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Results for “variable gene flow”

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At least 451 records · Page 25Linked to original sources
PubMed1995

Expression and loss of heterozygosity of c-met proto-oncogene in primary breast cancer.

The c-met proto-oncogene encodes the receptor to hepatocyte growth factor-scatter factor (HGF-SF), a mesenchyme-derived cytokine with cell-dissociating, invasion, and angiogenic properties. The expression of c-met in breast cancer is the subject of controversy; 111 primary breast cancers were examined for LOH of c-met by Southern blot electrophoresis. c-met expression was measured in a further 40 patients with breast cancer and in 8 patients with benign breast disease by flow cytometry. LOH of c-met was detected in only 4% of informative breast cancers. Expression of c-met was significantly greater in patients with breast cancer than in those with benign breast disease (P < 0.01, Mann-Whitney). There was no correlation however between increased c-met expression and clinicopathological prognostic variables. These results do not support the role of c-met as a tumour suppressor gene in breast cancer but suggest increased receptor expression in malignant breast disease. The significance of this increased expression in breast cancer is the subject of further investigation.

Blotting, Southern↗
PubMed1997

Cell display library for gene cloning of variable regions of human antibodies to hepatitis B surface antigen.

A novel cell display system was developed for cloning the variable region (V) genes of antigen-specific human antibodies. The system is based on an antibody library displayed on the surface of COS cells, using a plasmid vector designed to direct expression of membrane-bound antibodies. COS cells expressing antigen-specific antibodies were separated using a flow cytometer for their binding to a fluorescent dye-labeled antigen. To test the performance of this system. We cloned V genes of 4 antibodies directed against hepatitis B surface antigen (HBsAg) from a library prepared from peripheral blood lymphocytes of a vaccinated donor. These membrane-bound anti-HBsAg antibodies were easily converted to soluble forms, all of which showed a size similar to human serum IgG in SDS-PAGE and the same specific binding to HBsAg as membrane-bound forms in ELISA. All VH and VK gene segments of the 4 clones isolated in this study belonged to VHIII and VKI subgroups, respectively. These findings demonstrate the potential and selection capabilities of our cell display system for cloning the V genes of antigen-specific human antibodies.

Amino Acid Sequence↗
PubMed1999

Bispecific agents target endogenous murine T cells against human tumor xenografts.

A variety of immunological approaches to cancer treatment are currently being explored. These include strategies designed to enhance or redirect the activity of T cells against tumors. Bispecific antibodies comprise a class of agents capable of redirecting T cells by binding to a tumor antigen and the T-cell receptor (TCR). In vivo pre-clinical testing of bispecific antibodies against human tumors has to date been limited to the use of immunodeficient mice that receive the bispecific agent, activated human effector T cells, and human tumor cells. In this report, we show that TCR transgenic/RAG-1 knockout mice (TCR/RAG) serve as a unique model allowing endogenous T cells to be redirected against transplanted human tumors. The findings show that TCR/RAG mice (i) accepted transplants of human tumors, including the folate-receptor-positive tumor line KB; (ii) contained endogenous cytotoxic T lymphocytes that could be activated in vivo with an antigenic peptide recognized by the transgenic TCR; (iii) rejected human tumors after treatment with the activating peptide and bispecific agents that contained folic acid co-valently linked to an anti-TCR antibody. Successful rejection was achieved with folate conjugates of Fab or scFv fragments. Treatment with activating agents and bispecific conjugates resulted in the complete eradication of freshly transplanted tumors as well as significantly prolonging the survival of mice bearing established solid tumors. Our results highlight the importance of including T-cell-activating modalities in combination with bispecific antibodies. Additionally, we introduce a system that allows endogenous T cells to be redirected against human tumor xenografts and in which the T cells may be followed in vivo by use of a clonotypic marker.

Animals↗
PubMed2000

CD8(+) T cells in Hodgkin's disease tumor tissue are a polyclonal population with limited clonal expansion but little evidence of selection by antigen.

