Multi-scale models of local control of calcium induced calcium release.

Calcium-induced-calcium-release in cardiac myocytes is the release of Ca(2+) from the sarcoplasmic reticulum (SR) triggered by Ca(2+) entering the cell through L-type Ca(2+) channels. The Ca(2+) is released through ryanodine receptors which 'sense' local [Ca(2+)] in the small region (the diadic space) positioned between the t-tubules and the SR. The length-scale of a single diad is of the order of 10nm and the diffusion time-scale is of order of 1 micros with each cell containing approximately 10,000 diadic spaces which act independently. However, typically one is interested in Ca(2+) currents at the whole cell level and higher. This is a multi-scale problem and cannot be solved by direct computation. In this paper we develop a general framework for deriving approximate solutions of these models.

A simplified local control model of calcium-induced calcium release in cardiac ventricular myocytes.

Calcium (Ca2+)-induced Ca2+ release (CICR) in cardiac myocytes exhibits high gain and is graded. These properties result from local control of Ca2+ release. Existing local control models of Ca2+ release in which interactions between L-Type Ca2+ channels (LCCs) and ryanodine-sensitive Ca2+ release channels (RyRs) are simulated stochastically are able to reconstruct these properties, but only at high computational cost. Here we present a general analytical approach for deriving simplified models of local control of CICR, consisting of low-dimensional systems of coupled ordinary differential equations, from these more complex local control models in which LCC-RyR interactions are simulated stochastically. The resulting model, referred to as the coupled LCC-RyR gating model, successfully reproduces a range of experimental data, including L-Type Ca2+ current in response to voltage-clamp stimuli, inactivation of LCC current with and without Ca2+ release from the sarcoplasmic reticulum, voltage-dependence of excitation-contraction coupling gain, graded release, and the force-frequency relationship. The model does so with low computational cost.

Understanding the interface of HIV, trauma, post-traumatic stress disorder, and substance use and its implications for health outcomes.

Many individuals living with HIV have been exposed to some type of traumatic event during their lives and may be living with symptoms of post-traumatic stress disorder (PTSD). A substantial number of these individuals are also likely to show evidence of a co-morbid substance use disorder (SUD). There is reason to believe that the co-occurrence of HIV and PTSD or co-morbid PTSD and SUD (PTSD/SUD) may predict poorer health outcomes. There are several pathways through which PTSD or PTSD/SUD might adversely impact the health of individuals living with HIV, including participation in negative health behaviours, low levels of adherence to antiretroviral medications, and/or a direct, deleterious effect on immune function. Psychological interventions are needed to treat PTSD and PTSD/SUD in HIV-positive individuals, and reduce the negative impact of these conditions on health outcomes. This article will explore data on the prevalence of trauma exposure, PTSD, and PTSD/SUD among individuals living with HIV, the pathways through which these conditions might affect health, possible interventions for PTSD and PTSD/SUD for individuals living with HIV, and methods for integrating care for individuals with these disorders. Future directions for research related to HIV, PTSD, and PTSD/SUD will also be discussed.

Transgenic swine for biomedicine and agriculture.

Initial technologies for creating transgenic swine only permitted random integration of the construct. However, by combining the technology for homologous recombination in fetal somatic cells with that of nuclear transfer (NT), it is now possible to create specific modifications to the swine genome. The first such example is that of knocking out a gene that is responsible for hyperacute rejection (HAR) when organs from swine are transferred to primates. Because swine are widely used as models of human diseases, there are opportunities for genetic modification to alter these models or to create additional models of human disease. Unfortunately, some of the offspring resulting from NT have abnormal phenotypes. However, it appears that these abnormal phenotypes are a result of epigenetic modifications and, thus, are not transmitted to the offspring of the clones. Although the technique of producing animals with specific genetic modifications by NT has been achieved, improvements to the NT technique as well as improvements in the culture conditions for somatic cells and the techniques for genetic modification are still needed.

Molecular interactions between two long-QT syndrome gene products, HERG and KCNE2, rationalized by in vitro and in silico analysis.

