The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo.

The prospective fate of cells in the primitive streak was examined at early, mid and late stages of mouse gastrula development to determine the order of allocation of primitive streak cells to the mesoderm of the extraembryonic membranes and to the fetal tissues. At the early-streak stage, primitive streak cells contribute predominantly to tissues of the extraembryonic mesoderm as previously found. However, a surprising observation is that the erythropoietic precursors of the yolk sac emerge earlier than the bulk of the vitelline endothelium, which is formed continuously throughout gastrula development. This may suggest that the erythropoietic and the endothelial cell lineages may arise independently of one another. Furthermore, the extraembryonic mesoderm that is localized to the anterior and chorionic side of the yolk sac is recruited ahead of that destined for the posterior and amnionic side. For the mesodermal derivatives in the embryo, those destined for the rostral structures such as heart and forebrain mesoderm ingress through the primitive streak early during a narrow window of development. They are then followed by those for the rest of the cranial mesoderm and lastly the paraxial and lateral mesoderm of the trunk. Results of this study, which represent snapshots of the types of precursor cells in the primitive streak, have provided a better delineation of the timing of allocation of the various mesodermal lineages to specific compartments in the extraembryonic membranes and different locations in the embryonic anteroposterior axis.     

PACdb: PolyA Cleavage Site and 3'-UTR Database

SUMMARY: The PolyA Cleavage Site and 3'-UTR Database (PACdb) is a web-accessible database that catalogs putative 3'-processing sites and 3'-UTR sequences for multiple organisms. Sites have been identified primarily via expressed sequence tag-genome alignments, enabling delineation of both the specificities and heterogeneity of 3'-processing events. AVAILABILITY: By web browser or CGI: PACdb: http://harlequin.jax.org/pacdb/; AtPACdb: http://harlequin.jax.org/atpacdb/. SUPPLEMENTARY INFORMATION: Available online at http://harlequin.jax.org/pacdb/supplemental.php.     

Cell death mechanisms associated with G2 radiosensitivity in patients with prostate cancer and benign prostatic hyperplasia

Cells respond to genotoxic insults such as ionizing radiation by halting in the G2 phase of the cell cycle. Delayed cell death (mitotic death) can occur when the cell is released from G2, and specific spindle defects form endopolyploid cells (endoreduplication/tetraploidy). Enhanced G2 chromosomal radiosensitivity has been observed in many cancers and genomic instability syndromes, and it is manifested by radiation-induced chromatid aberrations observed in lymphocytes of patients. Here we compare the G2 chromosomal radiosensitivity in prostate patients with benign prostatic hyperplasia (BPH) or prostate cancer with disease-free controls. We also investigated whether there is a correlation between G2 chromosomal radiosensitivity and aneuploidy (tetraploidy and endoreduplication), which are indicative of mitotic cell death. The G2 assay was carried out on all human blood samples. Metaphase analysis was conducted on the harvested chromosomes by counting the number of aberrations and the mitotic errors (endoreduplication/tetraploidy) separately per 100 metaphases. A total of 1/14 of the controls were radiosensitive in G2 compared to 6/15 of the BPH patients and 15/17 of the prostate cancer patients. Radiation-induced mitotic inhibition was assessed to determine the efficacy of G2 checkpoint control in the prostate patients. There was no significant correlation of G2 radiosensitivity scores and mitotic inhibition in BPH patients (P = 0.057), in contrast to prostate cancer patients, who showed a small but significant positive correlation (P = 0.029). Furthermore, there was no significant correlation between G2 radiosensitivity scores of BPH patients and endoreduplication/ tetraploidy (P = 0.136), which contrasted with an extremely significant correlation observed in prostate cancer patients (P < 0.0001). In conclusion, cells from prostate cancer patients show increased sensitivity to the induction of G2 aberrations from ionizing radiation exposure but paradoxically show reduced mitotic indices and aneuploidy as a function of aberration frequency.     

Tropomodulin binds to filensin intermediate filaments

Tropomodulin (Tmod) is an actin filament pointed end capping protein found in the membrane skeleton of lens fiber cells. We demonstrate that Tmod4 is able to bind the lens-specific intermediate filament protein, filensin, in either co-sedimentation or solid phase binding assays in a saturable fashion, but with low affinity and stoichiometry. Furthermore, Tmod4 does not bind the 53 kDa rod domain of filensin, nor to CP49, the obligate assembly partner of filensin. Finally, the binding of filensin to Tmod4 does not inhibit the actin capping activity of Tmod4 in vitro, suggesting that the two functions are not mutually exclusive.     

