New Method for Visualization of Vascular Networks in Nonperfused Fixed Tissues

A new method of visualizing the angioarchitecture of tissues has been developed that uses blood components in nonperfused materials. Tissue blocks are fixed in 4% paraformaldehyde and cut with a vibratome into 50-60 μm sections. Endogenous peroxidase in red blood cells is then reduced in the presence of hydrogen peroxide with the resultant oxidation of the chromogen 3,3'-diaminobenzidine (DAB). This generates a dark, highly insoluble reaction product throughout the vascular system. The visualization of vascular components can be further enhanced by exposing the sections to peroxidase-conjugated IgG to increase the background staining of the blood plasma. The technique minimizes preparation artifact and permits the application of morphometric analytical methods, thus allowing parameters such as the volume density of the vascular bed to be quantified.     

Sodiumd-glucose cotransport in the gill of marine mussels: Studies with intact tissue and brush-border membrane vesicles

Summary Glucose transport was studied in marine mussels of the genusMytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na⁺-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na⁺-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na⁺, and concentrative glucose transport was observed in the presence of an inwardly directed Na⁺ gradient. There was a single saturable pathway for glucose uptake, with an apparentK _t of 3 m in BBMV and 9 m in intact gills. The kinetics of Na⁺ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na⁺ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with aK _i of 44 m. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na⁺-glucose cotransporters in other systems;d-glucose and -methyl glucopyranoside were the most effective inhibitors of Na⁺-glucose transport,d-galactose was intermediate in its inhibition, and there was little or no effect ofl-glucose,d-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na⁺-glucose uptake, with an apparentK _i of 154nm in BBMV and 21nm in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.     

Calcium influences the stability and conformation of rotavirus SAH glycoprotein VP7 expressed in Dictyostelium discoideum

We have previously reported expression of the rotavirus outer capsid glycoprotein, VP7, in the relatively new expression host, Dictyostelium discoideum. To optimise yields of recombinant VP7, we examined the role of Ca²⁺ since stability of both VP7 and mature rotavirus during a rotavirus infection are calcium-dependent. Low micromolar levels of free extracellular Ca²⁺ were required to maximise yields of VP7 in D. discoideum whilst levels of VP7 were reduced following depletion of intracellular Ca²⁺ reserves using A23187 and EGTA. Immunoblot analysis suggested that VP7 was being degraded in an intracellular compartment. Immunoprecipitation with a conformation-dependent neutralising antibody confirmed that EGTA-induced Ca²⁺ chelation alters the conformation of VP7. These results suggest that stability of VP7 is dependent on maintaining adequate levels of intracellular Ca²⁺ and that conformational changes in VP7 which occur following depletion of Ca²⁺ reserves induce rapid proteolysis of the protein. Since these results establish conditions for expressing optimal levels of VP7 in the correct conformation they have important implications for the development of a subunit vaccine based on recombinant VP7.     

Mapping of Unconventional Myosins in Mouse and Human

Myosins are molecular motors that move along filamentous actin. Seven classes of myosin are expressed in vertebrates: conventional myosin, or myosin-II, as well as the 6 unconventional myosin classes -I, -V, -VI, -VII, -IX, and -X. We have mapped in mouse 22 probes encompassing all known unconventional myosins and, as a result, have identified 16 potential unconventional myosin genes. These genes include 7 myosins-I, 2 myosins-V, 1 myosin-VI, 3 myosins-VII, 2 myosins-IX, and 1 myosin-X. The map location of 5 of these genes was identified in human chromosomes by fluorescencein situhybridization.     

Immunoaffinity Purification of Epitope-Tagged Human β2-Adrenergic Receptor to Homogeneity

To obtain large quantities of pure human β₂-adrenergic receptor (β₂-AR) needed for structural studies, an efficient method for β₂-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged β₂-AR was expressed in Sf9 cells with a specific activity of 5–20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-β-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60–70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of >30%. The purified receptor was concentrated to >1 mg/ml without loss of ligand binding activity.     

