EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3 end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14. The following Accession numbers were assigned to our deposited sequences: BF701840 – BF704551, BF708383, BF708386 – BF713604, BG322266 – BG322271, BI398567 – BI405235, BQ597354 – BQ605166.     

B-Type and C-Type Natriuretic Peptides Modify Norepinephrine Uptake in Discrete Encephalic Nuclei of the Rat

SUMMARY 1. We previously demonstrated that atrial natriuretic factor and B- and C-type natriuretic peptides (ANF, BNP, and CNP, respectively) modified catecholamine metabolism by increasing the neuronal uptake and decreasing the neuronal release of norepinephrine in the rat hypothalamus. The aim of the present work was to study the effects of natriuretic peptides BNP and CNP on norepinephrine uptake as an index of the amine metabolism in discrete areas and nuclei of the central nervous system (CNS) of the rat.2. Experiments were carried out in vitro using the punchout technique in diverse areas and nuclei of rat CNS. Results showed that 100 nM BNP and 1 nM CNP increased norepinephrine (NE) uptake in all brain areas and nuclei studied.3. Present results permit us to conclude that BNP and CNP regulate NE metabolism independently of the encephalic area or nucleus involved. In fact, NE uptake increased in nuclei related to the regullation of cardivascular activity as well as nuclei associated with endocrine metabolism and hydrosaline homeostasis. These observations suggest that BNP and CNP may be involved in the regulation of these physiological processes in an indirect manner through modifications of noradrenergic neurotransmission. Present findings provide futher support to the hypothesis that CNP would be the main natriuretic peptide in brain. Furthermore, previous as well as present results support the role of the natriureic peptides as neuromodulators of noradrenergic transmission at the presynaptic level.     

RESPONSE OF PACIFIC WALRUSES TO DISTURBANCES FROM CAPTURE AND HANDLING ACTIVITIES AT A HAUL-OUT IN BRISTOL BAY, ALASKA

Observations were made on herds of the Pacific walrus ( Odobenus rosmarus divergens ) to study their response during the capturing and handling of adult males in summer 1995 at a haul-out at Cape Peirce in southwestern Alaska. Three behaviors (alertness, displacement, and dispersal) were quantified from 16 capture sessions. Herd sizes ranged from 622 to 5,289 walruses. Handling of an immobilized walrus consisted of attempts to attach telemetry devices to the tusks and collect various biological samples. Handling activities resulted in an average of about 10-fold or greater levels of behavior in alertness, displacement, and dispersal than during precapture and darting periods. High levels of behavior usually occurred within the first 45 min of handling. In 8 of 10 capture sessions, walruses returned to predisturbance levels of behavior within 40 min of cessation of the handling disturbance. Alertness and displacement were moderately and negatively correlated with herd size during the handling period, which may reflect an effect of a threshold distance from the point of disturbance to responding individuals. Observations of walruses tagged with VHF radio transmitters indicated that the activities from a given capture session did not preclude tagged walruses from using the haul-out over a subsequent 11 -wk monitoring period. Moreover, non-tagged walruses continued to extensively use the haul-out during and after the period in which capture sessions were conducted.     

High genetic susceptibility to ethanol withdrawal predicts low ethanol consumption

C57BL/6J (B6) inbred mice are well known to drink large amounts of alcohol (ethanol) voluntarily and to have only modest ethanol-induced withdrawal under fixed dose conditions. In contrast, DBA/2J (D2) mice are ``teetotallers' and exhibit severe ethanol withdrawal. Speculation that an inverse genetic relationship existed between these two traits was substantiated by meta-analysis of existing data collected in multiple genetic models, including large panels of standard and recombinant inbred strains, their crosses, and selectively bred mouse lines. Despite methodological differences among laboratories in measurement of both preference drinking and withdrawal, a nearly universal finding was that genotypes consuming large amounts of 10% ethanol (calculated as g/kg/day) during two-bottle choice preference drinking were genetically predisposed to low withdrawal scores in independent studies after either acute or chronic ethanol treatment. Conversely, low-drinking genotypes had higher withdrawal severity scores. The genetic relationship appears to be strongest in populations derived from B6 and D2, where data from more genotypes (BXD RIs, B6D2F₂s, BXD RI F₁s, and B6D2F₂-derived selectively bred lines) were available for analysis. Gene mapping studies in these populations identified four chromosome regions [on Chromosomes (Chrs) 1, 2, 4, and 15] where genes might potentially influence both traits. Among genotypes with greater genetic diversity (for example, a panel of standard inbred strains or selectively bred lines), the relationship was less pronounced. Thus, reduced susceptibility to the development of high alcohol use may be supported by increased genetic susceptibility to ethanol withdrawal symptoms. Received: 15 September 1998 / Accepted: 8 October 1998     

