A new set of Arabidopsis expressed sequence tags from developing seeds. The metabolic pathway from carbohydrates to seed oil.

Large-scale single-pass sequencing of cDNAs from different plants has provided an extensive reservoir for the cloning of genes, the evaluation of tissue-specific gene expression, markers for map-based cloning, and the annotation of genomic sequences. Although as of January 2000 GenBank contained over 220,000 entries of expressed sequence tags (ESTs) from plants, most publicly available plant ESTs are derived from vegetative tissues and relatively few ESTs are specifically derived from developing seeds. However, important morphogenetic processes are exclusively associated with seed and embryo development and the metabolism of seeds is tailored toward the accumulation of economically valuable storage compounds such as oil. Here we describe a new set of ESTs from Arabidopsis, which has been derived from 5- to 13-d-old immature seeds. Close to 28,000 cDNAs have been screened by DNA/DNA hybridization and approximately 10,500 new Arabidopsis ESTs have been generated and analyzed using different bioinformatics tools. Approximately 40% of the ESTs currently have no match in dbEST, suggesting many represent mRNAs derived from genes that are specifically expressed in seeds. Although these data can be mined with many different biological questions in mind, this study emphasizes the import of photosynthate into developing embryos, its conversion into seed oil, and the regulation of this pathway.

Hybrid finite element-finite difference method for thermal analysis of blood vessels.

A hybrid finite-difference/finite-element technique for the thermal analysis of blood vessels embedded in perfused tissue has been developed and evaluated. This method provides efficient and accurate solutions to the conjugated heat transfer problem of convection by blood coupled to conduction in the tissue. The technique uses a previously developed 3D automatic meshing method for creating a finite element mesh in the tissue surrounding the vessels, coupled iteratively with a 1-D marching finite difference method for the interior of the vessels. This hybrid technique retains the flexibility and ease of automated finite-element meshing techniques for modelling the complex geometry of blood vessels and irregularly shaped tissues, and speeds the solution time by using a simple finite-difference method to calculate the bulk mean temperatures within all blood vessels. The use of the 1D finite-difference technique in the blood vessels also eliminates the large computer memory requirements needed to accurately solve large vessel network problems when fine FE meshes are used in the interior of vessels. The accuracy of the hybrid technique has been verified against previously verified numerical solutions. In summary, the hybrid technique combines the accuracy and flexibility found in automated finite-element techniques, with the speed and reduction of computational memory requirements associated with the 1D finite-difference technique, something which has not been done before. This method, thus, has the potential to provide accurate, flexible and relatively fast solutions for the thermal analysis of coupled perfusion/blood vessel problems, and large vessel network problems.

An accurate, convective energy equation based automated meshing technique for analysis of blood vessels and tissues.

An automated three-element meshing method for generating finite element based models for the accurate thermal analysis of blood vessels imbedded in tissue has been developed and evaluated. The meshing method places eight noded hexahedral elements inside the vessels where advective flows exist, and four noded tetrahedral elements in the surrounding tissue. The higher order hexahedrals are used where advective flow fields occur, since high accuracy is required and effective upwinding algorithms exist. Tetrahedral elements are placed in the remaining tissue region, since they are computationally more efficient and existing automatic tetrahedral mesh generators can be used. Five noded pyramid elements connect the hexahedrals and tetrahedrals. A convective energy equation (CEE) based finite element algorithm solves for the temperature distributions in the flowing blood, while a finite element formulation of a generalized conduction equation is used in the surrounding tissue. Use of the CEE allows accurate solutions to be obtained without the necessity of assuming ad hoc values for heat transfer coefficients. Comparisons of the predictions of the three-element model to analytical solutions show that the three-element model accurately simulates temperature fields. Energy balance checks show that the three-element model has small, acceptable errors. In summary, this method provides an accurate, automatic finite element gridding procedure for thermal analysis of irregularly shaped tissue regions that contain important blood vessels. At present, the models so generated are relatively large (in order to obtain accurate results) and are, thus, best used for providing accurate reference values for checking other approximate formulations to complicated, conjugated blood heat transfer problems.

