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PubMed2005

Biochemical and genetic characterization of 11 beta-hydroxysteroid dehydrogenase type 2 in low-renin essential hypertensives.

BACKGROUND: The 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) catalyzes the conversion of cortisol (F) to cortisone (E), avoiding the interaction of cortisol with the mineralocorticoid receptor. If it fails, cortisol will stimulate sodium and water reabsorption, increasing the intravascular volume that suppresses renin and secondarily increase the blood pressure. OBJECTIVE: To look for the possible contribution of a decreased ability of 11betaHSD2 to convert cortisol to its inactive metabolite cortisone in the pathogenesis of low renin hypertension (LREH). PATIENTS AND METHODS: We studied 64 LREH patients (plasma renin activity, PRA < 1 ng/ml per h), eighty normo-renin essential hypertensives (NREH) (PRA: 1-2.5 ng/ml per h) and 74 normotensives. Serum aldosterone (SA), F, E and serum F/E ratio was determined in all patients. A serum F/E ratio was considered high when it was higher than X + 2SD from the normotensive value. Cytosine-adenine (CA)-repeat microsatellite region in intron 1 of HSD11B2 gene was genotyped in all patients and normotensives volunteers. In 13 LREH with high F/E ratio we performed HSD11B2 gene sequencing. RESULTS: LREH had serum F/E ratio higher than NREH and normotensive controls (3.6 (2.9-4.3) versus 2.9 (2.2-4.3) versus 3.0 (2.4-3.7) (P = 0.004), respectively). We observed an inverse relation between F/E ratio and SA and PRA. In NREH and normotensives we did not find correlation between these variables. In the LREH subset the longer 155 bp CA-allele showed the highest serum F/E ratio. No mutations in coding region or short introns were found in LREH patients. CONCLUSION: In this study we show that low-renin essential hypertensives had increased serum cortisol/cortisone ratios as compared with normotensive subjects. This suggest that some essential hypertensives, with suppressed renin activity, may have an impairment in the cortisol inactivation catalyzed by the enzyme 11betaHSD2, whose low activity in LREH patients could be associated with the length of CA-repeat microsatellite in intron 1 of the HSD11B2 gene.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed2000

Clinicopathologic reassessment of primary cutaneous B-cell lymphomas with immunophenotypic and molecular genetic characterization.

Primary cutaneous B-cell lymphomas (PCBLs) may have particular clinicopathologic characteristics distinct from their lymph node-based counterparts. It has been suggested that PCBLs should have a separate classification system. The aim of this study was to determine whether the Revised European-American Lymphoid Neoplasms (REAL) classification is applicable to PCBL. Thirty-nine cases of PCBL from 36 patients, consisting of 20 men and 16 women (median age 66 yrs), were included in this study. Paraffin-section immunohistochemistry for CD3, CD5, CD10, CD20, CD43, Bcl-2, Bcl-6, and cyclin D1 was performed in all cases. Immunostaining for immunoglobulin light chains was also performed on cases histologically diagnosed as extranodal marginal zone lymphoma (MZL) and primary cutaneous B-cell lymphoma unclassifiable (PCBLu). Polymerase chain reaction (PCR) analysis of t(14;18) was performed in all cases. Immunoglobulin heavy chain gene rearrangement (VDJ) was tested by PCR on all follicle center lymphoma (FCL), MZL, and PCBLu cases. The 39 cases consisted of 15 (39%) FCLs, 13 (33%) diffuse large B-cell lymphomas (DLCL), 9 (23%) extranodal MZL, and 2 cases of PCBLu. Anatomically, 59% of PCBLs occurred in the head and neck, of which approximately 57% were FCL. Five of six cases presenting on the lower extremity were DLCL. Follow-up data was available from all 39 patients with a mean of 50.8 months. All but two patients are alive with or without disease at last contact. One patient with DLCL died of lung metastases and the other DLCL patient died of sepsis as a complication of therapy. In all 15 cases of FCL, CD10 and/or Bcl-6 expression supported the follicle center origin of the neoplastic cells. In contrast to previous reports, we found that 53% (8 of 15) of primary cutaneous FCL had either Bcl-2 protein expression or t(14;18). Our data indicate that many cases of primary cutaneous FCL have Bcl-2 alterations similar to their nodal counterpart. We found that 95% (37 of 39) of PCBLs could be classified according to the REAL classification, supporting its applicability in cutaneous lymphomas.

Adult↗

PubMed2006

Prevalence of Toxoplasma gondii in cats from Colombia, South America and genetic characterization of T. gondii isolates.

