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At least 1,009 records · Page 56Linked to original sources
PubMed1999

METH-1, a human ortholog of ADAMTS-1, and METH-2 are members of a new family of proteins with angio-inhibitory activity.

We have studied two related proteins that contain a repeated amino acid motif homologous to the anti-angiogenic type 1 repeats of thrombospondin-1 (TSP1). Complete sequence analysis revealed no other similarities with TSP1, but identified unique signal sequences, as well as metalloprotease and disintegrin-like domains in the NH(2) termini. We named these proteins METH-1 and METH-2 due to the novel combination of metalloprotease and thrombospondin domains. Overall amino acid sequence identity between METH-1 and METH-2 is 51. 7%, yet transcript distribution revealed non-overlapping patterns of expression in tissues and cultured cell lines. To characterize these proteins functionally, we isolated full-length cDNAs, produced recombinant protein, and generated antisera to the recombinant proteins. Both METH-1 and METH-2 represent single copy genes, which encode secreted and proteolytically processed proteins. METH proteins suppressed fibroblast growth factor-2-induced vascularization in the cornea pocket assay and inhibited vascular endothelial growth factor-induced angiogenesis in the chorioallantoic membrane assay. Suppression of vessel growth in both assays was considerably greater than that mediated by either thrombospondin-1 or endostatin on a molar basis. Consistent with an endothelial specific response, METH-1 and METH-2 were shown to inhibit endothelial cell proliferation, but not fibroblast or smooth muscle growth. We propose that METH-1 and METH-2 represent a new family of proteins with metalloprotease, disintegrin, and thrombospondin domains. The distinct distribution of each gene product suggests that each has evolved distinct regulatory mechanisms that potentially allow for fine control of activity during distinct physiological and pathological states.

ADAM Proteins↗
PubMed2002

Minimum-recombinant haplotyping in pedigrees.

This article presents a six-rule algorithm for the reconstruction of multiple minimum-recombinant haplotype configurations in pedigrees. The algorithm has three major features: First, it allows exhaustive search of all possible haplotype configurations under the criterion that there are minimum recombinants between markers. Second, its computational requirement is on the order of O(J(2)L(3)) in current implementation, where J is the family size and L is the number of marker loci under analysis. Third, it applies to various pedigree structures, with and without consanguinity relationship, and allows missing alleles to be imputed, during the haplotyping process, from their identical-by-descent copies. Haplotyping examples are provided using both published and simulated data sets.

Algorithms↗
PubMed1997

Evolution of the structural repertoire of the human V(H) and Vkappa germline genes.

Variable genes of human Ig are classified in families and clans which reflect the early events of gene duplication in the evolution of the locus. This organization in multiple copies of variable genes plus the somatic processes of recombination and hypermutation allows the immune system to generate an antibody repertoire of great diversity. At present the role that somatic processes play in the generation of that diversity is understood with some detail. It is a matter of hard controversy, however, which selective pressures have shaped the evolution of the germline genes of Ig and, consequently, what the role of this germline component in the generation of the antibody diversity actually is. Previous studies of our group have showed that the structural repertoire of Ig-determined by the canonical structures-is an important factor to determine the recognition properties of the antibodies. Complete knowledge of the sequences of the human V(H) and Vkappa loci is available to analyze the evolution of the structural repertoire of these loci. Two phylogenetic gene trees were built from the functional germline genes and the evolution of the structural repertoire was studied. We report that for both loci the canonical structures are not randomly distributed within the tree. Conversely, it is shown that the evolution of the structural repertoire follows a gradual process of diversification. This indicates a correlation between the evolution of genes and the structural repertoire, although important differences are found in the patterns of evolution of the structural repertoire between V(H) and Vkappa. Based on those results we propose a primordial structural repertoire for V(H) and Vkappa. The general properties and an outline of the three-dimensional structure of this primordial repertoire are given.

Amino Acid Sequence↗
PubMed1999

A putative regulatory element for carbon-source-dependent differentiation in Streptomyces griseus.