The Ross operation in a Jehovah's Witness: a paradigm for heart surgery in children without transfusion.

A 3-year-old 18 kg male child of the Jehovah's Witness faith presented with severe aortic regurgitation. A successful Ross procedure was performed using a pulmonary autograft, without the use of blood or blood product transfusion. Blood conservation strategy included: (1) preoperative treatment with recombinant human erythropoietin; (2) intraoperative strategies, including technical modifications to the Ross procedure, and the prophylactic use of fibrin glue; (3) utilization of a heparin-bonded cardiopulmonary bypass circuit and assisted venous drainage; and 4) the use of prebypass phlebotomy, cell-saving device and autotransfusion. The patient was discharged home on postoperative day 7 with a hemoglobin level of 11.9.

cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450.

Retinoids, including all-trans-retinoic acid (RA) and its stereoisomer 9-cis-RA play important roles in regulating gene expression, through interactions with nuclear receptors, during embryonic development and in the maintenance of adult epithelial tissues (Chambon, P. (1995) Rec. Prog. Horm. Res. 50, 317-32; Mangelsdorf, D. J., and Evans, R. M. (1995) Cell 83, 841-850; Petkovich, M. (1992) Annu. Rev. Nutr. 12, 443-471). Evidence suggests that 4-hydroxylation of RA inside the target cell limits its biological activity and initiates a degradative process of RA leading to its eventual elimination. However, 18-hydroxylation and glucuronidation may also be important steps in this process. In this paper, we describe the cloning and characterization of the first mammalian retinoic acid-inducible retinoic acid-metabolizing cytochrome P450 (hP450RAI), which belongs to a novel class of cytochromes (CYP26). We demonstrate that hP450RAI is responsible for generation of several hydroxylated forms of RA, including 4-OH-RA, 4-oxo-RA, and 18-OH-RA. We also show that hP450RAI mRNA expression is highly induced by RA in certain human tumor cell lines and further show that RA-inducible RA metabolism may correlate with P450RAI expression. We conclude that this enzyme plays a key role in RA metabolism, functioning in a feedback loop where RA levels are controlled in an autoregulatory manner.

Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase.

Retinoic acid (RA) metabolites of vitamin A are key regulators of gene expression involved in embryonic development and maintenance of epithelial tissues. The cellular effects of RA are dependent upon the complement of nuclear receptors expressed (RARs and RXRs), which transduce retinoid signals into transcriptional regulation, the presence of cellular retinoid-binding proteins (CRABP and CRBP), which may be involved in RA metabolism, and the activity of RA metabolizing enzymes. We have been using the zebrafish as a model to study these processes. To identify genes regulated by RA during exogenous RA exposure, we utilized mRNA differential display. We describe the isolation and characterization of a cDNA, P450RAI, encoding a novel member of the cytochrome P450 family. mRNA transcripts for P450RAI are expressed normally during gastrulation, and in a defined pattern in epithelial cells of the regenerating caudal fin in response to exogenous RA. In COS-1 cells transfected with the P450RAI cDNA, all-trans-RA is rapidly metabolized to more polar metabolites. We have identified 4-oxo-RA and 4-OH-RA as major metabolic products of this enzyme. P450RAI represents the first enzymatic component of RA metabolism to be isolated and characterized at the molecular level and provides key insight into regulation of retinoid homeostasis.

Gastrocolic fistula complicating benign gastric ulcer. A case report and review of the literature.

Gastrocolic fistula complicating gastric or colonic surgery is a well-recognized entity. It is extremely rare, however, to encounter this condition in patients with benign peptic ulceration who have not undergone previous abdominal surgery. Such a patient was seen recently at Addington Hospital, Durban. The diagnosis was confirmed by means of barium enema examination and subsequent successful surgical resection of the fistula was performed.