Cats are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete the environmentally-resistant oocysts. In the present study, prevalence of T. gondii was determined in serum, feces, and tissues of 170 unwanted cats from Colombia, South America. Antibodies to T. gondii were assayed by the modified agglutination test and found in 77 of 170 (45.2%) cats with titers of <1:5 in 93, 1:5 in eight, 1:10 in 17, 1:20 in 10, 1:40 in seven, 1:80 in four, 1:160 in eight, 1:320 in six, and 1:640 or higher in 17 cats. T. gondii oocysts were not found in feces of any cat as ascertained by bioassay in mice. Tissues (brain, heart, tongue) of 116 cats were bioassayed in mice or cats. T. gondii was isolated from tissues of 15 of the 42 cats with titers of 1:40 or higher and not from any of the 90 cats titers of 1:20 or lower. Of the 29 cats whose tissues were bioassayed individually, T. gondii was isolated from the tongues of nine, hearts of eight, and brains of five. Mice inoculated with tissues of 12 of 15 infected cats died of toxoplasmosis; with nine T. gondii isolates all infected mice died. Overall, 65 of 92 (70%) of T. gondii-infected mice died of toxoplasmosis. Genotyping of these 15 isolates using polymorphisms at the SAG1, SAG2, SAG3, BTUB, and GRA6 loci revealed that three isolates (TgCtCo1, 2, and 7) had Type I alleles and one isolate (TgCtCo8) had Type II allele at all five loci. Eleven isolates contained the combination of Type I and III alleles and were divided into three genotypes, with TgCtCo3,5,6,9,12,13 and 15 had alleles I, I, III, I and III, TgCtCo4,10,11 had alleles I, III, III, I and I, and TgCtCo14 had alleles I, III, III, III, and III, at loci SAG1, SAG2, SAG3, BTUB and GRA6, respectively. All infected mice from each group had identical genotype except one mouse infected with TgCtCo5 had a Type III allele at locus BTUB and a unique allele (u-1) at locus SAG1 indicating mixed infection for TgCtCo5, whereas the rest seven mice had a Type I alleles at both loci.

Agglutination Tests↗

PubMed1995

Morphological, ultrastructural, and genetic characterization of an unusual T-cell lymphoma in a patient with sinus histiocytosis with massive lymphadenopathy.

Sinus histiocytosis with massive lymphadenopathy (SHML) is a rare benign disease of unknown etiology. It is rarely associated with malignant lymphoma. This report documents the first case of a T-cell lymphoma, which developed in a patient with a 10-year history of SHML. The disease was complicated by hypereosinophilia and massive retroperitoneal lymphadenopathy. Histological examination of a cervical lymph node biopsy during the terminal phase identified a lymphoma composed of cells with morphological plasmacytoid features. Ultrastructurally, the tumor cells showed poorly developed cytoplasm, nuclei with peripheral chromatin clumping, and inconspicuous nucleoli. Cytogenetic studies showed two related clones. On immunohistochemical staining tumor cells were positive with monoclonal antibodies (mAb) CD3 and CD45RO. Southern blotting analysis identified clonal rearrangements in the T-cell receptor (TCR) alpha, beta and gamma genes. Thus, T-cell lineage of the tumor cells was established. In situ hybridization of interleukin-2 (IL-2) and interleukin-5 (IL-5) cDNA probes on tissue sections identified the synthesis of IL-5 by the eosinophils, suggesting an autocrine pathway of eosinophilopoiesis leading to hypereosinophilia in this patient.

Clone Cells↗

PubMed1990

Clinical, cytogenetic, and molecular genetic characterization of two unrelated patients with different duplications of 21q.

We present 2 patients with dup(21q). Patient MP01 had mild mental retardation, facial findings characteristic of Down syndrome (DS), and a terminal duplication of chromosome 21. His karyotype was 46,XY,dup(21) (q22.1-qter). Patient MP03 had mild mental retardation, minor anomalies not characteristic of DS, and a duplication of the proximal long arm of chromosome 21, karyotype 46,XX,dup(21) (q11.2-q21.2). The patients were studied with single-copy DNA sequences from 20 loci on chromosome 21 to characterize the extent of the duplicated regions at the DNA level. DNA loci from D21S55 to COL6A1 were triplicated in patient MP01 while loci from D21S13 to D21S8 were triplicated in patient MP03. Our results support the hypothesis of a critical region of chromosome 21, which in triplicate is responsible for many of the facial changes associated with DS. Other genes outside this region may also contribute to other abnormalities observed in DS.

Adult↗

PubMed1993

Molecular genetic characterization of an X-linked form of Leigh's syndrome.