To identify negative regulatory genes for cellular differentiation in Streptomyces griseus, DNA fragments repressing the normal developmental processes were cloned on a high-copy-number plasmid. One of these DNA fragments markedly repressed aerial mycelium and spore formation on solid media containing glucose or galactose, but not on media containing maltose or mannitol. The fragment contained three complete ORFs; precise subcloning revealed that a 249 bp fragment located in the promoter region between ORF1 and ORF3 was sufficient for repression. Quantification of the promoter activities by using a thermostable malate dehydrogenase gene as a reporter showed that the promoter for ORF3 (P(ORF3)) maintained high activity in mycelia grown in the presence of glucose but lost activity rapidly in maltose medium. P(ORF3) activity increased markedly when the promoter sequence was introduced on a high-copy-number plasmid. The results suggested that carbon-source-dependent deactivation of P(ORF3) mediated by a transcriptional repressor may initiate differentiation in S. griseus.

Amino Acid Sequence↗
PubMed1991

L-pilin variants of Neisseria gonorrhoeae MS11.

Phase- and antigenic variation of pilin expression in Neisseria gonorrhoeae is based on the genetic exchange between silent pilin genes (pilS) and the pilin expression locus (pilE). Similarly, the non-piliated L-variants of strain MS11, which show an increased resistance to certain antibiotics, are the result of recombination with the pilE locus. However, this recombination is atypical in that pilE(L) carries a tandem arrangement of a complete pilin gene and additional partial pilin genes under the control of the same pilE promoter. Since the two pilin gene copies are tandemly arranged and are often in the same translational frame, oversized pilin molecules are produced, which do not assemble into pili. The tandem gene copies introduced in a pilE(L) locus originate from silent loci where they are already joint. Upon reversion to the P+ phenotype the L-variants lose one pilin gene copy from the pilE(L) in a process reminiscent of the deletion events that otherwise lead to the formation of the non-revertible and non-piliated Pn mutants of MS11 gonococci. Thus deletion of pilin genes from pilE can be regarded as a third mechanism of pilin variation in gonococci.

Amino Acid Sequence↗
PubMed2005

Highly sensitive SIV plasma viral load assay: practical considerations, realistic performance expectations, and application to reverse engineering of vaccines for AIDS.

As new assay methods for quantitative reverse transcription-polymerase chain reaction (RT-PCR), such as real time RT-PCR techniques, approach theoretical limits of per reaction sensitivity, further increments in the sensitivity of measurements of viral load can only be achieved by increasing the amount of input RNA per reaction. We describe a robust, convenient, rapid integrated approach for specimen preparation and real time RT-PCR assay for plasma simian immunodeficiency virus (SIV) RNA viral load that provides a threshold sensitivity of 10 copy Eq/ml, and tolerates less than optimally processed specimens. The method provides accurate quantitation of viral load for the SIV virus isolates in common use for non-human primate studies. We demonstrate the utility of the method in sensitively tracking viral load in an animal showing effective control of viral replication to levels below the threshold for quantitation in conventional assays.

AIDS Vaccines↗
PubMed2001

Making sense of eukaryotic DNA replication origins.

DNA replication is the process by which cells make one complete copy of their genetic information before cell division. In bacteria, readily identifiable DNA sequences constitute the start sites or origins of DNA replication. In eukaryotes, replication origins have been difficult to identify. In some systems, any DNA sequence can promote replication, but other systems require specific DNA sequences. Despite these disparities, the proteins that regulate replication are highly conserved from yeast to humans. The resolution may lie in a current model for once-per-cell-cycle regulation of eukaryotic replication that does not require defined origin sequences. This model implies that the specification of precise origins is a response to selective pressures that transcend those of once-per-cell-cycle replication, such as the coordination of replication with other chromosomal functions. Viewed in this context, the locations of origins may be an integral part of the functional organization of eukaryotic chromosomes.

Animals↗
PubMed1989

Genetic reconstitution of the high-affinity L-arabinose transport system.