The cardiac delayed rectifier potassium current mediates repolarization of the action potential and underlies the QT interval of the ECG. Mutations in either of the two molecular components of the rapid delayed rectifier (I(K,r)), HERG and KCNE2, have been linked to heritable or acquired long-QT syndrome. Mechanisms whereby mutations of KCNE2 produce fatal cardiac arrhythmias characteristic of long-QT syndrome remain unclear. In this study, we characterize functional interactions between HERG and KCNE2 with a view to defining underlying mechanisms for action potential prolongation and long-QT syndrome. Whereas coexpression of hKCNE2 with HERG alters both kinetics and density of ionic current, incorporation of these effects into a quantitative model of the action potential predicts that only changes in current density significantly affect repolarization. Thus, the primary functional consequence of hKCNE2 on action potential morphology is through modulation of I(K,r) density, as predicted by the model. Mutations associated with long-QT syndrome that result only in modest changes of gating kinetics may be epiphenomena or may modulate action potential repolarization via interaction with alternative pore-forming potassium channel alpha subunits.

Role of the calcium-independent transient outward current I(to1) in shaping action potential morphology and duration.

The Kv4.3-encoded current (I:(Kv4.3)) has been identified as the major component of the voltage-dependent Ca(2+)-independent transient outward current (I:(to1)) in human and canine ventricular cells. Experimental evidence supports a correlation between I:(to1) density and prominence of the phase 1 notch; however, the role of I:(to1) in modulating action potential duration (APD) remains unclear. To help resolve this role, Markov state models of the human and canine Kv4.3- and Kv1.4-encoded currents at 35 degrees C are developed on the basis of experimental measurements. A model of canine I:(to1) is formulated as the combination of these Kv4.3 and Kv1.4 currents and is incorporated into an existing canine ventricular myocyte model. Simulations demonstrate strong coupling between L-type Ca(2+) current and I:(Kv4.3) and predict a bimodal relationship between I:(Kv4.3) density and APD whereby perturbations in I:(Kv4.3) density may produce either prolongation or shortening of APD, depending on baseline I:(to1) current level.

How do children's eating patterns and food choices change over time? Results from a cohort study.

PURPOSE: The purpose of this study was to investigate the change in eating patterns and food choices in a cohort of students as they moved from the third to the eighth grade. DESIGN: Dietary behaviors and food choices were abstracted from 24-hour recalls and compared across grade levels. SETTING: The setting for the study was elementary and junior high schools. SUBJECTS: The subjects were a cohort of 291 students from Minnesota. MEASURES: Individual 24-hour dietary recalls from a cohort of students were collected in the third, fifth and eighth grades. RESULTS: As students moved from elementary to junior high and middle school, their consumption of breakfast, fruits, vegetables, and milk decreased. Soft drink consumption increased. In the third grade, nearly 99% of the cohort reported eating breakfast; by the eighth grade, 85% reported eating breakfast. Fruit consumption fell by 41% between the third and the eighth grades while vegetable consumption fell by 25%. The proportion of beverage coming from soft drinks more than tripled between the third and the eighth grades with concomitant reductions in milk and fruit juice consumption. CONCLUSIONS: Family, school, and community-wide efforts are needed to promote healthful eating patterns and food choices among adolescents. Our research indicates that nutrition education is needed in the elementary and middle school years. In addition, we need to work on improving teens' social and physical environments to encourage and facilitate their choice of healthy foods.

Production of transgenic porcine blastocysts by nuclear transfer.

In this study the in vitro development of porcine nuclear transfer (NT) embryos was investigated. Transgenic fetal fibroblast cells that were frozen after 5 days of serum starvation were injected immediately after thawing into enucleated metaphase II (MII) oocytes. Reconstructed embryos were activated by incubation in 200 microM thimerosal followed by a 30-min treatment of 8 mM DTT. The embryos were subsequently cultured in NCSU23, supplemented with 4 mg/ml BSA for 7 days. The actual cleavage rate (embryos showing > or =2 nuclei) in 6 replicates was 33% (ranging from 15% to 50%). Three blastocysts with cell numbers of 14, 15, and 18 were obtained. The blastocyst rate was significantly lower for NT embryos as opposed to parthenogenetically activated embryos (1% vs. 5%; P<0.05). The neomycin-resistance gene was amplified by PCR in all three NT embryos, indicating their origin from the injected transgenic fibroblasts. Efforts are now being directed in improvements in the nuclear transfer technology, whereby viable fetuses or offspring can be produced from these NT-embryos.

The design of a visual display for the presentation of statistical quality control information to operators on the plant floor.