We report a patient with necrotizing encephalomyelopathy (Leigh's syndrome) associated with a deficiency of pyruvate dehydrogenase complex activity. The underlying mutation is an A to C transversion in the pyruvate dehydrogenase complex E1 alpha subunit gene. As the E1 alpha subunit is encoded on the X chromosome, this observation confirms that some patients with Leigh's syndrome may potentially exhibit X-linked inheritance.

Amino Acid Sequence↗

PubMed1992

Cytogenetic and molecular genetic characterization of papillary thyroid carcinomas.

A combined cytogenetic and molecular analysis was performed on 11 cases of papillary thyroid carcinoma. A simple karyotypic abnormality was detected in five tumors, whereas six had no apparent chromosome change. In four of five rearranged cases the presence of a specific chromosomal abnormality involving chromosome 10 (cases 1 and 2) and chromosome 1 (cases 3 and 4) was associated with the rearrangement of two protooncogenes: RET and NTRKI (formerly trk), respectively, with different donor genes. Moreover, the chromosomal localization of the involved genes and the type of chromosomal change observed suggested that RET and NTRKI activation occurred by intrachromosomal rearrangements. The six cases with normal karyotype did not show RET or NTRKI activation. These findings suggest that a combined cytogenetic and molecular approach would be useful in understanding the pathogenesis of thyroid neoplasia.

Blotting, Southern↗

PubMed2004

Genetic characterization and structural analysis of VHL Spanish families to define genotype-phenotype correlations.

Von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations in the VHL gene. This gene, located in the 3p25-26 chromosome, is a tumor suppressor gene associated with the inhibition of angiogenesis and apoptosis, cell cycle exit, fibronectin matrix assembly, and proteolysis. To define the molecular basis of VHL in a Spanish population, we studied 33 patients suspected of suffering familial or de novo VHL disease and two familial pheochromocytoma cases. Sequence analysis of the coding regions of the VHL gene revealed germline sequence variants in 68.7% (24 out of 35) of the patients, and four of them presented with undescribed germline alterations: g.5429-5430insG, p.Leu128Arg, p.Tyr175Cys, and p.Tyr175Asn. For the remaining 11 patients who showed negative for point mutations, we performed Southern blot analysis and detected gross rearrangements in eight cases (22.8% of the index cases). Our results support the relevance of VHL gene analysis in familial pheochromocytoma cases and also in those with no familial history. In order to investigate the relevance of different amino acid changes in the VHL phenotype, we also analyzed the genotype-phenotype correlations using structural analysis to assess protein stability and complexes. The association of clear cell renal carcinoma (CCRC) development with a relatively high loss of structural stability in pVHL missense-mutants was consistent. Structural stability data in the genotype-phenotype correlations therefore provides us with a better understanding of VHL clinical implications. It is also a suitable approach to the evaluation of unknown significance changes.

Adenocarcinoma, Clear Cell↗

PubMed1983

Biochemical and genetical characterization of a fiber-defective temperature-sensitive mutant of type 2 adenovirus.

The adenovirus type 2 fiber mutant H2 ts 125 synthesized an unstable, temperature-sensitive fiber polypeptide with an apparent mol. wt. smaller by 2500 than the wild-type (62 K). The polypeptide of 59.5 K was found to be stable at the permissive temperature (33 degrees C). H2 ts 125 fiber synthesized in reticulocyte lysates had the same apparent mol. wt. of 59.5 K as the mutant fiber produced in vivo. Neither structural nor functional differences between wild-type and mutant fibers were detected in the N-terminal and C-terminal sequences, excluding the occurrence of a new initiation or termination codon. Restriction analysis of H2 ts 125 DNA also ruled out the hypothesis of a deletion mutant. The 59.5 K mutant fiber unit was normally glycosyated, N-acetylated, assembled into 6S oligomeric fiber and incorporated into virions. DNA sequencing of the H2 ts 125 fiber gene revealed two point mutations at nucleotides 3970 (C*TT leads to T*TT) and 4958 (GC*T leads to GT*T), corresponding to two amino acid changes at positions 105 and 434, respectively. The 105 mutation consisted of a conservative change Leu leads to Phe; the 434 interchange was Ala leads to Val, usually considered as nonconservative. The possibility of a donor site for splicing created by the mutation at codon GTT was eliminated on the basis of S1 nuclease analysis data. All these results suggested that either one or both mutations concerned highly organized domain(s) of the fiber polypeptide chain, resulting in aberrant mobility in SDS-polyacrylamide gels and temperature-sensitivity.

Acetylation↗

PubMed1994

Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation.