Sex differences in children’s investment in peers

It is hypothesized from within an evolutionary framework that females should be less invested in peer relations than males. Investment was operationalized as enjoyment in Study 1 and as preference for interaction in Study 2. In the first study, four- and six-year-old children’s enjoyment of peer interaction was observed in 26 groups of same-sex peers. Girls were rated as enjoying their interactions significantly less than boys. In the second study, six- and nine-year-old children were interviewed about the individuals with whom they spend time in their homes and neighborhoods and about the individuals who participate in their favorite activities. The proportion of individuals named by children who were peers was significantly lower for girls than boys both in children’s neighborhoods and in children’s favorite activities. Results strongly support the hypothesis that females and males have evolved differential preferences for interaction with peers.     

3D parallel coordinate systems--a new data visualization method in the context of microscopy-based multicolor tissue cytometry.

BACKGROUND: Presentation of multiple interactions is of vital importance in the new field of cytomics. Quantitative analysis of multi- and polychromatic stained cells in tissue will serve as a basis for medical diagnosis and prediction of disease in forthcoming years. A major problem associated with huge interdependent data sets is visualization. Therefore, alternative and easy-to-handle strategies for data visualization as well as data meta-evaluation (population analysis, cross-correlation, co-expression analysis) were developed. METHODS: To facilitate human comprehension of complex data, 3D parallel coordinate systems have been developed and used in automated microscopy-based multicolor tissue cytometry (MMTC). Frozen sections of human skin were stained using the combination anti-CD45-PE, anti-CD14-APC, and SytoxGreen as well as the appropriate single and double negative controls. Stained sections were analyzed using automated confocal laser microscopy and semiquantitative MMTC-analysis with TissueQuest 2.0. The 3D parallel coordinate plots are generated from semiquantitative immunofluorescent data of single cells. The 2D and 3D parallel coordinate plots were produced by further processing using the Matlab environment (Mathworks, USA). RESULTS: Current techniques in data visualization primarily utilize scattergrams, where two parameters are plotted against each other on linear or logarithmic scales. However, data evaluation on cartesian x/y-scattergrams is, in general, only of limited value in multiparameter analysis. Dot plots suffer from serious problems, and in particular, do not meet the requirements of polychromatic high-context tissue cytometry of millions of cells. The 3D parallel coordinate plot replaces the vast amount of scattergrams that are usually needed for the cross-correlation analysis. As a result, the scientist is able to perform the data meta-evaluation by using one single plot. On the basis of 2D parallel coordinate systems, a density isosurface is created for representing the event population in an intuitive way. CONCLUSIONS: The proposed method opens new possibilities to represent and explore multidimensional data in the perspective of cytomics and other life sciences, e.g., DNA chip array technology. Current protocols in immunofluorescence permit simultaneous staining of up to 17 markers. Showing the cross-correlation between these markers requires 136 scattergrams, which is a prohibitively high number. The improved data visualization method allows the observation of such complex patterns in only one 3D plot and could take advantage of the latest developments in 3D imaging.     