We applied human-centered design methodologies to enhance the presentation of product quality information to operators on a manufacturing plant floor. First, an initial visual display concept that integrated a pictorial representation of a product with standard graphical and tabular information about the product's quality was refined through iterative design and testing. A preliminary study was then conducted to determine the specific features of such a display (termed a pictorial control chart) from among eight candidate detail designs. Finally, a formal study was conducted to compare the performance of operators using this refined pictorial control chart design with their performance using a conventional control chart. Operators completed a quality control task in significantly less time using the pictorial control chart. There were no significant differences in the number of errors committed with the two charts. Subjective measures showed a significant preference for the pictorial control chart. Actual or potential applications of this research include the development of quality control tools that are useful to and usable by operators on the manufacturing plant floor.

The use of advanced technology for visual inspection training.

In the past, training with traditional methods was shown to improve inspection performance. However, advances in technology have automated training and revolutionized the way training will be delivered in the future. Examples of such technology include computer-based simulators, digital interactive video, computer-based training, and intelligent tutoring systems. Despite the lower cost and increased availability of computer technology, the application of advanced technology to training within the manufacturing industry and specifically for inspection has been limited. In this vein, a case study is presented which shows how advanced technology along with our basic knowledge of training principles, can be used to develop a computer-based training program for a contact lens inspection task. Improvements due to computer-based inspection training were measured in an evaluation study and are reported.

A prospective cohort study of intake of calcium, vitamin D, and other micronutrients in relation to incidence of rectal cancer among postmenopausal women.

To investigate whether high intakes of calcium and other micronutrients (carotene, retinol, and vitamins C, D, and E) are related to reduced risks of rectal cancer, we analyzed data from a large cohort study of postmenopausal Iowa women who responded to a mailed survey in 1986. After 9 years of follow-up, 144 incident rectal cancer cases were ascertained among the 34,702 women at risk. Intake levels of micronutrients at baseline were derived from self-reported data on vitamin supplements and dietary intake of 127 foods included in a semiquantitative food frequency questionnaire. After adjustment for total energy intake and other potential confounding factors, a dose-response inverse association was observed between total calcium intake and the risk of rectal cancer: adjusted relative risks (RRs) were 1.00, 0.90, and 0.59 (trend test, P = 0.02) from the lowest to the highest calcium intake tertiles. High intakes of dietary and supplement calcium were both related to a slightly reduced risk of rectal cancer, but neither of the trend tests was statistically significant. Reduced risks of rectal cancer were also observed for high intake of carotene and vitamins A, C, and D, although none of the associations were statistically significant. For vitamin D, the adjusted RRs were 1.00, 0.71, and 0.76 (trend test, P = 0.20) for increasing intake tertiles. Compared with women who consumed low levels of both total calcium and vitamin D, those in the highest intake group of both nutrients were at a 45% reduced risk of rectal cancer (RR, 0.55; 95% confidence interval, 0.32-0.93). This study supports the hypothesis that high intake of calcium and possibly other micronutrients may be beneficial in the prevention of rectal cancer.

Identification of a full-length cDNA for an endogenous retrovirus of miniature swine.

Endogenous retroviruses of swine are a concern in the use of pig-derived tissues for xenotransplantation into humans. The nucleotide sequence of porcine endogenous retrovirus taken from lymphocytes of miniature swine (PERV-MSL) has been characterized. PERV-MSL is a type C retrovirus of 8,132 bp with the greatest nucleic acid sequence identity to gibbon ape leukemia virus and murine leukemia virus. Constitutive production of PERV-MSL RNA has been detected in normal leukocytes and in multiple organs of swine. The copy numbers of full-length PERV sequences per genome (approximately 8 to 15) vary among swine strains. The open reading frames for gag, pol, and env in PERV-MSL have over 99% amino acid sequence identity to those of Tsukuba-1 retrovirus and are highly homologous to those of endogenous retrovirus of cell line PK15 (PK15-ERV). Most of the differences in the predicted amino acid sequences of PK15-ERV and PERV-MSL are in the SU (cell attachment) region of env. The existence of these PERV clones will enable studies of infection by endogenous retroviruses in xenotransplantation.

Current and future prospects for xenotransplantation.

