Localization of laminin alpha4-chain in developing and adult human tissues.

Recent studies suggest important functions for laminin-8 (Ln-8; alpha4beta1gamma1) in vascular and blood cell biology, but its distribution in human tissues has remained elusive. We have raised a monoclonal antibody (MAb) FC10, and by enzyme-linked immunoassay (EIA) and Western blotting techniques we show that it recognizes the human Ln alpha4-chain. Immunoreactivity for the Ln alpha4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the Ln alpha4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of Ln alpha4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Ln alpha4- and Ln alpha2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric glands and acini of pancreas. Cultured human pulmonary artery endothelial (HPAE) cells produced Ln alpha4-chain as M(r) 180,000 and 200,000 doublet and rapidly deposited it to the growth substratum. In cell-free extracellular matrices of human kidney and lung, Ln alpha4-chain was found as M(r) 180,000 protein.     

Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization.

The transmission of Escherichia coli O157:H7 from manure-contaminated soil and irrigation water to lettuce plants was demonstrated using laser scanning confocal microscopy, epifluorescence microscopy, and recovery of viable cells from the inner tissues of plants. E. coli O157:H7 migrated to internal locations in plant tissue and was thus protected from the action of sanitizing agents by virtue of its inaccessibility. Experiments demonstrate that E. coli O157:H7 can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant.     

Late Serpukhovian (Namurian A) microfacies and carbonate microfossils from the Carboniferous of Nötsch (Austria)

Summary The Carboniferous of N?tsch (Austria), divided into Erlachgraben, Badstub and N?tsch Formations, is composed of a thick sequence of dominantly siliciclastic deepsea sediments. Intercalated marly and silty limestones in the upper Erlachgraben Formation consist of bioclastic wackestones and algal wackestones/packstones which contain a diverse fossil assemblage of formainifers, algae and pseudo-algae. These microfossils are accurately described and documented, and three species of algae are established:Principia fluegeli n. sp.,Paraepimastopora noetschensis n. sp., andNanopora pseudofragilissima n. sp. Based on the occurrence of both important species of the foraminifers Lasiodiscoidea (Howchinia gibba andEolasiodiscus dilatatus), and also on the presence ofEndothyranopsis plana, of the lastEarlandia ex. gr.vulgaris and of the firstEostaffella exp gr.postmosquensis, the upper Erlachgraben Formation is dated as late Serpukhovian (goniatite biozone E 2 of the Namurian A; Arnsbergian stage, corresponding to the Zapaltyubinsky of the standard Russian sequence; foraminiferal biozones 18 or Cf 7 of Belgium, or Cf 16 of the Donbass). Compared to the Pyrenees and the Donbass region, the algal flora of the Carboniferous of N?tsch seems to be relatively endemic. Algae and foraminifers originally inhabited a shallow carbonate ramp and were transported and redeposited in a deep-water environment by gravity flows. The formainifers most probably migrated from the Donbass region along the shelf of a narrow seaway to N?tsch.     

A critical epitope for substrate recognition by the nucleosome remodeling ATPase ISWI

The ATPase ISWI is the catalytic core of several nucleosome remodeling complexes, which are able to alter histone–DNA interactions within nucleosomes such that the sliding of histone octamers on DNA is facilitated. Dynamic nucleosome repositioning may be involved in the assembly of chromatin with regularly spaced nucleosomes and accessible regulatory sequence elements. The mechanism that underlies nucleosome sliding is largely unresolved. We recently discovered that the N-terminal ‘tail’ of histone H4 is critical for nucleosome remodeling by ISWI. If deleted, nucleosomes are no longer recognized as substrates and do not stimulate the ATPase activity of ISWI. We show here that the H4 tail is part of a more complex recognition epitope which is destroyed by grafting the H4 N-terminus onto other histones. We mapped the H4 tail requirement to a hydrophilic patch consisting of the amino acids R₁₇H₁₈R₁₉ localized at the base of the tail. These residues have been shown earlier to contact nucleosomal DNA, suggesting that ISWI recognizes an ‘epitope’ consisting of the DNA-bound H4 tail. Consistent with this hypothesis, the ISWI ATPase is stimulated by isolated H4 tail peptides ISWI only in the presence of DNA. Acetylation of the adjacent K₁₂ and K₁₆ residues impairs substrate recognition by ISWI.     