Expression plasmids containing various portions of araFGH operon sequences were assayed for their ability to facilitate the high-affinity L-arabinose transport process in a strain lacking the chromosomal copy of this operon. Accumulation studies demonstrated that the specific induction of all three operon coding sequences was necessary to restore high-affinity L-arabinose transport. Kinetic analysis of this genetically reconstituted transport system indicated that it functions with essentially wild-type parameters. Therefore, L-arabinose-binding protein-mediated transport appears to require only two inducible membrane-associated components (araG and araH) in addition to the binding protein (araF).

Arabinose↗
PubMed2004

Agrobacterium-mediated transformation of Aspergillus awamori in the absence of full-length VirD2, VirC2, or VirE2 leads to insertion of aberrant T-DNA structures.

Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.

Aspergillus↗
PubMed2005

Development of multiplex PCRs for detection of common viral pathogens and agents of congenital infections.

Potential causes of congenital infection include Toxoplasma gondii and viruses such as cytomegalovirus (CMV), enterovirus, hepatitis C virus, herpes simplex virus types 1 and 2 (HSV-1 and -2), human herpesvirus types 6, 7, and 8, lymphocytic choriomeningitis virus, parvovirus, rubella virus, and varicella-zoster virus. Testing for each of these agents using nucleic acid tests is time consuming and the availability of clinical samples such as amniotic fluid or neonatal blood is often limited. The aim of this study was to develop multiplex PCRs (mPCRs) for detection of DNA and RNA agents in the investigation of congenital infection and an mPCR for the viruses most commonly requested in a diagnostic virology laboratory (CMV, Epstein-Barr virus, enterovirus, HSV-1, HSV-2, and varicella-zoster virus). The assays were assessed using known pathogen-positive tissues (cultures, placentae, plasma, and amniotic fluid) and limits of detection were determined for all the agents studied using serial dilutions of plasmid targets. Nested PCR was performed as the most sensitive assay currently available, and detection of the amplicons using hybridization to labeled probes and enzyme-linked immunosorbent assay detection was incorporated into three of the four assays. This allowed detection of 10 to 10(2) copies of each agent in the samples processed. In several patients, an unexpected infection was diagnosed, including a case of encephalitis where HSV was the initial clinical suspicion but CMV was detected. In the majority of these cases the alternative agent could be confirmed using reference culture, serology, or fluorescence methods and was of relevance to clinical care of the patient. The methods described here provide useful techniques for diagnosing congenital infections and a paradigm for assessment of new multiplex PCRs for use in the diagnostic laboratory.

Amniotic Fluid↗
PubMed1987

Insertions of up to 17 amino acids into a region of alpha-tubulin do not disrupt function in vivo.

Microtubules in yeasts are essential components of the mitotic and meiotic spindle and are necessary for nuclear movement during cell division and mating. The yeast Saccharomyces cerevisiae has two alpha-tubulin genes, TUB1 and TUB3, either of which alone is sufficient for these processes when present in a high enough copy number. Comparisons of sequences from several species reveals the presence of a variable region near the amino terminus of alpha-tubulin proteins. We perturbed the structure of this region in TUB3 by inserting into it 3, 9, or 17 amino acids and tested the ability of these altered proteins to function as the only alpha-tubulin protein in yeast cells. We found that each of these altered proteins was sufficient on its own for mitotic growth, mating, and methods of yeast. We conclude that this region can tolerate considerable variation without losing any of the highly conserved functions of alpha-tubulin. Our results suggest that variability in this region occurs because it can be tolerated, not because it specifies an important function for the protein.

Amino Acid Sequence↗
PubMed1988

Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei.

Paramecium tetraurelia can be transformed by microinjection of cloned serotype A gene sequences into the macronucleus. Transformants are detected by their ability to express serotype A surface antigen from the injected templates. After injection, the DNA is converted from a supercoiled form to a linear form by cleavage at nonrandom sites. The linear form appears to replicate autonomously as a unit-length molecule and is present in transformants at high copy number. The injected DNA is further processed by the addition of paramecium-type telomeric sequences to the termini of the linear DNA. To examine the fate of injected linear DNA molecules, plasmid pSA14SB DNA containing the A gene was cleaved into two linear pieces, a 14-kilobase (kb) piece containing the A gene and flanking sequences and a 2.2-kb piece consisting of the procaryotic vector. In transformants expressing the A gene, we observed that two linear DNA species were present which correspond to the two species injected. Both species had Paramecium telomerelike sequences added to their termini. For the 2.2-kb DNA, we show that the site of addition of the telomerelike sequences is directly at one terminus and within one nucleotide of the other terminus. These results indicate that injected procaryotic DNA is capable of autonomous replication in Paramecium macronuclei and that telomeric addition in the macronucleus does not require specific recognition sequences.

