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PubMed2001

Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts.

Recently fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. However, most investigators have applied steady or pulsing flow profiles rather than oscillatory fluid flow, which occurs in vivo because of mechanical loading. Here oscillatory fluid flow was demonstrated to be a potentially important physical signal for loading-induced changes in bone cell metabolism. We selected three well known biological response variables including intracellular calcium (Ca(2+)i), mitogen-activated protein kinase (MAPK) activity, and osteopontin (OPN) mRNA levels to examine the response of MC3T3-E1 osteoblastic cells to oscillatory fluid flow with shear stresses ranging from 2 to -2 Newtons/m(2) at 1 Hz, which is in the range expected to occur during routine physical activities. Our results showed that within 1 min, oscillatory flow induced cell Ca(2+)i mobilization, whereas two MAPKs (ERK and p38) were activated over a 2-h time frame. However, there was no activation of JNK. Furthermore 2 h of oscillatory fluid flow increased steady-state OPN mRNA expression levels by approximately 4-fold, 24 h after exposure to fluid flow. The presence of both ERK and p38 inhibitors and thapsigargin completely abolished the effect of oscillatory flow on steady-state OPN mRNA levels. In addition, experiments using a variety of pharmacological agents suggest that oscillatory flow induces Ca(2+)i mobilization via the L-type voltage-operated calcium channel and the inositol 1,4,5-trisphosphate pathway.

3T3 Cells↗
PubMed2004

Experimental and numerical studies of adenovirus delivery to outflow tissues of perfused human anterior segments.

PURPOSE: To investigate the efficacy of two different methods of adenoviral transfer of genes to trabecular meshwork (TM) and Schlemm's canal (SC) cells in cultured human anterior segments, using both experimental and numerical analyses. METHODS: Replication-deficient adenoviruses having coding sequence for beta-galactosidase (beta-gal) under the control of the cytomegalovirus promoter were used. Efficiency of gene transfer over time was verified by infecting cultured human TM cells and assaying for beta-gal activity. Next, ostensibly normal paired human eyes were prepared by standard techniques and perfused for 2 to 5 days to measure baseline facilities. Eyes were then infected by one of two methods: standard transcorneal puncture, or injection into a 1 mm diameter silastic segment of supply tubing immediately upstream of the perfusion dish. In both cases, the nominal total dose was 2 x 10(8) viral particles. Five days after viral injection, eyes were harvested and fixed, and wedges from each of four quadrants were examined histologically. Sections were assayed for beta-gal activity and/or stained with toluidine blue. In a parallel study, flow and viral transport within perfused anterior segments were numerically simulated for conditions that approximated those used experimentally. RESULTS: Eyes receiving viral particles by transcorneal injection showed variable levels of beta-gal activity and highly variable TM cellular morphology, ranging from excellent preservation to cellular lysis. Eyes receiving an equivalent viral dose via the supply tubing showed higher transfer efficiency, as judged by almost complete TM cell loss (indicative of viral toxicity) and intense extracellular beta-gal activity from the residual cytoplasm. At lower doses (1/3 to 1/1000 of that used in transcorneal injection) beta-gal activity was still present, while TM cell morphology was good at the lower viral doses. Computer modeling showed that the region beneath the cornea was nearly stagnant, and consequently virus introduced into this region by transcorneal injection was delivered very slowly to the TM. This caused the effective delivered viral dose to be low and sensitively dependent on the volume and shape of the transcorneally injected virus bolus. CONCLUSIONS: Injection of adenovirus into supply tubing led to consistent delivery of reporter gene and approximately 300-fold greater efficiency of gene transfer compared to the transcorneal injection method, and is therefore the preferred method for introducing viral particles into perfused anterior segments. These findings were consistent with computer modeling of flow and mass transport in perfused anterior segments. Although these quantitative results are specific to adenovirus, this general trend should hold for a wide range of perfused compounds.

Adenoviruses, Human↗
PubMed2005

A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting.

Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50,000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression variability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP) gene fused to the Discosoma red fluorescent protein (DsRed) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells containing inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were successfully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation.

Cell Separation↗
PubMed1996

Expression of BCL-2 proto-oncogene in adult acute lymphoblastic leukemia.

The products of the BCL-2 gene prolong survival of lymphohematopoietic cells by inhibition of programmed cell death. We studied bcl-2 protein expression in a series of 43 adult acute lymphoblastic leukemia (ALL) at diagnosis, using a specific monoclonal antibody and flow cytometry. All samples expressed bcl-2 with a mean percentage of positive cells of 77.9. The level of bcl-2 in positive cells expressed as mean equivalent of soluble fluorescence (MESF) was highly variable ranging from 5 x 10(3) to 552 x 10(3) (mean +/- s.d.: 96.5 +/- 109 x 10(3)). Neither the percentage of positive cells nor bcl-2 MESF levels were correlated with initial characteristics including blood counts, immunological phenotype, or cytogenetics. The survival of leukemic cells maintained in cytokine-free liquid culture was not correlated with bcl-2 expression. However, cells from ALL with higher white blood cell (WBC) counts, with t(9;22) translocation, or expressing myeloid surface antigens exhibited significantly longer survival in this culture system. The outcome after intensive chemotherapy did not differ according to bcl-2 expression. Factors associated with poor outcome included WBC counts, presence of t(9;22) translocation, presence of myeloid antigens and prolonged survival of cultured cells. These results indicate that high levels of bcl-2 are not associated with distinct clinical or biological characteristics in ALL.

Adolescent↗
PubMed1996

The genetic and molecular dissection of a prototypic circadian system.

A great deal is known about this archetypal circadian system, and it is likely that Neurospora will represent the first circadian system in which it will be possible to provide a complete description of the flow of information from the photoreceptor, through the components of oscillator, out to a terminal aspect of regulation. In Neurospora the strongest case has been made for there being a state variable of clock identified (Hall, 1995), it has now been shown that light resetting of the clock is mediated by the rapid light induction of the gene encoding this state variable, and a number of defined clock-regulated output genes have been identified, in two of which the clock-specific parts of the promoters have been localized. In addition to the importance of these factoids themselves, our efforts towards understanding of this system has allowed the development of tools and paradigms (e.g. Loros et al., 1989; Loros and Dunlap, 1991; Aronson et al., 1994a) that will help to pave the way for proving the identity of clock components in more complex systems, for understanding how clocks are regulated by entraining factors, and for showing how time information eventually is used to regulate the behaviors of clock cells, and of whole organisms.

Animals↗
PubMed1998

Population genetic structure, phylogeography and spawning philopatry in walleye (Stizostedion vitreum) from mitochondrial DNA control region sequences.

Mitochondrial (mt) DNA control region sequences were used to test the genetic and phylogeographic structure of walleye Stizostedion vitreum populations at different geographical scales: among spawning sites, lake basins, lakes, and putative glacial refugia in the Great Lakes region. Sequencing 199 walleye revealed nucleotide substitutions and tandemly repeated sequences that varied in copy number, as well as in sequence composition, in approximately 1200 bp of the mtDNA control region. Variable numbers of copies of an 11-bp tandem repeat showed no geographical patterning and were not used in further analyses. Substitutions in the other areas of the control region yielded 19 haplotypes, revealing phylogeographic structure and significant differences among glacial refugia, lakes, basins and some spawning sites. Differences among spawning populations were consistent with reduced gene flow, philopatry and possible natal homing. Analysis of spawning populations showed consistency of genotypic frequencies among years and between males and females, supporting philopatry in both sexes. The unglaciated plateau in southern Ohio, USA housed a very different haplotype that diverged prior to the Missouri, Mississippi and Atlantic glacial refugia types. Haplotypes from the three refugia colonized the Great Lakes after retreat of the Wisconsin glaciers, and their present distribution reflects the geography of their prior isolation and differential colonization. Populations that became associated with spawning localities appear to have diverged further due to philopatry, resulting in fine-scale phylogeographic structuring.