The effect of interleukin-6 on L-selectin levels on polymorphonuclear leukocytes

Interleukin-6 (IL-6) shortens the transit time of polymorphonuclear leukocytes (PMN) through the marrow and accelerates their release into the circulation. In contrast to other inflammatory stimuli, this response is associated with a decrease in L-selectin levels on circulating PMN. The present study was designed to determine the effect of IL-6 on L-selectin levels of PMN in rabbits. Recombinant human IL-6 (2 microg/kg) caused a decrease in L-selectin levels on circulating PMN 3 to 12 h after treatment (P < 0.05). L-selectin levels decreased on PMN already in the circulation for up to 4 h (P < 0.05), on PMN released from the marrow posttreatment for up to 12 h (P < 0.01) and on PMN in the marrow for up to 6 h (P < 0.05) after IL-6 treatment. We conclude that IL-6 decreases L-selectin levels on circulating PMN by demarginating PMN with low levels of L-selectin and by releasing PMN from the marrow with low levels of L-selectin. We postulate that this prolonged downregulation of L-selectin on circulating PMN could influence their recruitment into inflammatory sites.     

Evaluation of a computer-based approach to teaching acid/base physiology

Because acid/base physiology is a difficult subject for most medical and veterinary students, the first author designed a software program, Acid/Base Primer, that would help students with this topic. The Acid/Base Primer was designed and evaluated within a conceptual framework of basic educational principles. Seventy-five first-year veterinary students (of 81; 93% response rate) participated in this study. Students took both a pre- and posttest of content understanding. After completing the Acid/Base Primer in pairs, each student filled out a survey evaluating the features of the program and describing his/her use and experience of it. Four pairs of students participated in interviews that elaborated on the surveys. Scores improved from 53 +/- 2% on the pretest to 74 +/- 1% on an immediate posttest. On surveys and in interviews, students reported that the program helped them construct their own understanding of acid/base physiology and prompted discussions in pairs of students when individual understandings differed. The case-based format provided anchors and a high degree of relevance. Repetition of concepts helped students develop a more complex network of understanding. Questions in the program served to scaffold the learning process by providing direction, accentuating the relevant features of the cases, and provoking discussion. Guidelines for software development were generated on the basis of the findings and relevant educational literature.     

The effect of oxygen and benzoquinone concentration on the reversible spectrophotometric changes accompanying the photooxidation of porphyrins at low pH values

Oxygen has little or no effect on the spectrophotometric changes accompanying the reversible photooxidation of porphyrins by benzoquinone at low pH values. High concentrations of benzoquinone inhibit these reversible changes. Spectral evidence is presented to indicate that the porphyrin may be undergoing different photooxidative processes at different pH values.     

Comparison of the small heat shock proteins αB-crystallin, MKBP, HSP25, HSP20, and cvHSP in heart and skeletal muscle

Seven members of the small heat shock protein (sHSP) family are exceptional with respect to their constitutive high abundance in muscle tissue. It has been suggested that sHSPs displaying chaperone-like properties may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. In the present study five sHSPs (B-crystallin, MKBP, HSP25, HSP20, and cvHSP) were investigated with respect to similarities and differences of their expression in heart and skeletal muscle under normal and ischemic conditions. In ischemic heart and skeletal muscle these five sHSPs translocated from cytosol to the Z-/I-area of myofibrils. Myofibrillar binding of all sHSPs was very tight and resisted for the most part extraction with 1 M NaSCN or 1 M urea. MKBP and HSP20 became extracted by 1 M NaSCN to a significant extent indicating that these two sHSPs may bind partially to actin-associated proteins which were completely extracted by this treatment. Ultrastructural localization of B-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes B-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of B-crystallin in cardiomyocytes versus skeletal muscle fibers. Further differences of the properties of sHSPs could be observed regarding fiber type distribution of sHSPs. Thus sHSPs form a complex stress–response system in striated muscle tissue with some common as well as some distinct functions in different muscle types.