Assessing the Effects of Multiple Stressors on the Recruitment of Fruit Harvested Trees in a Tropical Dry Forest,Western Ghats,India

The harvest of non-timber forest products (NTFPs), together with other sources of anthropogenic disturbance, impact plant populations greatly. Despite this, conservation research on NTFPs typically focuses on harvest alone, ignoring possible confounding effects of other anthropogenic and ecological factors. Disentangling anthropogenic disturbances is critical in regions such as India’s Western Ghats, a biodiversity hotspot with high human density. Identifying strategies that permit both use and conservation of resources is essential to preserving biodiversity while meeting local needs. We assessed the effects of NTFP harvesting (fruit harvest from canopy and lopping of branches for fruit) in combination with other common anthropogenic disturbances (cattle grazing, fire frequency and distance from village), in order to identify which stressors have greater effects on recruitment of three tropical dry forest fruit tree species. Specifically, we assessed the structure of 54 populations of Phyllanthus emblica, P. indofischeri and Terminalia chebula spread across the Nilgiri Biosphere Reserve, Western Ghats to ask: (1) How are populations recruiting? and (2) What anthropogenic disturbance and environmental factors, specifically forest type and elevation, are the most important predictors of recruitment status? We combined participatory research with an information-theoretic model-averaging approach to determine which factors most affect population structure and recruitment status. Our models illustrate that for T. chebula, high fire frequency and high fruit harvest intensity decreased the proportion of saplings, while lopping branches or stems to obtain fruit increased it. For Phyllanthus spp, recruitment was significantly lower in plots with more frequent fire. Indices of recruitment of both species were significantly higher for plots in more open-canopy environments of savanna woodlands than in dry forests. Our research illustrates an approach for identifying which factors are most important in limiting recruitment of NTFP populations and other plant species that may be in decline, in order to design effective management strategies.     

Sample Preconcentration Utilizing Nanofractures Generated by Junction Gap Breakdown Assisted by Self-Assembled Monolayer of Gold Nanoparticles

The preconcentration of proteins with low concentrations can be used to increase the sensitivity and accuracy of detection. A nonlinear electrokinetic flow is induced in a nanofluidic channel due to the overlap of electrical double layers, resulting in the fast accumulation of proteins, referred to as the exclusion-enrichment effect. The proposed chip for protein preconcentration was fabricated using simple standard soft lithography with a polydimethylsiloxane replica. This study extends our previous paper, in which gold nanoparticles were manually deposited onto the surface of a protein preconcentrator. In the present work, nanofractures were formed by utilizing the self-assembly of gold-nanoparticle-assisted electric breakdown. This reliable method for nanofracture formation, involving self-assembled monolayers of nanoparticles at the junction gap between microchannels, also decreases the required electric breakdown voltage. The experimental results reveal that a high concentration factor of 1.5×10⁴ for a protein sample with an extremely low concentration of 1 nM was achieved in 30 min by using the proposed chip, which is faster than our previously proposed chip at the same conditions. Moreover, an immunoassay of bovine serum albumin (BSA) and anti-BSA was carried out to demonstrate the applicability of the proposed chip.     

Magnetic Resonance Imaging Allows the Evaluation of Tissue Damage and Regeneration in a Mouse Model of Critical Limb Ischemia

Magnetic resonance imaging (MRI) provides non-invasive, repetitive measures in the same individual, allowing the study of a physio-pathological event over time. In this study, we tested the performance of 7 Tesla multi-parametric MRI to monitor the dynamic changes of mouse skeletal muscle injury and regeneration upon acute ischemia induced by femoral artery dissection. T2-mapping (T2 relaxation time), diffusion-tensor imaging (Fractional Anisotropy) and perfusion by Dynamic Contrast-Enhanced MRI (K-trans) were measured and imaging results were correlated with histological morphometric analysis in both Gastrocnemius and Tibialis anterior muscles. We found that tissue damage positively correlated with T2-relaxation time, while myofiber regeneration and capillary density positively correlated with Fractional Anisotropy. Interestingly, K-trans positively correlated with capillary density. Accordingly, repeated MRI measurements between day 1 and day 28 after surgery in ischemic muscles showed that: 1) T2-relaxation time rapidly increased upon ischemia and then gradually declined, returning almost to basal level in the last phases of the regeneration process; 2) Fractional Anisotropy dropped upon ischemic damage induction and then recovered along with muscle regeneration and neoangiogenesis; 3) K-trans reached a minimum upon ischemia, then progressively recovered. Overall, Gastrocnemius and Tibialis anterior muscles displayed similar patterns of MRI parameters dynamic, with more marked responses and less variability in Tibialis anterior. We conclude that MRI provides quantitative information about both tissue damage after ischemia and the subsequent vascular and muscle regeneration, accounting for the differences between subjects and, within the same individual, between different muscles.