Animals↗
PubMed2006

Do ants enhance diversification in lycaenid butterflies? Phylogeographic evidence from a model myrmecophile, Jalmenus evagoras.

The ant-tended Australian butterfly, Jalmenus evagoras, has been a model system for studying the ecology and evolution of mutualism. A phylogeographic analysis of mitochondrial DNA cytochrome oxidase I sequences from 242 butterflies (615 bp) and 66 attendant ants (585 bp) from 22 populations was carried out to explore the relationship between ant association and butterfly population structure. This analysis revealed 12 closely related butterfly haplotypes in three distinct clades roughly corresponding to three allopatric subpopulations of the butterflies. Minimal genetic diversity and widespread haplotypes within biogeographical regions suggest high levels of matrilineal gene flow. Attendant ants are significantly more diverse than was previously thought, with at least seven well-defined clades corresponding to independent morphological determinations, distributed throughout the range of the butterflies. Nested analysis of molecular variance showed that biogeography, host plant, and ant associate all contribute significantly in explaining variation in butterfly genetic diversity, but these variables are not independent of one another. Major influences appear to come from fragmentation due to large-scale biogeographical barriers, and diversification following a shift in habitat preference. A consequence of such a shift could be codiversification of the butterfly with habitat-adapted ants, resulting in apparent phylogenetic concordance between butterflies and ants. The implications of these results are discussed in terms of possible effects of ant attendance on the diversification of Lycaenidae as a whole.

Animals↗
PubMed1989

Diversity in T-cell receptor gamma gene usage in intestinal epithelium.

The intraepithelial cells of the murine small intestine include a significant number of CD3+ T cells that use T-cell receptor gamma genes rather than T-cell receptor beta genes. As with other sites of T-cell receptor gamma expression, combinatorial diversity is limited, but there is junctional diversity, and this, together with the specific variable region gamma gene segments used, distinguishes gamma gene expression in the gut epithelium from that in cells derived from the dermal epithelium. The restriction of productive gamma gene expression largely to one V-J-C (V, variable; J, joining; C, constant) gene combination may result from nonproductive joining of other V-J combinations and from productively rearranged genes rendered nonfunctional by incorrect splicing.

Animals↗
PubMed1996

A novel and efficient route for the isolation of antibodies that recognise T cell receptor V alpha(s).

Studies of the T cell repertoire have been hindered by the lack of antibodies that recognise V region families, particularly for V alpha regions. In this report, single chain Fv (scFv) fragments have been isolated that recognise both recombinant V alpha(s) and native V alpha(s) on the surface of T cells. Mice have been immunised with purified soluble T cell receptors (TCRs) and antibody heavy and light chain variable domain (VH and VL, respectively) genes isolated from splenocytes using the polymerase chain reaction (PCR). The VH and VL genes have been assembled as scFv gene libraries and a bacteriophage display system used to isolate scFvs that recognise a soluble V alpha. Five scFvs have been purified and characterised in detail using enzyme-linked immunosorbent assays (ELISAs) and flow cytometry. Three of these five scFvs recognise native V alpha(s) on the surface of T cell hybridomas. This method therefore offers a rapid route to the generation of scFvs that recognise native TCRs and can readily be extended to the production of anti-human TCR antibodies for use in therapy and diagnosis.

Amino Acid Sequence↗
PubMed1998

Temporal changes in PO2 of R3230AC tumors in Fischer-344 rats.