Animals↗
PubMed1997

Effects of multiple estrogen responsive elements, their spacing, and location on estrogen response of reporter genes.

Most highly estrogen-responsive genes possess multiple estrogen-responsive elements (EREs) that act synergistically to activate expression. Synergism between EREs appears to depend on structural features of the EREs and the promoter. To examine the activation process, we cloned single or multiple tandem copies of the consensus ERE into reporter plasmids. These plasmids contained either a chloramphenicol acetyl transferase reporter gene driven by a minimal promoter or a luciferase reporter gene driven by the Simian virus 40 (SV40) promoter. Using MCF-7 human breast cancer cells, we demonstrate that synergism among EREs depends on the number of EREs, their spacing, and the distance of the EREs from the promoter. The induction capacity of EREs falls off slowly with distance from the promoter. Remarkably, multiple EREs can induce effectively and synergize even when they are located more than 2000 nucleotides from the promoter. For EREs located immediately upstream of the promoter, both the distance separating the EREs and the distance to the promoter have to be optimal for synergy. Altering either distance changes the response from synergistic to additive. For distant EREs, presumed to interact by a looping mechanism at the promoter, the length of DNA between the EREs and the promoter is not critical. Synergy among closely spaced EREs that are far from the promoter only requires an optimal distance separating the ERE centers of symmetry. Interestingly, very widely separated EREs can also synergize, presumably also because of their ability to interact by looping. The estrogen response from single or multiple tandem copies of ERE half-palindromes near the SV40 promoter was also tested. The negligible induction capacity of a single half-site was not significantly increased in multiple sites. The biological role of half-EREs is not apparent in the system employed here.

Animals↗
PubMedDate not supplied

Reduction in p53 gene dosage diminishes differentiation capacity of osteoblasts.

BACKGROUND: Loss of p53 function is seen in a large number of human tumors including osteosarcoma, and in previous studies we have demonstrated a close relationship between wild type p53 and osteoblasts differentiation. In the present study we attempted to understand the relationship of bone growth and differentiation to p53 gene dosage by studying calvarial osteoblasts obtained from p53 deficient mice. RESULTS: Growth rules were higher in p53 -/- than p53 +/- osteoblasts when passaged for different lenght of time. However, loss of the remaining wt allele occurred early during culturing of the p53 +/- cells, and these cells became immortalized with growth rates and doubling times reaching values higher than the p53 -/- homozygotes. Early passage osteoblasts were used to study differentiation properties of these osteoblasts in vitro by exposing them to differentiation promoting media. While osteocalcin, a marker protein of differentiated osteoblasts, was synthesized in both cell types, p53 -/- cells consistently showed higher levels of osteocalcin activity, and in long term cultures formed more bone spicules that p53 +/- cells. Basal osteocalcin promoter activity was similar in both cell types, but transient transfection of wt p53 into the cells produced a larger activation of the promoter in p53 +/- cells, than in p53 -/- cells. Sections of long bones from neonatal p53 +/- and -/- were examined histologically, and no gross abnormalities were detected. TUNEL staining of the bones showed apoptotic cells in the growth plate and lining cells of both p53 genotypes, but apoptosis in hypertrophic chondrocytes was seen only in p53 +/- bones. CONCLUSION: Overall our studies show that osteoblastic differentiation becomes a p53 dependent process only when at least one functional copy of the p53 gene is present during development.

Animals↗
PubMedDate not supplied

[Socio-cultural alientation and heroin addiction].