A minor component (about 25%) of lymphocytes in Hodgkin's disease (HD) are CD8(+) T cells. It is unclear whether the presence of these cells reflects an antitumor cytotoxic response. The goal of the present study was to investigate clonal composition and the T cell receptor (TCR) beta repertoire of the CD8(+) T cell population in HD. Single CD8(+) cells were micromanipulated from frozen tissue sections of lymph nodes affected by primary HD and subjected to single target amplification of TCRbeta gene rearrangements. Sequence analysis of the V region genes revealed the presence of expanded CD8(+) T cell clones in all three cases analyzed. Most of these clonal expansions accounted for less than 10% of the CD8(+) T cell population. In one case, 30% of the CD8(+) T cells belonged to one or two clones. Comparison of V region sequences, however, did not provide evidence that the micromanipulated CD8(+) cells were sampled from a population that was selected for particular antigen specificities. No obvious biases in TCR Vbeta and Jbeta gene segment usage or CDR3 length distribution were found. Similarities of CDR3 amino acid sequences as found in selected CDR3 structures were rare. These results suggest that, like CD4(+) T cells, CD8(+) T cells may also be recruited into the tumor tissue in an antigen-nonspecific manner.

CD8-Positive T-Lymphocytes↗
PubMed2000

Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan.

Vascular endothelial cells produce nitric oxide (NO), which contributes to the regulation of blood pressure and regional blood flow. Although Endothelial NO synthase (eNOS) gene polymorphisms have been shown to have a positive association with coronary artery disease, the linkage between eNOS gene polymorphisms and hypertension has been controversial. In the present study, therefore, we identified genotypes for Glu298Asp and variable number tandem repeats in intron 4 (4b/a) in 183 hypertensive and 193 normotensive populations. The Glu298Asp variant had a significant association with hypertension (odds ratio, 1.8; 95% confidence interval, 1.1-3.0). The allele frequencies of 298Asp for Glu298 in hypertensive patients were significantly higher than those in normotensive subjects (0.128 vs 0.080, p<0.05). Diastolic and mean arterial blood pressures were significantly higher in hypertensive subjects with the 298Asp allele than those without the variant allele (p<0.05). However, disequilibrium of 4b/a polymorphism was absent between these two groups. These results suggest that the Glu298Asp variant may be a genetic susceptibility factor for hypertension.

Aged↗
PubMed2001

Population genetics of the hispid cotton rat (Sigmodon hispidus): patterns of genetic diversity at the major histocompatibility complex.

The hispid cotton rat, Sigmodon hispidus, is a common rodent widely distributed across the southern USA and south into South America. To characterize major histocompatibility complex (MHC) diversity in this species and to elucidate large-scale patterns of genetic partitioning, we examined MHC genetic variability within and among 13 localities, including a disjunct population in Arizona and a population from Costa Rica that may represent an undescribed species. We also tested the hypothesis that populations within the USA are at equilibrium with regard to gene flow and genetic drift, resulting in isolation-by-distance. Using single-strand conformation polymorphism (SSCP) analysis we identified 25 alleles from 246 individuals. Gene diversity within populations ranged from 0.000 to 0.908. Analysis of molecular variance (AMOVA) revealed that 83.7% of observed variation was accounted for by within-population diversity and 16.3% was accounted for by among-population divergence. The disjunct population in Arizona was fixed for a single allele. The Costa Rican population was quite divergent based on allelic composition and was the only population with unique alleles. Within the main portion of the geographical distribution of S. hispidus in the USA there was considerable divergence among some populations; however, there was no significant pattern of isolation-by-distance overall (P = 0.090). Based on the significant divergence of the only sampled population to its east, the Mississippi River appears to represent a substantial barrier to gene flow.

Alleles↗
PubMed2001

Antennal sensilla patterns indicate geographic and ecotopic variability among Triatoma infestans (Hemiptera: Reduviidae) populations.

Antennal sensilla patterns were used to explore intraspecific variability among Triatoma infestans (Klug) from domestic habitats in Argentina and Bolivia, and from sylvatic habitats in Bolivia. The sensilla pattern was effective in distinguishing individuals at sexual, ecotopic, and geographic levels, and supported the idea of a lack of gene flow between sylvatic and domestic habitats.