PURPOSE: The purpose of this study was to characterize the kinetics of hypoxia-reoxygenation in a murine tumor. Information on the prevalence and kinetics of this process are lacking in solid tumors, although there are data on blood flow fluctuation. MATERIALS AND METHODS: Oxygen tension (pO2) was monitored at one position in 1 cm diameter R3230Ac tumors of Fischer-344 rats, using 10-12 microm diameter recessed-tip polarographic electrodes. Data were collected continuously at a sampling frequency of 25 Hz for 30-90 min. Mean arterial blood pressure (MAP) and heart rate were also monitored. RESULTS: Temporal fluctuations in pO2 were observed in all 13 experiments. To assess the potential for hypoxia-reoxygenation, two threshold pO2 values were chosen (5 and 10 mmHg), and the number and duration of intervals that measurements resided below the thresholds was quantitated. In some experiments, the measurements did not fluctuate across the threshold values and, instead, either remained above or below them throughout the observation period. The percentage of sites that did not fluctuate across the thresholds was 38 and 61% for the 10- and 5-mmHg values, respectively. For the remaining studies, fluctuations above and below the thresholds of hypoxia ranged around 4-7 events per h. There were wide variations in the duration of hypoxic episodes, ranging from less than 1 to more than 40 min. The percentage time that measurements were below the hypoxic thresholds was also variable, ranging from 30-90%. CONCLUSIONS: These results, taken with the already published data on temporal instability in human and murine tumor blood flow, suggest that intermittent hypoxia is a common phenomenon in tumors. Future studies will focus on the underlying mechanisms that contribute to this process, because it has important implications for radiation and chemotherapy and, perhaps, gene regulation in tumors.

Animals↗
PubMed2005

Temporal mitochondrial DNA variation in the Basque Country: influence of post-neolithic events.

The Basque population has been considered an outlier in a large number of genetic studies, due to its hypothesized antiquity and greater genetic isolation. The present paper deals with an analysis of the mtDNA variability of the historical population of Aldaieta (VI-VII c. AD; Basque Country) which, together with genetic data existing for other prehistoric populations of the Basque Country (4,500-5,000 YBP), permits an appraisal of the hypotheses proposed for the origin of the genetic differentiation of the Basque population. Given that this is an aDNA study, application has been made both of standard precautions, to avoid contamination, and of authentication criteria (analysis of duplicates, replication in an independent laboratory, quantification of target DNA, sequencing and cloning of PCR products). The variability of the mtDNA haplogroups of the historical population of Aldaieta falls within the range of the present-day populations of Europe's Atlantic fringe, whereas the prehistoric populations of the Basque Country display clear differentiation in relation to all others. Consequently, we suggest that between 5,000-1,500 YBP approximately, there may have been gene flow amongst the western European populations that homogenised mtDNA lineages.

Base Sequence↗
PubMed1999

Independent evolution of HIV type 1 in different brain regions.

HIV-1-associated brain pathology exhibits regional variability and we therefore studied the genetic differences in the V1-V5 domains of the HIV env gene in up to four regions of brain (frontal lobe, basal ganglia, medial temporal lobe, and nonmedial temporal lobe) from three patients. We found that in each separate brain region HIV-1 forms different quasispecies and that there is little gene flow among these regions. In further support of brain region-specific evolution of HIV-1, we analyzed amino acid signatures in these clones. In addition to known amino acid signatures associated with macrophage tropism and the lack of syncytium formation, we found 15 majority amino acid signature patterns from the V1-V5 env sequences associated with the neuroanatomical regions analyzed from the three individuals. Furthermore, on average, intrabrain genetic distances for the HIV-1 env were estimated to be much smaller than genetic distances between brain regions. Specific strains of HIV-1 may be neurotropic or neuroinvasive (replication preference in brain tissue) and may contribute to pathology, cognitive loss, and neuropsychiatric disease.

Adult↗
PubMed2026

Species distribution models predict genome-wide polymorphism and gene flow in an endangered amphibian.