Heroin addiction is approached in this paper as a sociological process which takes place as a refusal of, or alienation from, the conventional social system, with, at the same time, attraction towards an alienated world, which gives recognition and identity to the subject. Three phases are identifiable in such process: 1) The subject is unable to copy or identify with an adult sexual model (normally, the parent of the same sex). This may reach the level of rejectal of the model. 2) The subject does not succeed in obtaining legitimate goals and benefits. This failure may only be imaginary, but involves a condition of subjective suffering. 3) The subject cannot establish good and significant relationships with nonaddicted peers, and consquently lives in isolation. The three phases are evident in a study carried out by the Author on 750 eddicts from New York and Puerto Rico. The integration into the deviant addict culture, which follows the triple failure outlined above, must be treated with training and resocialization techniques.

Adolescent↗
PubMedDate not supplied

[Detection of intragenomic polymorphism in the LR2 region of human intergenic ribosomal spacer].

It is a common point of view today that tandem ribosomal genes are subject to concerted evolution, a process that promotes homogeneity among the many copies through the mechanisms of unequal homologous exchange and gene conversion. These mechanisms can lead to opposite results: they can correct and eliminate new variants and they also can promote the spread of new gene variants throughout individual gene clusters among homologous and non homologous chromosomes. A number of experiments performed to decide which of these mechanisms is more important have yielded contradictory answers to this question. In this work we have cloned and partially sequenced 36 PCR-amplified copies of the human rIGS segment 22763-23523 apart from the transcription start point, obtained from the individual genome. This segment is enriched by (G)n and (AG)n clusters and enters into 2kb LR2 element of the human rIGS. Comparative analysis showed that absolutely identical sequences are absent among 36 inserts sequenced. The 26 clones differed only on a number of repeating elements in the paralogous (G)n and (AG)n clusters. The last ten clones differed not only on a number of cluster repeating elements, but contained insertions and deletions of distinct sizes, which do not correspond to the human rIGS polymorphism variants described earlier.

Base Sequence↗
PubMed1994

Human chromogranin A gene. Molecular cloning, structural analysis, and neuroendocrine cell-specific expression.

Chromogranin A (CgA) is an acidic glycoprotein, which is widely expressed in endocrine and neuroendocrine cells. It plays multiple important roles in the process of regulated hormone secretion. The single copy human CgA gene was isolated from a human fetal liver gene library. The gene spans 15 kilobases and contains 8 exons. Exon I encodes the 5'-noncoding region and the majority of the signal peptide coding region. Exons II-V collectively encode the highly conserved amino-terminal domain (the beta-granin sequence). Exon VI encodes a variable domain within which is the chromostatin sequence, and exon VII encodes another variable domain, which contains the pancreastatin sequence. Exon VIII encodes the highly conserved carboxyl-terminal domain and the 3'-noncoding region. The human gene promoter has a consensus TATA box, cAMP response element, and Sp-I sequence. 2.3 kilobases of the upstream regulatory region of the human CgA gene directed efficient transcription of a reporter chloramphenicol acetyltransferase gene in several neuroendocrine cell lines, including human medullary thyroid C-cell tumor, mouse pituitary corticotroph, rat pituitary tumor, and rat pheochromocytoma. The promoter was virtually inactive in nonneuroendocrine cell lines. Transient transfection studies with deleted promoter constructs showed that sequences lying between -55 and +32 base pairs relative to the transcription initiation site, containing the consensus cyclic AMP response element and TATA box, were sufficient for neuroendocrine cell-specific expression.

Amino Acid Sequence↗
PubMed1996

Turnaround time of in-patient discharge letters: a simple system of audit.

This user-friendly audit system, successfully developed and implemented in a district general hospital for use across all clinical specialties, is able to calculate the turnaround time of final discharge letters and as a consequence identify any areas of delay between patients' discharge and the letter being mailed. By serially monitoring both individual consultant and specialty performance, the system can provide valuable information on the discharge letter process to Purchaser and Provider alike. A copy of the audit system together with details of audit design and methodology is available to interested clinicians, managers and audit facilitators.

Communication↗