The transplantation of organs and tissues between animal species, or xenotransplantation, is the focus of a growing field of research, owing primarily to the increasing shortage of allogeneic donor organs. The pig stands out as the most suitable donor animal for humans; however, xenografts (e.g. pig organs) used for human transplantation are normally destroyed by the host within minutes by hyperacute xenograft rejection. An improved understanding of the immune recognition and rejection of xenografts has resulted in new therapies that can partially overcome hyperacute rejection (HAR), delayed xenograft rejection (DXR) or acute vascular xenograft rejection. Strategies to diminish immunogenicity following xenotransplantation can be divided into two approaches: those directed at the recipient (e.g. antibodies or complement depletion or inhibition and tolerance induction) and those directed at the donor (e.g. transgenic modifications to express human complement-regulatory proteins or removal or displacement of alphaGal epitopes). DXR is likely to be controlled by transgenic inhibition of endothelial cell activation (e.g. inhibition of NF-kappaB). Transgenic pigs required for xenotransplantation will soon be generated at a greater efficiency and precision using nuclear transfer and cloning when compared to pronuclear injection. Of greater significance is that nuclear transfer offers the ability to target gene insertion selectively to specific gene loci and to delete specific genes in the pig. Experimental pig-to-primate organ xenotransplantation is currently under way, and results show increased transplant function from minutes to days and weeks. The final therapeutic regimen that allows survival of a discordant xenograft is likely to involve a combination of 'modified' functional genes in the donor organ, the development of immunological tolerance to pig antigens and administration of novel therapeutic agents, including immunosuppressants, that can control natural killer (NK) cell and monocyte mediated responses.

The use of tolerance for transplantation across xenogeneic barriers.

The success of human organ transplantation as a clinical treatment has created a conundrum for the transplant community. It has caused a shortage of human donor organs and uncovered problems of chronic immunosuppression in those lucky enough to receive organ transplants due to their use of chronic immunosuppressive drugs. Our aim is to attempt to approach both issues by establishing specific transplantation tolerance to pig organ grafts.

The behaviour of human vitronectin in vivo: effects of complement activation, conformation and phosphorylation.

We examined the behaviour in vivo of native, specifically phosphorylated, and multimeric vitronectin to determine the effects of these modifications on its turnover, distribution and molecular behaviour. In normal rabbits, the plasma half-life (T1/2) of antigenically detected vitronectin was 8.00 +/- 1.26 h (mean +/- s.d.), with a fractional catabolic rate (FCR) of 18.77 +/- 1.57%/h and extravascular/intravascular ratio (EV/IV) of 1.00 (0.48-1.60, median and range). For vitronectin selectively phosphorylated by protein kinase A, T1/2 was 8.87 +/- 0.48 h, with a significantly smaller FCR of 10.85 +/- 0.71%/h (P < 0.005) and an EV/IV of 0.28 (0.15-0.36) (P < 0.05 compared with antigenically detected vitronectin). In vitro, phosphorylation had no effect on the affinity of vitronectin for heparin-Sepharose, while complement activation with cobra venom factor (CVF) led to a two-fold enrichment of 32P-vitronectin within the SC5b-9 complex. In vivo CVF caused a rapid decrease in the circulating levels of 32P-vitronectin and was accompanied by the prompt appearance of a high mol. wt species consistent with SC5b-9. Despite specific incorporation of 32P-vitronectin into SC5b-9, both forms of the molecule had similar inhibitory effects on C9-mediated haemolysis of EAC1-7 cells. Urea-activated vitronectin was rapidly cleared from circulation with less than 15% remaining after 1 h while protein-bound label accumulated in the spleen, lung and liver. These results demonstrate that vitronectin is a rapidly metabolized protein whose in vivo behaviour is markedly altered when phosphorylated or activated to form multimers and SC5b-9.

Definition of the human T-cell epitopes of Fel d 1, the major allergen of the domestic cat.

BACKGROUND: A heterodimeric acidic glycoprotein (Fel d 1) has been defined as the major allergen of the domestic cat. Because T-cell help is required for the initiation and maintenance of allergic responses, it is of importance to determine the T-cell-reactive regions of the Fel d 1 molecule. METHODS: Overlapping peptides corresponding to the two chains of Fel d 1 were tested in proliferation assays on polyclonal T-cell lines and for the ability to bind Fel d 1-specific IgE in ELISA and histamine release assays. RESULTS: Assay of T-cell lines derived from 53 subjects allergic to cats demonstrated that the majority of T-cell reactivity is found in chain 1 of Fel d 1. Two peptides (Fel-1 and Fel-2) containing major epitopes, alone or as a mixture, efficiently activated T cells and exhibited minimal detectable reactivity with IgE by ELISA or histamine release assay. CONCLUSIONS: Two Fel d 1 peptides containing major T-cell epitopes have been identified, have been shown to bind minimal Fel d 1-specific IgE, and are now being tested for the ability to decrease T-cell responses in patients with cat allergy as a new form of immunotherapy.