Deformylase performs an essential step in the maturation of proteins in eubacteria, by removing the formyl group from the N-terminal methionine residue of ribosome-synthesized polypeptides. In spite of this important role in translation, the enzyme had so far eluded characterization because of its instability. We report the isolation of the deformylase gene of Escherichia coli, def, by overexpression of a genomic library from a high-copy-number plasmid and selection for utilization of the substrate analogue formyl-leucyl-methionine as a source of methionine. The def gene encodes a 169 amino acid polypeptide that bears no obvious resemblance to other known proteins. It forms an operon with the fmt gene, that encodes the initiator methionyl-tRNA(i) transformylase, which was recently characterized (Guillon et al., J. Bacteriol., 174, 4294-4301, 1992). This operon was mapped at min 72 of the E. coli chromosome. The def gene could be inactivated if the fmt gene was also inactivated, or if biosynthesis of N10-formyl-tetrahydrofolate, the formyl donor in methionyl-tRNA(i) transformylation, was blocked by trimethoprim. These findings designate deformylase as a target for antibacterial chemotherapy.

Acyltransferases↗

PubMed2004

Genetic characterization of Pompeii and Herculaneum Equidae buried by Vesuvius in 79 AD.

DNA extracted from the skeletons of five equids discovered in a Pompeii stable and of a horse found in Herculaneum was investigated. Amino acid racemization level was consistent with the presence of DNA. Post-mortem base modifications were excluded by sequencing a 146 bp fragment of the 16S rRNA mitochondrial gene. Sequencing of a 370 bp fragment of mitochondrial (mt)DNA control region allowed the construction of a phylogenetic tree that, along with sequencing of nuclear genes (epsilon globin, gamma interferon, and p53) fragments, gave us the possibility to address some questions puzzling archaeologists. What animals-donkeys, horses, or crossbreeds-were they? And, given they had been evidently assigned to one specific job, were they all akin or were they animals with different mitochondrial haplotypes? The conclusions provided by molecular analysis show that the Pompeii remains are those of horses and mules. Furthermore one of the equids (CAV5) seems to belong to a haplotype, which is either not yet documented in the GenBank or has since disappeared. As its characteristics closely recall those of donkeys, which is the out group chosen to construct the tree, that appears to have evolved within the Equidae family much earlier than horses, this assumption seems to be nearer the truth.

Animals↗

PubMed1994

Biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child.

The coronavirus strain HECV-4408 was isolated from diarrhea fluid of a 6-year-old child with acute diarrhea and propagated in human rectal tumor (HRT-18) cells. Electron microscopy revealed coronavirus particles in the diarrhea fluid sample and the infected HRT-18 cell cultures. This virus possessed hemagglutinating and acetylesterase activities and caused cytopathic effects in HRT-18 cells but not in MDBK, GBK and FE cells. One of four S-specific monoclonal antibodies reacted in Western blots with HECV-4408, BCV-L9 and BCV-LY138 but not with HCV-OC43, and two reacted with BCV-L9 but not with HECV-4408, BCV-LY138 and HCV-OC43. One S-specific and two N-specific monoclonal antibodies reacted with all of these strains. cDNA encompassing the 3' 8.5 kb of the viral RNA genome was isolated by reverse transcription followed by polymerase chain reaction amplification had size and restriction endonuclease patterns similar to those of BCV-L9 and BCV-LY138. In contrast, the M gene of HCV-OC43 differed in restriction patterns from HECV-4408 and BCV. A genomic deletion located between the S and M within the non-structural genes of HCV-OC43 was not detected in HECV-4408. DNA sequence analyses of the S and HE genes revealed more than 99% nucleotide and deduced amino acid homologies between HECV-4408 and the virulent wild-type BCV. Forty-nine nucleotide and 22 amino acid differences were found between the HE genes of HECV-4408 and HCV-OC43, while only 16 nucleotide and 3 amino acid differences occurred between the HE genes of HECV-4408 and BCV-LY138. We thus conclude that the strain HECV-4408 is a hemagglutinating enteric coronavirus that is biologically, antigenically and genomically more closely related to the virulent BCV-LY138 than to HCV-OC43.

Acetylesterase↗

PubMed1995

Biochemical and genetic characterization of a replication protein A dependent DNA helicase from the yeast, Saccharomyces cerevisiae.

We have purified a DNA dependent ATPase/DNA helicase, DNA helicase B, from S. cerevisiae. Helicase B was a 129-kDa polypeptide. The ATPase activity of helicase B was strongly DNA dependent. The DNA helicase activity was stimulated by yeast replication protein A, indicating a probable function in DNA replication. Helicase B showed a 5'-->3' polarity of movement. Protein sequencing indicated that helicase B was identical to a hypothetical 127-kDa polypeptide encoded by yORF61, located 5' upstream of the BMH1 locus in chromosome V. The protein sequence contained a "type I ATP/GTP binding motif" and other helicase-like motifs and the expressed protein exhibited helicase activity. Thus, we concluded that yORF61 is the gene for helicase B and will be referred to as HCSB.

