Bioluminescence detection of ATP release mechanisms in epithelia.

Autocrine and paracrine release of and extracellular signaling by ATP is a ubiquitous cell biological and physiological process. Despite this knowledge, the mechanisms and physiological roles of cellular ATP release are unknown. We tested the hypothesis that epithelia release ATP under basal and stimulated conditions by using a newly designed and highly sensitive assay for bioluminescence detection of ATP released from polarized epithelial monolayers. This bioluminescence assay measures ATP released from cystic fibrosis (CF) and non-CF human epithelial monolayers in a reduced serum medium through catalysis of the luciferase-luciferin reaction, yielding a photon of light collected by a luminometer. This novel assay measures ATP released into the apical or basolateral medium surrounding epithelia. Of relevance to CF, CF epithelia fail to release ATP across the apical membrane under basal conditions. Moreover, hypotonicity is an extracellular signal that stimulates ATP release into both compartments of non-CF epithelia in a reversible manner; the response to hypotonicity is also lost in CF epithelia. The bioluminescence detection assay for ATP released from epithelia and other cells will be useful in the study of extracellular nucleotide signaling in physiological and pathophysiological paradigms. Taken together, these results suggest that extracellular ATP may be a constant regulator of epithelial cell function under basal conditions and an autocrine regulator of cell volume under hypotonic conditions, two functions that may be lost in CF and contribute to CF pathophysiology.

Expression and RNA splicing of the maize glutathione S-transferase Bronze2 gene is regulated by cadmium and other stresses.

The Bronze2 (Bz2) gene in maize (Zea mays) encodes a glutathione S-transferase that performs the last genetically defined step in anthocyanin biosynthesis--tagging anthocyanin precursors with glutathione, allowing for recognition and entry of anthocyanins into the vacuole. Here we show that Bz2 gene expression is highly induced by heavy metals such as cadmium. Treatment of maize seedlings with cadmium results in a 20-fold increase in Bz2 message accumulation and a 50-fold increase in the presence of the unspliced, intron-containing transcript. The increase in message levels during cadmium stress appears to result, at least in part, from activation of an alternative mRNA start site approximately 200 nucleotides upstream of the normal start site; this site is not used in unstressed or heat-stressed tissues. The effect of cadmium on the RNA splicing of Bz2 seems to be specific: splicing of other intron-containing maize genes, including a maize actin gene under the control of the cadmium-inducible Bz2 promoter, is unaffected by cadmium stress. Conversely, Bz2 intron splicing is not affected by other stress conditions that induce Bz2 gene expression, such as abscisic acid, auxin, or cold stress. Surprisingly, the increase in Bz2 mRNA during cadmium stress does not result in an increase in Bz2 glutathione S-transferase activity. We propose that an alternative protein may be encoded by Bz2 that has a role during responses to heavy metals.

A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2.

Glutathione S-transferases (GSTs) are enzymes that detoxify heterocyclic compounds (xenobiotics) by covalently linking glutathione to the substrate, forming a glutathione S-conjugate. A glutathione pump in the vacuolar membrane of barley actively sequesters herbicide-glutathione S-conjugates; glutathionation allows recognition and entry of the conjugates into vacuoles. The protein encoded by the Bronze-2 gene in maize performs the last genetically defined step in anthocyanin biosynthesis, resulting in the deposition of red and purple pigments in the vacuoles of maize tissues. We show here that Bz2 encodes a GST with activity in maize, transformed Arabidopsis thaliana plants and Escherichia coli. We demonstrate that anthocyanins extracted from maize protoplasts expressing BZ2 are conjugated with glutathione, and that vanadate, a known inhibitor of the glutathione pump in plant vacuolar membranes, inhibits the accumulation of anthocyanins in the vacuole. These results provide a biochemical function for BZ2, and suggest a common mechanism for the ability of plants to sequester structurally similar but functionally diverse molecules in the vacuole.

Extending a clinical repository to include multiple sites.

With the consolidation of health care organizations and services, a clinical repository comprising data from a single site is no longer sufficient. Individual patient data are now spread across multiple sites comprising a single enterprise. Users require an integrated view, or at least a common view, of these clinical data across multiple sites. Many issues arise when one tries to merge data from multiple, distinct organizations into an existing schema. We have addressed these issues while extending our clinical repository for Barnes Hospital with data from Jewish Hospital, both of which are members of the recently formed BJC Health System. We describe the architecture of our existing repository, approaches and issues in extending this repository to include multiple sites, and the specific issues we addressed in our system.

Creating temporal abstractions in three clinical information systems.

Modern clinical information system developers recognize the need to associate temporal information with clinical data. However, specific clinical systems capture different temporal features using a variety of data modeling techniques. Two commonly used methods to represent temporal information are point-based events and interval-based durations. We recently implemented a rule-based expert system for drug dose monitoring on three clinical information systems. The expert system requires both static drug dosing information (drug name, amount, route, frequency) and temporal dosing information (duration of therapy, renewals, restarts). Our design goal was to use the same expert system code on all three information systems by defining a common database schema to hide differences in the original systems' data models. Although we have been successful in mapping clinical data from these three source systems into a unified temporal data representation, we describe how differences in handling time within the three clinical systems made this goal difficult to achieve.