Species distribution models (SDMs) are widely used to predict habitat suitability but their usefulness and accuracy for inferring population health is still debated. Here, we evaluate whether SDM-derived relative habitat suitability (RHS) predicts genome-wide genetic diversity and connectivity-which are key proxies for population health and the functional integrity of landscapes. We addressed this issue in the Yellow-bellied toad (Bombina variegata), an endangered amphibian species with limited dispersal. We combined hierarchical SDMs, integrating both continental-level bioclimatic data and regional-level landscape variables, with genome-wide SNP data from 404 individuals sampled across 92 sites in southeastern France. We then used a multi-scale modelling framework to test the effect of bioclimatic (BRHS) and landscape (LRHS) habitat suitability on observed heterozygosity and pairwise genetic differentiation, accounting for heterogeneous genetic drift using gravity models. Our results show that both BRHS and LRHS are significant predictors of heterozygosity, with their effects expressed at different spatial scales-11 km and 3 km for BRHS and LRHS, respectively. Connectivity patterns also widely varied depending on scale and were best explained by gravity models integrating LRHS, BRHS, and local heterozygosity, underscoring the combined role of landscape resistance and population size in shaping patterns of genetic differentiation. These findings show that SDMs, when carefully calibrated and interpreted, can provide proxies for genetic diversity and landscape resistance in species with limited dispersal.

Journal Article↗
PubMed2001

Dermal neurovascular dysfunction in type 2 diabetes.

OBJECTIVE: To review evidence for a relationship between dermal neurovascular dysfunction and other components of the metabolic syndrome of type 2 diabetes. RESEARCH DESIGN AND METHODS: We review and present data supporting concepts relating dermal neurovascular function to prediabetes and the metabolic syndrome. Skin blood flow can be easily measured by laser Doppler techniques. RESULTS: Heat and gravity have been shown to have specific neural, nitrergic, and independent mediators to regulate skin blood flow. We describe data showing that this new tool identifies dermal neurovascular dysfunction in the majority of type 2 diabetic patients. The defect in skin vasodilation is detectable before the development of diabetes and is partially correctable with insulin sensitizers. This defect is associated with C-fiber dysfunction (i.e., the dermal neurovascular unit) and coexists with variables of the insulin resistance syndrome. The defect most likely results from an imbalance among the endogenous vasodilator compound nitric oxide, the vasodilator neuropeptides substance P and calcitonin gene-related peptide, and the vasoconstrictors angiotensin II and endothelin. Hypertension per se increases skin vasodilation and does not impair the responses to gravity, which is opposite to that of diabetes, suggesting that the effects of diabetes override and counteract those of hypertension. CONCLUSIONS: These observations suggest that dermal neurovascular function is largely regulated by peripheral C-fiber neurons and that dysregulation may be a component of the metabolic syndrome associated with type 2 diabetes.

Diabetes Mellitus, Type 2↗
PubMed1991

An evolutionary perspective on salt, hypertension, and human genetic variability.

Natural selection for electrolyte conservation has likely been the norm throughout human evolutionary history. However, the current patterns of excessive dietary salt intake create the potential for salt overload. Under these conditions, hypertension may be considered an expected pathological response to an evolutionarily new constraint. The transatlantic Middle Passage may have created a genetic bottleneck for salt conservation in African-Americans. Although the initial consequences of this important historical event probably constricted genetic variability and further magnified the potential for salt-sensitive hypertension, the Middle Passage undoubtedly also served as a more generalized major source of environmental stress and may have stimulated subsequent hereditary diversity in the survivors of this holocaust and their descendants. Accelerated rates of mutation, genetic recombination, and transposable genetic elements in conjunction with enhanced opportunities for gene flow, new selective pressures, and drift have all contributed to the tremendous heterogeneity of contemporary African-Americans. It is unlikely that a single genetic event, even of the severity of the Middle Passage, can account comprehensively for the apparent susceptibility of this macroethnic group to high blood pressure and hypertension.

Biological Evolution↗
PubMed2006

Efficiency of T-cell receptor expression in dual-specific T cells is controlled by the intrinsic qualities of the TCR chains within the TCR-CD3 complex.

Genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T-cell immunity toward viral infections and malignancies. We previously demonstrated redirected antileukemic reactivity of cytomegalovirus (CMV)-specific T cells by transfer of minor histocompatibility antigen HA-2-specific T-cell receptors (TCRs). HA-2-TCR-transferred CMV-specific T cells were potent effectors against HA-2-expressing leukemic cells, as well as CMV-expressing cells. Functional activity of these T cells correlated with TCR cell-surface expression. In the present study we analyzed which properties of transferred and endogenous TCRs are crucial for efficient cell-surface expression. We demonstrate that expression of the introduced TCR is not a random process but is determined by characteristics of both the introduced and the endogenously expressed TCR. The efficiency of TCR cell-surface expression is controlled by the intrinsic quality of the TCR complex. In addition, we demonstrate that chimeric TCRs can be formed and that efficiency of TCR expression is independent of whether TCRs are retrovirally introduced or naturally expressed. In conclusion, introduced, endogenous, and chimeric TCRs compete for cell-surface expression in favor of the TCR-CD3 complex with best-pairing properties.

Amino Acid Sequence↗
PubMedDate not supplied

[Genetic control of vectorial competence in Aedes mosquitoes].

The transmission of pathogens by arthropods is dependent on the relationships that exist between the pathogen, the invertebrate host (the vector) and the vertebrate host, each of which is influenced by environmental variations. Particular attention is given to the knowledge of intrinsic factors and the mechanisms controlling the ability of vectors to transmit pathogens (viruses or parasites). Polymorphism in the expression of susceptibility to oral infection has been shown to occur among geographical samples of mosquitoes. It has been proven that intraspecific variations in vector competence are controlled by one or more genes and expressed in variable proportions within a mosquito population. Recent advances in molecular biology have facilitated accessibility of nucleic acid sequence data. These new techniques allow one to analyse the genotype distribution within and among populations. Population genetic studies are currently used to understand the evolution of species differentiation and provide indications on genetic relationship among field vector populations. Estimations of gene flow with respect to vector capacity have provided rich insight into vector species complexes. Knowledge of intraspecies variation is important for the understanding of vector transmission, disease epidemiology and disease control. In this article, two examples are presented to illustrate the contribution of population genetic studies to the understanding of epidemiology of arthropod-borne diseases: Aedes polyneniensis, a vector of human lymphatic filariasis and Aedes aegypti, the vector of dengue viruses.

Aedes↗
PubMed1996

B7-1 gene transfer into human cancer cells by infection with an adenovirus-B7 (Ad-B7) expression vector.

BACKGROUND: Transfection of the costimulatory molecule B7-1 into some murine tumors can increase antitumor immunity and eradicate tumor growth. The purpose of this work was to construct an adenovirus-B7 (Ad-B7) expression vector and study B7-1 gene transfer into human cancer cells. METHODS: The human B7-1 cDNA was ligated into an expression cassette containing the human cytomegalovirus immediate early gene promoter and then inserted into the E1 region of the Ad5 genome by homologous recombination. The resulting Ad-B7 vector was used to infect established cancer cell lines and freshly resected cancers. Resected tumors were disaggregated into single cell suspensions by mechanical mincing and enzymatic digestion. Surface expression of B7-1 after infection was verified by flow cytometry. RESULTS: Expression kinetics in three cell lines showed that infected cells began to express B7-1 within 24 h. The proportion of B7-1+ cells continued to increase during the next 48 h, after which expression remained relatively constant during the next 5 days (up to 98% B7-1+ cells). Fresh tumor cells from various cancers displayed similar kinetics, but with greater variability in the proportion of cells expressing B7-1 (13% to 95% B7-1+ cells). Cancers which were successfully infected included 3 colorectal adenocarcinomas, 2 leiomyosarcomas, 2 lung squamous cell carcinomas, and 1 renal cell carcinoma. CONCLUSIONS: The Ad-B7 vector is a rapid and efficient means of gene transfer which does not require host cell proliferation. The ultimate objective is to engineer autologous tumors to express B7-1 and vaccinate cancer patients in an adjuvant or palliative setting.

Adenocarcinoma↗