Animals↗
PubMed2001

Temporal patterns of genetic variation for resistance and infectivity in a Daphnia-microparasite system.

Theoretical studies have indicated that the population genetics of host-parasite interactions may be highly dynamic. with parasites perpetually adapting to common host genotypes and hosts evolving resistance to common parasite genotypes. The present study examined temporal variation in resistance of hosts and infectivity of parasites within three populations of Daphnia magna infected with the sterilizing bacterium Pasteuria ramosa. Parasite isolates and host clones were collected in each of two years (1997, 1998) from one population; in two other populations, hosts were collected from both years, but parasites from only the first year. We then performed infection experiments (separately for each population) that exposed hosts to parasites from the same year or made combinations involving hosts and parasites from different years. In two populations, patterns were consistent with the evolution of host resistance: either infectivity or the speed with which parasites sterilized hosts declined from 1997 to 1998. In another population, infectivity, virulence, and parasite spore production did not vary among host-year or parasite-year. For this population, we also detected strong within-population genetic variation for resistance. Thus, in this case, genetic variability for fitness-related traits apparently did not translate into evolutionary change. We discuss a number of reasons why genetic change may not occur as expected in parasite-host systems, including negative correlations between resistance and other traits, gene flow, or that the dynamic process itself may obscure the detection of gene frequency changes.

Animals↗
PubMed2006

Regulatory mechanisms of gene expression: complexity with elements of deterministic chaos.

Linear models based on proportionality between variables have been commonly applied in biology and medicine but in many cases they do not describe correctly the complex relationships of living organisms and now are being replaced by nonlinear theories of deterministic chaos. Recent advances in molecular biology and genome sequencing may lead to a simplistic view that all life processes in a cell, or in the whole organism, are strictly and in a linear fashion controlled by genes. In reality, the existing phenotype arises from a complex interaction of the genome and various environmental factors. Regulation of gene expression in the animal organism occurs at the level of epigenetic DNA modification, RNA transcription, mRNA translation, and many additional alterations of nascent proteins. The process of transcription is highly complicated and includes hundreds of transcription factors, enhancers and silencers, as well as various species of low molecular mass RNAs. In addition, alternative splicing or mRNA editing can generate a family of polypeptides from a single gene. Rearrangement of coding DNA sequences during somatic recombination is the source of great variability in the structure of immunoglobulins and some other proteins. The process of rearrangement of immunoglobulin genes, or such phenomena as parental imprinting of some genes, appear to occur in a random fashion. Therefore, it seems that the mechanism of genetic information flow from DNA to mature proteins does not fit the category of linear relationship based on simple reductionism or hard determinism but would be probably better described by nonlinear models, such as deterministic chaos.

Alleles↗
PubMed2004

Distinction between wild and cultivated enset (Ensete ventricosum) gene pools in Ethiopia using RAPD markers.

In southwest Ethiopia, the cultivation area of Ensete ventricosum (enset) overlaps with the natural distribution area of this species. Analyses of genetic diversity were undertaken using RAPD to provide information for conservation strategies as well as evidence of possible gene flow between the different gene pools, which can be of interest for future improvement of cultivated enset. The extent of RAPD variation in wild enset was investigated in 5 populations in the Bonga area (Kefficho administrative region) and 9 cultivated clones. Comparisons were also made with some Musa samples of potential relevance for crop improvement. Nine oligonucleotide primers amplified 72 polymorphic loci. Population differentiation was estimated with the Shannon index (G'(ST)=0.10), Nei's G(ST) (0.12) and AMOVA (Phi(ST)=0.12), and appears to be relatively low when compared with outbreeding, perennial species in general. Cluster analysis (UPGMA) and principal component analysis (PCA) similarly indicated low population differentiation, and also demonstrated that cultivated clones essentially clustered distinctly from wild enset samples, suggesting that the present-day cultivated enset clones have been introduced to domestication from a limited number of wild progenitors. In addition, subsequent gene flow between wild and cultivated enset may have been prohibited by differences between modes of propagation and harvesting time; cultivated enset is propagated vegetatively through sucker production and the plant is generally harvested before maturity or flower set, thereby hindering pollination by wild enset or vice versa. A significant correlation was not found between genetic and geographical distances. The relatively high total RAPD diversity suggests that wild enset populations in the Bonga area harbour genetic variability which could potentially act as a source for useful or rare genes in the improvement of cultivated enset. As expected, E. ventricosum was clearly differentiated from the analysed Musa samples, that clustered in accordance with the present morphology- and molecular marker-based taxonomy of the genus.