Adenosine Triphosphatases↗

PubMed1993

Molecular genetic characterization of maple syrup urine disease in European families.

Maple syrup urine disease results from defects in the branched chain alpha-ketoacid dehydrogenase complex. Cells from seven German, three Turkish, and two Italian families including five consanguineous matings were analyzed for the causative mutations. Enzyme assays were used to confirm the initial clinical diagnosis of all probands. Immunoblots of mitochondrial proteins from these probands revealed reduced expression of the E1 alpha and beta proteins of the complex. Previous studies showed that interaction of alpha and beta was necessary to stabilize both proteins so that defects in either protein can result in decreased presence of both. The E1 alpha Y393N mutation common in the Mennonite population that results in diminished amounts of both alpha and beta proteins was not the cause of the reduction in these European patients.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1993

Derivation of a scale-independent parameter which characterizes genetic sequence comparisons.

A new method of quantifying the similarity between genetic sequences is presented. The method makes use of the finding that sequence comparisons expressed in binary vector form have an associated scale-independent parameter, D. This parameter is represented in the function M(S,n) = (N) (en/enD), where S is the vector, n represents the window size which is allowed to vary, N is a constant, and D is the scale-independent measure of homology. By comparing two sequences using this method, a unimodal, symmetric distribution of D values associated with the frameshifted vectors is obtained. The degree of sequence similarity is determined by the distribution of these parameters. A set of sequences of evolutionary interest coding for glyceraldehyde-3-phosphate dehydrogenases and mammalian insulins is compared using this methodology. The results confirm evolutionary tree distances calculated using different procedures. Since a z score can be calculated for each comparison, the method allows for the rapid identification of sequence homologies ranked according to the probability of occurrence. This unique scale-independent measure of similarity allows contrasts and comparisons between any two sequence fragments using all available order information.

Actins↗

PubMed1993

Molecular and genetic characterization of the Drosophila tartan gene.

Here we report the discovery and characterization of the Drosophila tartan gene. tartan is transcribed in an unusual embryonic pattern of intersecting stripes which are generated in response to the anterior-posterior and dorsal-ventral regulatory systems. tartan encodes a putative transmembrane protein containing extracellular leucine-rich repeats characteristic of numerous cell surface receptors and adhesion proteins. Its expression is correlated with aspects of segmentation and neurogenesis, including the formation of neuroblasts, sensory mother cells, and peripheral nerves. Mutants homozygous for a recessive lethal tartan loss-function allele exhibit defects in the position and number of cells within peripheral sense organs, the routing of peripheral nerves, and the organization of commissures within the central nervous system. Mutants are also defective in muscle organization. These results suggest that tartan is required for cell surface interactions important for normal organization of epidermal and subepidermal structures.

Amino Acid Sequence↗

PubMed1998

Genetic characterization of the murine Ym1 gene and identification of a cluster of highly homologous genes.

The murine Ym1 gene belongs to a family of mammalian genes that are homologous to the chitinases from lower organisms, such as insects, nematodes, bacteria, and plants. All of these homologous mammalian proteins (except one member) have no demonstrable chitinase activity and therefore cannot be considered chitinases. The biological functions of these proteins remain unknown. However, these proteins may function through binding to carbohydrate polymers. In an effort to characterize the murine Ym1 gene better, we have delineated its genomic structure. We mapped the chromosomal location of the Ym1 gene to a central region of mouse chromosome 3 (in the region syntenic to human chromosome 1p13) and that of another murine chitinase-like gene, Brp39, to a central region of mouse chromosome 1 (in the region syntenic to human chromosome 1q31). In addition, we identified several genes highly homologous to Ym1 that cluster at the Ym1 locus. We show that these genes are expressed selectively in different mouse tissues.

Amino Acid Sequence↗

PubMed2002

Detection and genetic characterization of canine distemper virus (CDV) from free-ranging red foxes in Italy.

Fragments of the genes encoding the haemoagglutinin (H) and the nucleocapsid protein (N) of a canine distemper (CDV)-like virus affecting a red fox (Vulpes vulpes) were sequenced and analysed. The CDV-like virus detected in the fox was found to be not dissimilar, in both the H and N gene, from other CDVs spreading in Italy, as well as all over the world, and phylogenetic analysis on the H protein-encoding gene allowed to include all the Italian CDVs in the H European genotype.

Animals↗