Supporting ad-hoc queries in an integrated clinical database.

Whether caring for patients or conducting research, medical decision-makers need access to clinical data. To fulfill that need, commercial software developers have produced a wide range of database query tools that differ greatly in functionality and cost. Generally, tools that have a greater ability to conceal database complexity from the user also require more effort for administrative setup. We describe a cost-effective, commercially-available query tool that requires no special setup to perform most simple queries, yet can be customized to satisfy users' more complex querying requirements.

A probability database for decision-analytic models of coronary revascularization procedures.

The time required to extract probabilities from medical literature is a primary reason decision analysis is not used more frequently for individual patient management decisions. Objective clinical trial information from the medical literature for one management decision was placed in a database which provided probabilities when queried. The database was tested with decision-analytic models of specific patient cases from the medical literature. Performance was assessed in terms of the ability to select trials which resembled the patients' individual characteristics, the number of trials providing probabilities for a given outcome, and the number of follow-up points available for that outcome. The timely assistance the database provides in expediting literature review and synthesis could enable the more common use of decision analysis in management decisions for individual patients.

Antidromic activation of a peptidergic pathway in the limbic system of the male rat.

Stimulation of the medial amygdaloid nucleus (AME) produces a long-latency and long-lasting inhibition of pyramidal cells in both the dorsal and the ventral hippocampus. The inhibition is blocked by a specific antagonist to vasopressin, which is a candidate neurotransmitter in the system. Antidromic activation of the AME from the hippocampus occurs with a latency suggestive of the conduction velocity of small diameter unmyelinated fibers. Immunocytochemistry for vasopressin reveals small diameter, unmyelinated immunoreactive fibers in the vicinity of the stimulating electrode in the hippocampus, and immunoreactive cell bodies in the vicinity of the recording electrode in the AME.

Unifying heterogeneous distributed clinical data in a relational database.

Access to clinical data which are distributed among multiple satellite information systems is crucial to delivering better care and reducing costs in many hospitals and medical centers. An integrated view of these data is needed to reduce the effort of users requiring data from multiple systems. We have addressed the issue of distributed data integration while developing both production and research decision-support applications. We describe an ideal integration solution, obstacles to realizing this solution, and our integration requirements and architecture. Our focus is a description of our specific schema and data integration techniques. We conclude with an analysis of our approach.

Characterization of two maize HSP90 heat shock protein genes: expression during heat shock, embryogenesis, and pollen development.

We have isolated two genes from Zea mays encoding proteins of 82 and 81 kD that are highly homologous to the Drosophila 83-kD heat shock protein gene and have analyzed the structure and pattern of expression of these two genes during heat shock and development. Southern blot analysis and hybrid select translations indicate that the highly homologous hsp82 and hsp81 genes are members of a small multigene family composed of at least two and perhaps three or more gene family members. The deduced amino acid sequence of these proteins based on the nucleotide sequence of the coding regions shows 64-88% amino acid homology to other hsp90 family genes from human, yeast, Drosophila, and Arabidopsis. The promoter regions of both the hsp82 and hsp81 genes contain several heat shock elements (HSEs), which are putative binding sites for heat shock transcription factor (HSF) commonly found in the promoters of other heat shock genes. Gene-specific oligonucleotide probes were synthesized and used to examine the mRNA expression patterns of the hsp81 and hsp82 genes during heat shock, embryogenesis, and pollen development. The hsp81 gene is only mildly heat inducible in leaf tissue, but is strongly expressed in the absence of heat shock during the pre-meiotic and meiotic prophase stages of pollen development and in embryos, as well as in heat-shocked embryos and tassels. The hsp82 gene shows strong heat inducibility at heat-shock temperatures (37-42 degrees C) and in heat shocked embryos and tassels but is only weakly expressed in the absence of heat shock. Promoter-GUS reporter gene fusions made and analyzed by transient expression assays in Black Mexican Sweet (BMS) Maize protoplasts also indicate that the hsp82 and hsp81 are regulated differentially. The hsp82 promoter confers strong heat-inducible expression of the GUS reporter gene in heat-treated cells (60- to 80-fold over control levels), whereas the hsp81 promoter is only weakly heat inducible (5- to 10-fold over control levels).

A peptidergic circuit for reproductive behavior.

A projection from the medial amygdaloid nucleus to the hippocampus and septum probably uses vasopressin as a transmitter. The nucleus synthesizes vasopressin and activation of the nucleus has a hippocampal effect that is completely blocked by a vasopressin antagonist. The afferent and efferent projections of this peptidergic nucleus suggest a possible role for the system in sexual behavior. Stimulation of the nucleus inhibits the output of the hippocampus in both genders and reorganizes behavior for a period of 15-20 min. In males, the effect of peptidergic activation is to produce a behavior that resembles the post-ejaculatory interval in coitus. This state is characterized by an EEG that resembles slow-wave sleep and by ultrasonic vocalizations at a characteristic frequency of 22 kHz. Castration in either gender causes depletion of the peptide from the target fields and eliminates the peptidergic signal in the hippocampus after about 15 weeks. The effects of castration in males can be reversed by testosterone replacement. The fluctuation of estrogen levels in rat plasma during the estrus cycle happens too quickly to impact the peptidergic system, and thus there is no significant change in the strength of the peptidergic signal among the proestrus, estrus, metestrus and diestrus stages. This fact permits study of the physiology of the system without concern for stage of estrus but does not permit conclusions regarding its function in females.