DNA, Plant↗
PubMed2006

Common sequence variations in the P2Y12 and CYP3A5 genes do not explain the variability in the inhibitory effects of clopidogrel therapy.

The efficacy of the platelet P2Y12 receptor antagonist clopidogrel, which undergoes cytochrome-mediated metabolism to its active form, shows marked inter-individual variability. We investigated whether polymorphic variations in the P2Y12 gene, which have been linked to platelet aggregation phenotypes, or the cytochrome P450 3A5 gene 6986G > A polymorphism, which largely determines CYP3A5 expression, influence the response to clopidogrel therapy. Fifty-four patients listed for elective percutaneous coronary intervention were studied using ADP-induced optical aggregometry, whole-blood single platelet counting (WBSPC) aggregometry, and flow-cytometric analysis of platelet P-selectin expression and platelet-monocyte conjugate formation. Platelet reactivity was measured at baseline, 4 h post clopidogrel 300 mg, and 10 and 28 days following clopidogrel 75 mg daily. A further 55 patients were studied using ADP-induced WBSPC at baseline and 4 h post clopidogrel 600 mg. Patients were genotyped for P2Y12 haplotype and the CYP3A5 6986G > A single nucleotide polymorphism. Neither genotype was found to significantly influence the inhibition of platelet responses by either clopidogrel regimen. In conclusion, common sequence variations within the P2Y12 and CYP3A5 genes do not contribute any major effect to the inter-patient variability in clopidogrel efficacy.

Clopidogrel↗
PubMed2003

Memory B cells producing somatically mutated antiphospholipid antibodies are present in healthy individuals.

Antiphospholipid antibodies (aPLs) are associated with thrombosis and recurrent abortions during autoimmune pathologies, but they are also produced in healthy individuals and during infectious diseases. To analyze the possible links between physiologic and pathologic aPLs, it is of importance to characterize normal aPL production. We took advantage of the known tropism of Epstein-Barr virus (EBV) for B cells in general, and memory B cells in particular, during primary infectious mononucleosis (IMN) in 3 patients to get access to anticardiolipin (aCL)-producing B cells. Flow cytometry analysis of these cells showed that, depending on the patient, 10% to 60% of immunoglobulin M (IgM) aCL-producing B cells express the CD27 marker of memory B cells. Single cell sorting of aCL B cells, followed by single cell reverse transcription-polymerase chain reaction (RT-PCR) amplification of their immunoglobulin variable region genes, showed that some of these cells produce mutated forms of aCL antibodies, confirming their memory B-cell origin. Considering that, during primary IMN, EBV infects and expands already pre-existing memory B cells, we conclude that healthy individuals have a discrete pool of aCL memory cells able to produce mutated forms of antibodies. The implications of this new information are discussed in light of different hypotheses regarding the origin of aCL.

Adolescent↗
PubMed2026

Understanding Genomic Landscapes of Differentiation in Round-Tailed Horned Lizards (Phrynosoma modestum).

