Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potentials role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed. © 1995 John Wiley & Sons, Inc.     

Immunohistochemical distribution of S-100 protein and type IV collagen in human embryonic and fetal sympathetic neuroblasts

Summary The expression and distribution of S-100 protein and type IV collagen was studied immunohistochemically in sympathetic neuroblasts from the paravertebral region to the adrenal glands in human embryos and fetuses ranging from 7 to 12 weeks gestational age. Prom 7 weeks gestational age, S-100 protein was detected in round or oval cells mingling with sympathetic neuroblasts, and in spindle-shaped cells forming a continuous layer around them. The latter S-100 protein-positive cells were found in contact with the Schwann cells of nerve fibres entering the groups of sympathetic neuroblasts. Staining for type IV collagen showed that all groups of sympathetic neuroblasts were surrounded by a continuous basement membrane. By examining serial sections stained for type IV collagen and S-100 protein, a continuous basement membrane was found along the distribution pattern of the peripheral S-100 protein-positive spindle cells. The morphology of these cells, and their relationships with Schwann cells and with the basement membrane of the sympathetic neuroblasts, indicated that they were Schwann-like cells probably capable of synthesizing a continuous basement membrane separating the neuroblasts from the adjacent tissues. In contrast, the round or oval S-100 protein-positive cells, in contact with the sympathetic neuroblasts and not associated with nerve fibres, were considered as sustentacular or sustentacular precursor cells. At week 7 gestational age, the peri-adrenal sympathetic neuroblasts and their sustentacular and Schwann-like cells started to invade the adrenal glands and mingled with the adrenal cortical cells. These findings suggest the extra-adrenal origin of the sustentacular cells in embryonic and fetal adrenal glands.     

Inhibition and killing of fungi by the polyamine oxidase-polyamine system

Both components of the polyamine oxidase (PAO)-polyamine system are known to be present in phagocytes and have thus been postulated to contribute to the antimicrobial activity of these cells. Therefore, the effects of the PAO-polyamine system on three medically important opportunistic fungi were examined. Yeasts of Cryptococcus neoformans, but not Candida albicans blastoconidia or Aspergillus fumigatus conidia, were efficiently killed by the system. Two putative end products of the system, hydrogen peroxide and acrolein, both killed C. neoformans at concentrations attainable with the whole system. However, catalase failed to inhibit activity of the whole system, making hydrogen peroxide an unlikely mediator of killing. Although C. albicans blastoconidia and A. fumigatus conidia were not killed by the PAO-polyamine system, germ tube formation by the former, and hyphal growth by the latter, were markedly inhibited. These data establish that the PAO-polyamine system possesses antifungal activity.     

Pure red cell hypoplasia associated with long-arm deletion of chromosome 5

Summary A further instance of the new hematologic entity of pure red cell hypoplasia with deletion of the long arm of a chromosome 5(5q-) was seen in a 59-year-old white male. Examination of the bone marrow showed depressed erythroid cells with normal granulocytic and megakaryocytic precursors. Chromosomal analysis showed deletion of a chromosome 5 without any apparent translocation. This karyotype may be associated with acute leukemia.     

Origins and Evolution of the Etruscans’ mtDNA

The Etruscan culture is documented in Etruria, Central Italy, from the 8^th to the 1^st century BC. For more than 2,000 years there has been disagreement on the Etruscans’ biological origins, whether local or in Anatolia. Genetic affinities with both Tuscan and Anatolian populations have been reported, but so far all attempts have failed to fit the Etruscans’ and modern populations in the same genealogy. We extracted and typed the hypervariable region of mitochondrial DNA of 14 individuals buried in two Etruscan necropoleis, analyzing them along with other Etruscan and Medieval samples, and 4,910 contemporary individuals from the Mediterranean basin. Comparing ancient (30 Etruscans, 27 Medieval individuals) and modern DNA sequences (370 Tuscans), with the results of millions of computer simulations, we show that the Etruscans can be considered ancestral, with a high degree of confidence, to the current inhabitants of Casentino and Volterra, but not to the general contemporary population of the former Etruscan homeland. By further considering two Anatolian samples (35 and 123 individuals) we could estimate that the genetic links between Tuscany and Anatolia date back to at least 5,000 years ago, strongly suggesting that the Etruscan culture developed locally, and not as an immediate consequence of immigration from the Eastern Mediterranean shores.     

A biomimetic motility assay provides insight into the mechanism of actin-based motility

Abiomimetic motility assay is used to analyze the mechanism of force production by site-directed polymerization of actin. Polystyrene microspheres, functionalized in a controlled fashion by the N-WASP protein, the ubiquitous activator of Arp2/3 complex, undergo actin-based propulsion in a medium that consists of five pure proteins. We have analyzed the dependence of velocity on N-WASP surface density, on the concentration of capping protein, and on external force. Movement was not slowed down by increasing the diameter of the beads (0.2 to 3 microm) nor by increasing the viscosity of the medium by 10(5)-fold. This important result shows that forces due to actin polymerization are balanced by internal forces due to transient attachment of filament ends at the surface. These forces are greater than the viscous drag. Using Alexa488-labeled Arp2/3, we show that Arp2/3 is incorporated in the actin tail like G-actin by barbed end branching of filaments at the bead surface, not by side branching, and that filaments are more densely branched upon increasing gelsolin concentration. These data support models in which the rates of filament branching and capping control velocity, and autocatalytic branching of filament ends, rather than filament nucleation, occurs at the particle surface.     

A quantitative approach to analyze binding diffusion kinetics by confocal FRAP

Most of the important types of interactions that occur in cells can be characterized as binding-diffusion type processes, and can be quantified by kinetic rate constants such as diffusion coefficients (D) and binding rate constants (k_on and k_off). Confocal FRAP is a potentially important tool for the quantitative analysis of intracellular binding-diffusion kinetics, but how to dependably extract accurate kinetic constants from such analyses is still an open question. To this end, in this study, we developed what we believe is a new analytical model for confocal FRAP-based measurements of intracellular binding-diffusion processes, based on a closed-form equation of the FRAP formula for a spot photobleach geometry. This approach incorporates a binding diffusion model that allows for diffusion of both the unbound and bound species, and also compensates for binding diffusion that occurs during photobleaching, a critical consideration in confocal FRAP analysis. In addition, to address the problem of parametric multiplicity, we propose a scheme to reduce the number of fitting parameters in the effective diffusion subregime when D's for the bound and unbound species are known. We validate this method by measuring kinetic rate constants for the CAAX-mediated binding of Ras to membranes of the endoplasmic reticulum, obtaining binding constants of k_on ∼ 255/s and k_off ∼ 31/s.