A method for publishing genomic maps.

We describe a method for the creation, manipulation and publication of physical human genome maps. Employing an "intelligent" document interface metaphor, our system uses a commercially available programmable document production system as an interface to primary genetic data resources and externally created mapping algorithms. Our document architecture distinguishes between primary data (usually obtained through database queries) and both manual and programmatic manipulations on these data. Our document architecture can be extended to accommodate a wide range of genetic and physical mapping problems. Because our approach is based on a widely available document preparation product, the principal focus of activity remains centered on the production of genomic maps that can be made available in a wide range of paper and electronic formats.

Rapid transcriptional regulation of the Cab and pEA207 gene families in peas by blue light in the absence of cytoplasmic protein synthesis.

We have analyzed the fluence-response and time-course characteristics, and the requirements for protein synthesis, for the blue-light (BL) regulated transcription of the nuclear-coded Cab, pEA207, and pEA25 gene families in pea (Pisum sativum L.). Fluence-response curves indicate two BL responses: a blue low-fluence (BLF) response with a threshold at or below 10(-1) µmol·m(-2) of BL and a blue high-fluence (BHF) response with a threshold between 10(1) and 10(3) µmol·m(-2) of BL. Excitation of the photomorphogenic system responsible for the BLF response results in increased Cab, and decreased pEA25, transcription. Excitation of the photomorphogenic system responsible for the BHF response results in decreased pEA207 transcription and induction of turnover for Cab RNA and pEA207 RNA. Altered rates of transcription for the Cab and pEA207 gene families are apparent within 15 min of BL treatment and remain in effect for at least 24 h. The effect of BL on pEA25 transcription is not apparent until 3-5 h after the BL treatment. Cycloheximide, an inhibitor of cytoplasmic protein synthesis, has no effect on the altered rates of pEA207 and Cab transcription. We conclude from these results that the BLF response for Cab transcription and the BHF response for pEA207 transcription probably occurs without the expression of intervening regulatory genes coded within the nucleus or the translation of pre-existing transcripts derived from nuclear-coded genes.

Blue-Light Regulation of Specific Transcript Levels in Pisum sativum: Fluence-Response, Time-Course, and Reciprocity Characteristics.

The expression of many nuclear genes in plants is light regulated. We have examined the fluence-response, time-course, and reciprocity characteristics of four nuclear, blue-light-regulated transcripts in Pisum sativum L. var Alaska: Cab RNA, pEA207 RNA, pEA215 RNA, and pEA25 RNA. To avoid complications due to blue-light-induced transformation of phytochrome, we have adapted the procedure of measuring blue-light-induced changes in steady-state-RNA levels in seedlings grown in continuous red light. The fluence-response curves for Cab and pEA215 RNA are bell-shaped, with peak accumulations at 10(2) and 10(1) micromoles per square meter, respectively. No response is observed at 10(4) micromoles per square meter. pEA25 RNA has threshold and saturation fluences below 10(-1) micromoles per square meter. pEA207 RNA has a threshold at 10(2) micromoles per square meter and saturates above 10(4) micromoles per square meter. pEA215 and Cab RNA start to increase within 1 hour after the 10(1) micromoles per square meter pulse, and finish accumulation by 5 hours. The decrease in pEA207 RNA in response to 10(4) micromoles per square meter and pEA25 RNA in response to 10(1) or 10(4) micromoles per square meter starts between 3 and 5 hours after the blue-light pulse. The Bunsen-Roscoe Law of Reciprocity is followed in the Cab, pEE215, pEA25, and pEA207 RNA responses to 10(1) and 10(4) micromoles per square meter.

Blue-light regulation of transcription for nuclear genes in pea.

The expression of many nuclear genes in plants is light regulated. Previous investigations have shown that the excitation of phytochrome can affect transcription rate and steady-state RNA levels for several of these genes. No direct demonstration of the effects of blue light has been reported. We have identified several nuclear genes in pea whose transcription rate and steady-state RNA levels are affected by a single pulse of blue light. Pea seedlings, grown in continuous red light to saturate any phytochrome response, were treated with a single pulse (10(3) mumol.m(-2)) of blue light 6 days after planting. The blue-light treatment resulted in an increase in the steady-state RNA level and rate of transcription for the Cab (chlorophyll a/b binding protein) gene family and a decrease in the steady-state RNA level and the rate of transcription for the gene families corresponding to cDNA clones pEA25 and pEA207. Changes in the rate of transcription for Cab and pEA207 are apparent within 3 hr of the blue-light treatment.