Population divergence is promoted by divergent selection and inhibited by gene flow, but the mechanisms of and relationship between these two processes remain poorly understood. Developing a well-informed hypothesis of the selective pressures underlying divergence in a natural population requires a thorough understanding of both species structure and demographic history. In this study, we assess whole-genome sequences of round-tailed horned lizards (Phrynosoma modestum) from throughout the species range and combine phylogenetic analyses with genomic landscape scans to understand how current genetic diversity has been influenced by demographic histories and evolutionary pressures. Maximum likelihood (ML) phylogenetic analysis supports two lineages within the species, corresponding to a North/South population divide that developed around 7&#x2005;million years ago (Ma) and displays little migration. However, intermediate genealogical divergence index values between the two lineages ultimately leave us unable to recommend a full taxonomic distinction. Genome-wide scans of population genetic statistics identified islands of divergence exhibiting differentiation patterns linked to models of reproductive isolation and within-population selection. Significantly negative values of Tajima's D and positive selection statistics in these islands offer support for selection acting on P. modestum, but patterns may also stem from recent population expansions. We posit that selection within populations has played a large role in shaping genomic divergence across the species' range. Taken together, our results provide perspective into how variable selective pressures shape the genomics of two divergent populations currently maintaining species integrity, despite significant signatures of geographic structure and divergence.

Animals↗
PubMed1993

V gamma gene usage in peripheral blood gamma delta T cells.

The majority (50-90%) of gamma delta T cells in the peripheral blood of adult individuals expresses a T-cell receptor (TCR) which uses V gamma 9 and V delta 2 as variable elements. Little is known about the distribution of other V gamma gene elements in the remaining 10-50% of gamma delta T cells. Here we have studied the V gamma gene expression in peripheral blood gamma delta T cells by 3-color flow cytometry analysis applying established monoclonal antibodies (mAb) directed against V gamma 9 and V gamma 4, as well as a novel mAb directed against V gamma 2, V gamma 3 and V gamma 4. On average, 79.9% of gamma delta T cells expressed V gamma 9, 11.9% V gamma 2/V gamma 3, 4.4% V gamma 4, and 7.5% one of the remaining V gamma 5, V gamma 8, V gamma 10 or V gamma 11 elements. There were remarkable variations in the gamma delta subset composition between individual donors. The majority (69.8%) of V gamma 2/V gamma 3/V gamma 4-bearing cells co-expressed V delta 1, while on average only 17.8% of V gamma 2/V gamma 3/V gamma 4-bearing cells co-expressed V delta 2. This is in contrast to V gamma 9-bearing gamma delta T cells, of which 83.1% used V delta 2 and only 12.7% V delta 1. Taken together, this data identifies V gamma 2/V gamma 3 as the second most frequently used set of V gamma elements in human peripheral blood gamma delta T cells.

Adult↗
PubMed1994

Selective expression of V beta families by T cells in the blood and salivary gland infiltrate of patients with primary Sjögren's syndrome.

OBJECTIVE: The T cell infiltration of the salivary gland of patients with Sjögren's syndrome (SS) has been implicated in the pathogenic process of the disease. We examined the representation of V beta subsets in the blood and salivary tissue of patients with SS. METHODS: Circulating T cells from 12 patients and paired samples of blood and labial salivary glands obtained from 8 patients were studied. A panel of monoclonal antibodies directed against the variable region of the T cell receptor was used to enumerate the cells expressing V beta families in the peripheral blood by flow cytometry, and in tissue sections by immunofluorescence. RESULTS: We found an increase of cells bearing V beta 2 family gene products in the circulation, and an increase in both V beta 2 and V beta 8 in the salivary gland infiltrate of patients with SS. No significant difference was noted between the 5 DR3+ patients and 7 DR3- patients studied with regard to the V beta families seen. CONCLUSION: Our data are consistent with a role for specific T cell families in the pathogenesis of SS.

Adult↗
PubMed1991

Effects of prolonged infusion of human alpha calcitonin gene-related peptide on hemodynamics, renal blood flow and hormone levels in congestive heart failure.

