/4/5 clade of elastic fibulins branched out at the sequence level after relaxation of selective constraints immediately after the two gene duplication events in quick succession that originated the individual vertebrate Fibulin-3, -4, and -5 genes. Divergence took place mostly in the Fibulin-5 branch, at the atypical first EGF module and at the fibulin-like C-terminal region. Differentiation in gene expression further split Fibulin-5 from the other elastic fibulins, likely contributing to its nonredundant role in elastic fiber assembly.

Evaluation of 15 functional candidate genes for association with chronic otitis media with effusion and/or recurrent otitis media (COME/ROM).

DNA sequence variants in genes involved in the innate immune response and secondary response to infection may confer susceptibility to chronic otitis media with effusion and/or recurrent otitis media (COME/ROM). We evaluated single nucleotide polymorphisms (SNPs) in 15 functional candidate genes. A total of 99 SNPs were successfully genotyped on the Sequenom platform in 142 families (618 subjects) from the Minnesota COME/ROM Family Study. Data were analyzed for association with COME/ROM using the Generalized Disequilibrium Test (GDT). Sex and age at exam were adjusted as covariates, relatedness was accounted for, and genotype differences from all phenotypically discordant relative pairs were utilized to measure the evidence of association between COME/ROM and each SNP. SNP rs2735733 in the region of the mucin 5, subtypes A/C gene (MUC5AC) exhibited nominal evidence for association with COME/ROM (P = 0.002). Two additional SNPs from this region had P values<0.05. Other variants exhibiting associations with COME/ROM at P<0.05 included the SCN1B SNP rs8100085 (P = 0.013), SFTPD SNP rs1051246 (P = 0.039) and TLR4 SNP rs2770146 (P = 0.038). However, none of these associations replicated in an independent sample of COME/ROM families. The candidate gene variants examined do not appear to make a major contribution to COME/ROM susceptibility, despite a priori evidence from functional or animal model studies for a role in COME/ROM pathology.

Functional evolution of the microfibril-associated glycoproteins

The microfibril-associated glycoproteins (MAGPs) are cysteine-rich low molecular weight components of the fibrillin-based microfibrillar complex. MAGPs are evolutionarily conserved in vertebrates and have important roles in microfibril and elastic fiber structure, homeostasis, and vascular development. Two MAGPs, designated MAGP1 and MAGP2, are encoded in the mammalian genome. Although MAGP sequences have been identified in several vertebrate species, the extent of conservation and evolutionary history of the MAGPs in vertebrates is unknown. Sequence similarity searches of nucleotide and protein databases identified the first homologs of MAGP1 in monotremes, birds, elasmobranchs and agnathans, and the first MAGP2 genes in marsupials, birds and teleosts. A model for MAGP evolution is presented. Phylogenetic analysis identified the ancient origin of MAGP1 and the evolution of MAGP2 from a gene duplication event early in vertebrate evolution. Phylogenomic analysis shows conservation of synteny between teleosts and tetrapods and suggests a multigene duplication event. The MAGP2 gene has evolved rapidly as an innovation in the bony vertebrate lineage. Estimates of functional divergence and complex nucleotide substitution models suggest that the divergence of MAGP2 took place by relaxation of selective constraints; and that MAGP1 has consistently been constrained by strong purifying selection. Correlated evolution between MAGP1 and the developmental regulator, Notch1, may explain some of the selective forces acting on MAGP2.

A simple procedure for large-scale purification of plasmid DNA.

We report a simple, rapid and reliable procedure for large-scale purification of plasmid DNA from non-amplified bacterial cultures. It is a modification of the boiling method of Holmes and Quigley [Anal. Biochem. 114 (1981) 193-197] and involves gel-filtration chromatography using Sephacryl S-1000 for final purification of plasmid DNA. This method does not require CsCl gradients and the recovered plasmids are free of RNA and chromosomal DNA, are supercoiled, retain their biological activity, and are suitable for restriction analysis.