Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.     

Parthenogenesis in Xenopus eggs injected with centrosomes from synchronized human lymphoid cells

In Xenopus eggs, normal development requires the participation of the centrosome provided by the sperm. Injection of foreign centrosomes purified from exponentially growing mammalian cells enables the eggs to undertake parthenogenesis. In order to know whether such a complementation required centrosomes already committed to duplication, we have prepared centrosomes from human cells synchronized at different stages of the cell cycle (G0, G1, G2). We show that the three types of centrosome possess a similar parthenogenetic activity and conclude that duplication of heterologous centrosome can be triggered in Xenopus eggs.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Noncompetitive enzyme immunoassay for the measurement of bronchial inhibitor in biological fluids

An enzyme-linked immunosorbent assay (ELISA) of bronchial inhibitor using rabbit antibronchial inhibitor antibody-coated polystyrene balls as the solid-phase antibody and peroxidase-labeled antibody as the conjugate is described. A crude antibody fraction is used for coating the solid phase. The assay can be run within 8 h and gives reproducible results in the range of 6 to 60 micrograms/l of bronchial inhibitor (mean within-run coefficient of variation, 7%). It can detect bronchial inhibitor concentrations as low as 2 micrograms/l (10(-10) M) and recovery of varying amounts of bronchial inhibitor added to bronchial liquids is greater than 90%. This enzyme immunoassay appears to be a convenient way to quantify bronchial inhibitor in biological fluids such as serum, sputum, or bronchoalveolar lavage fluid.     

Structural and functional determinants in adenovirus type 2 penton base recombinant protein.

Discrete domains involved in structural and functional properties of adenovirus type 2 (Ad2) penton base were investigated with site-directed mutagenesis of the recombinant protein expressed in baculovirus-infected cells. Seventeen substitution mutants were generated and phenotyped for various functions in insect and human cells as follows. (i) Pentamerization of the penton base protein was found to be dependent on three amino acid side chains, the indole ring of Trp119, the hydroxylic group of Tyr553, and the basic group of Lys556. (ii) Arg254, Cys432, and Trp439, the stretch of basic residues at positions 547 to 556, and Arg340 of the RGD motif played a critical role in stable fiber-penton base interactions in vivo. (iii) Nuclear localization of penton base in Sf9 cells was negatively affected in mutants W119H or W165H, and, to a lesser extent, by substitutions in the consensus polybasic signal at positions 547 to 549. (iv) Penton base mutants were also assayed for HeLa cell binding, cell detachment, plasmid DNA internalization, and Ad-mediated gene delivery. The results obtained suggested that the previously identified integrin-binding motifs RGD340 and LDV287 were functionally and/or topologically related to other discrete regions which include Trp119, Trp165, Cys246, Cys432, and Trp439, all of which were involved in penton base-cell surface recognition, endocytosis, and postendocytotic steps of the virus life cycle.     

Differential proliferation rates generate patterns of mechanical tension that orient tissue growth

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal–distal (P–D) axis by the Dachsous‐Fat‐Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P–D axis despite strong Dachs polarization. Here, we show that these circumferential divisions are oriented by circumferential mechanical forces that influence cell shapes and thus orient the mitotic spindle. We propose that this circumferential pattern of force is not generated locally by polarized constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localized overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape.     

Association between pygmy right whales (Caperea marginata) and areas of high marine productivity off Australia and New Zealand

Abstract

Opportunistic sightings and strandings of Caperea marginata (n=196) from the vicinity of Australia and New Zealand (1884 to early 2007) were used to relate geographic and temporal patterns to oceanographic and broad-scale climatic variability. Records were not uniformly distributed along the coast and more (69%) were from Australia than New Zealand. Seven coastal whale ‘hotspots’ were identified which accounted for 61% of records with locality data. Half of the hotspot records were from southeast (37) and northwest (20) Tasmania—others each had 9–15 events. Upwelling and/or high zooplankton abundance has been documented near all whale hotspots. Records of C. marginata occurred in all months, with 75% in spring and summer. Inter-annual variability showed broad agreement between increased whale records (usually in spring/summer) and strongly positive ‘Niño 3.4’ during 1980–1995 but not thereafter. Coastal upwelling and productivity increase during climatic phenomena such as El Niño and are likely to be quickly beneficial to plankton-feeding whales such as C. marginata.