We have previously demonstrated that short-term infusion of calcitonin gene-related peptide (CGRP) has beneficial effects in congestive heart failure. The effects of prolonged infusion of CGRP on hemodynamic functions, plasma hormones and renal blood flow were studied in 9 patients with congestive heart failure (New York Heart Association class III or IV, ejection fraction less than 35%). Hemodynamic variables were measured at 30-minute intervals for 8 hours during CGRP infusion (8 ng/kg/min) and for 2 hours after discontinuation. CGRP caused a decrease in right atrial (28%, p less than 0.05), pulmonary artery (22%, p less than 0.02), pulmonary artery wedge (37%, p less than 0.001) and systemic arterial (18%, p less than 0.05) pressures. Systemic vascular resistance decreased more than pulmonary vascular resistance. Cardiac output (72%, p less than 0.001) and stroke volume (60%, p less than 0.02) increased. Heart rate did not change. There was no evidence of tolerance throughout the infusion. The hemodynamic effects were lost within 30 minutes of stopping CGRP. Renal blood flow (34%, p less than 0.01) and glomerular filtration rate (43%, p less than 0.01) increased. Atrial natriuretic peptide decreased (p less than 0.05), while plasma cortisol (p less than 0.02) increased. Plasma epinephrine, norepinephrine, renin activity, aldosterone and growth hormone were unchanged. It is concluded that in patients with severe congestive heart failure, CGRP has sustained beneficial effects on hemodynamic functions and has no adverse effects on hormones. Unlike many other vasodilators, CGRP also increases renal blood flow and glomerular filtration.

Calcitonin Gene-Related Peptide↗
PubMed1995

Cell cycle regulation and subcellular localization of the major human uracil-DNA glycosylase.

The subcellular localization of the human DNA-repair enzyme uracil-DNA glycosylase from the UNG gene has been studied using flow cytometry and laser scanning confocal microscopy of freely cycling HeLa S3 cells. A two-parameter flow cytometric analysis using propidium iodide and UNG-specific antibodies demonstrated that total cellular UNG increased during the G1-phase and was approximately doubled in early S-phase compared to early G1. The UNG level was stable during the S-phase and increased further during G2, reaching a 2.8-fold level compared to early G1. This factor included differences in cell size and staining variabilities. These findings were confirmed using two-parameter confocal analysis of UNG/DNA and UNG/mitochondria at different stages of the cell cycle. Although the major fraction of UNG was associated with nuclei, we also observed distinctive staining associated with mitochondria and a more diffuse staining probably reflecting UNG in the cytosol. Furthermore, very little UNG staining was observed in nucleoli. The UNG level in different cell compartments varied at different stages of the cell cycle, and this variation was most pronounced in the nuclei. These results demonstrate that the gene product from the UNG gene is located within three subcellular compartments and that the distribution between these compartments varies during the cell cycle.

Cell Cycle↗
PubMed2004

Decline of surface MHC I by adenoviral gene transfer of anti-MHC I intrabodies in human endothelial cells-new perspectives for the generation of universal donor cells for tissue transplantation.

BACKGROUND: The seeding of small-calibre vascular polytetrafluoroethylene (PTFE) grafts with endothelial cells provides an increase in biocompatibility of the graft surface. The harvest and ex vivo culture of autologous endothelial cells is highly delicate. Allogeneic human umbilical vein endothelial cells (HUVEC) could be a potential cell source-however, rejection might occur due to major histocompatibility complex (MHC) I mismatches. Lowering cell surface MHC I expression on endothelial cells by gene transfer of an anti-MHC I intrabody might reduce graft failure. The intrabody consists of a single-chain variable fragment (sFv) of an anti-MHC I antibody, carrying a terminal KDEL sequence to retain the molecule together with the MHC I inside the endoplasmic reticulum. METHODS: Adenoviral gene transfer was used to express the intrabody in HUVEC. The MHC I surface expression was measured 48 h after transduction by flow cytometry. Functional effects of the intrabody expression were analyzed in a calcein release cytotoxicity assay. RESULTS: A transduction efficiency of more than 95% with EGFP-adenovirus indicates a sufficient gene transfer into HUVEC. Intrabody-adenovirus-transduced HUVEC show a massive reduction in MHC I surface expression creating almost a complete 'knockout' phenotype. Stimulation with inflammatory cytokines could not overcome this effect. The cell lysis of anti-MHC I intrabody-expressing HUVEC in a cytotoxicity assay is reduced when compared with the level of the MHC mismatched control. CONCLUSIONS: Our data indicate that HUVEC with reduced levels of MHC I might be used as universal donor cells for the seeding of vascular grafts.

Adenoviridae↗