Methylation at arginine 17 of histone H3 is linked to gene activation

The nuclear hormone receptor co-activator CARM1 has the potential to methylate histone H3 at arginine residues in vitro. The methyltransferase activity of CARM1 is necessary for its co-activator functions in transient transfection assays. However, the role of this methyltransferase in vivo is unclear, given that methylation of arginines is not easily detectable on histones. We have raised an antibody that specifically recognizes methylated arginine 17 (R17) of histone H3, the major site of methylation by CARM1. Using this antibody we show that methylated R17 exists in vivo. Chromatin immunoprecipitation analysis shows that R17 methylation on histone H3 is dramatically upregulated when the estrogen receptor-regulated pS2 gene is activated. Coincident with the appearance of methylated R17, CARM1 is found associated with the histones on the pS2 gene. Together these results demonstrate that CARM1 is recruited to an active promoter and that CARM1-mediated R17 methylation on histone H3 takes place in vivo during this active state.     

Cellular and cis-regulation of En-2 expression in the mandibular arch.

Investigations into early muscle development have focused primarily on somite derived cells. Cranial mesoderm does not undergo somitogenesis, and muscle formation in this region is less well understood. In the present study, we have focused upon the expression of engrailed in mandibular arch myoblasts. We demonstrate that En-2 is expressed in mandibular arch myoblasts of the mouse. The activity of the En-2 enhancer is maintained in several functionally related muscles that arise from the first arch. Through the use of reporter transgenics, we demonstrate that local cell-cell interactions are important in maintaining En-2 expression in the mandibular arch cells. En-2 enhancer activity in the first arch requires a combination of cis-acting sequences that includes a motif which is identical to one found in the Otx2 enhancer and which is sufficient to direct expression in the first arch. These data support the notion that cranial muscle development is regulated by local cell-cell interactions which distinguish distinct anatomical and functional muscle groups.     

Dual signalling by different octopamine receptors converges on adenylate cyclase in Sf9 cells.

The Sf9 insect cell line, derived from Spodoptera frugiperda, was used to study the regulation of adenylate cyclase (AC) activity by octopamine receptors. The cyclic AMP (cAMP) production was stimulated in a concentration-dependent manner by octopamine. Octopamine also elicited a rise in cytosolic Ca(2+). The Ca(2+) elevation was independent of the cAMP elevation whereas the cAMP elevation was partially inhibited by removal of Ca(2+). The antagonistic effects of a series of compounds were tested on both responses. Phentolamine inhibited both responses with similar potency. Two of the tested compounds, MK-912 and RS 79948, were over 1000-fold more potent in blocking the Ca(2+) response. Ionomycin, a Ca(2+) ionophore, or activation of the heterologously expressed muscarinic M(3) receptors in the cells did not alone stimulate cAMP production. However, a Ca(2+) elevation potentiated cAMP production in the presence of a primary stimulant such as forskolin or activated G(s) proteins. This type of regulation of AC is different from previously identified Ca(2+)-sensitive AC isoforms. For comparison the Ca(2+)/calmodulin-activated type I AC was expressed and demonstrated to be stimulated directly by an increase in Ca(2+). Together the results demonstrate that octopamine can synergistically regulate the AC activity via two different receptors in Sf9 cells.     

Telomere Dysfunction Triggers Developmentally Regulated Germ Cell Apoptosis

Telomere dysfunction results in fertility defects in a number of organisms. Although data from fission yeast and Caenorhabditis elegans suggests that telomere dysfunction manifests itself primarily as defects in proper meiotic chromosome segregation, it is unclear how mammalian telomere dysfunction results in germ cell death. To investigate the specific effects of telomere dysfunction on mammalian germ cell development, we examined the meiotic progression and germ cell apoptosis in late generation telomerase null mice. Our results indicate that chromosome asynapsis and missegregation are not the cause of infertility in mice with shortened telomeres. Rather, telomere dysfunction is recognized at the onset of meiosis, and cells with telomeric defects are removed from the germ cell precursor pool. This germ cell telomere surveillance may be an important mechanism to protect against the transmission of dysfunctional telomeres and chromosomal abnormalities.     

Movement and sound generation by the toadfish swimbladder

Although sound-producing (sonic) muscles attached to fish swimbladders are the fastest known vertebrate muscles, the functional requirement for such extreme speed has never been addressed. We measured movement of the swimbladder caused by sonic muscle stimulation in the oyster toadfish Opsanus tau and related it to major features of the sound waveform. The movement pattern is complex and produces sound inefficiently because the sides and bottom of the bladder move in opposite in and out directions, and both movement and sound decay rapidly. Sound amplitude is related to speed of swimbladder movement, and slow movements do not produce perceptible sound. Peak sound amplitude overlaps fundamental frequencies of the male's mating call because of muscle mechanics and not the natural frequency of the bladder. These findings suggest that rapid muscle speed evolved to generate sound from an inefficient highly damped system.