The advantage of long-distance clonal spreading in highly disturbed habitats

Summary Classical theory states that cover of annual plants should increase relative to perennials as disturbance frequency increases. However, it has been suggested that long-distance clonal spreading can allow some perennial plants to survive in highly disturbed areas by quickly spreading into disturbed patches. To evaluate these hypotheses, we analysed data of plant distributions in two different ecosystems, a barrier island and a short-grass steppe. The disturbances studied were sand deposition during storms (overwash) on the barrier island and grazing by cattle in the short-grass steppe. In each case the disturbance frequency varied over the ecosystem; we categorized different areas in terms of their disturbance frequencies. All plant species in each area were categorized as one of four plant life forms (1) annual or biennial, (2) herbaceous perennial without long-distance clonal spreading (3) herbaceous perennial with long-distance clonal spreading (i.e guerilla form) and (4) woody plant. Percentage cover of each plant life form in each disturbance frequency category was calculated. In both ecosystems, (1) there was an increase in the relative cover of annuals as one moved from areas of low to moderate disturbance frequencies, but then a decrease in cover of annuals as one moved into the areas of highest disturbance frequency and (2) the guerilla forms showed the greatest relative increase in cover from moderately to highly disturbed areas. The combination of two factors can explain this pattern: (1) long-distance clonal spreading effectively reduces the time to colonization of recently disturbed sites and (2) effects of the disturbances in these two systems are probably more severe for seeds than for stems. We illustrate these effects using a spatially explicit simulation model of the population dynamics of plants in a disturbed landscape.     

Germ-Line Mutations in the von Hippel–Lindau Tumor-Suppressor Gene Are Similar to Somatic von Hippel–Lindau Aberrations in Sporadic Renal Cell Carcinoma

von Hippel–Lindau (VHL) disease is a hereditary tumor syndrome predisposing to multifocal bilateral renal cell carcinomas (RCCs), pheochromocytomas, and pancreatic tumors, as well as angiomas and hemangioblastomas of the CNS. A candidate gene for VHL was recently identified, which led to the isolation of a partial cDNA clone with extended open reading frame, without significant homology to known genes or obvious functional motifs, except for an acidic pentamer repeat domain. To further characterize the functional domains of the VHL gene and assess its involvement in hereditary and nonhereditary tumors, we performed mutation analyses and studied its expression in normal and tumor tissue. We identified germ-line mutations in 39% of VHL disease families. Moreover, 33% of sporadic RCCs and all (6/6) sporadic RCC cell lines analyzed showed mutations within the VHL gene. Both germ-line and somatic mutations included deletions, insertions, splice-site mutations, and missense and nonsense mutations, all of which clustered at the 3' end of the corresponding partial VHL cDNA open reading frame, including an alternatively spliced exon 123 nt in length, suggesting functionally important domains encoded by the VHL gene in this region. Over 180 sporadic tumors of other types have shown no detectable base changes within the presumed coding sequence of the VHL gene to date. We conclude that the gene causing VHL has an important and specific role in the etiology of sporadic RCCs, acts as a recessive tumor-suppressor gene, and appears to encode important functional domains within the 3' end of the known open reading frame.     

Effect of biocatalyst activity changes in continuous reactions in the gas phase

Summary In the gas phase bioreactions, continuous production rate depends on the biocatalyst activity and complete dehydration causes the biocatalyst to lose most of its activity. To overcome these difficulties, a theoretical method is suggested along with the new design of biocatalyst. This will be applicable and helpful for the optimization of the gas phase continuous bioreaction.Nomenclature C_A ethanol concentration [mol/mL] - C_P acetaldehyde concentration [mol/mL] - X_P acetaldehyde composition     

Voltage-Gated Potassium Channel Genes Are Clustered in Paralogous Regions of the Mouse Genome

Cloning of the Drosophila Shaker gene established that a neurological phenotype including locomotor dysfunction can be caused by a mutation in a voltage-gated potassium (K) channel gene. Shaker sequences have been used to isolate a large family of related K channel genes from both flies and mammals. Toward elucidating the evolutionary relationship between loci and the potential causal connection that K channels may have to mammalian genetic disorders, we report here the genetic mapping of 12-16 different murine, voltage-gated K channel genes. We find that multiple genes, in some cases from distantly related K channel subfamilies, occur in clusters in the mouse genome. These mapping results suggest that the K channel gene subfamilies arose through ancient localized gene duplication events, followed by chromosomal duplications and rearrangements as well as further gene duplication. We also note that several neurologic disorders of both mouse and human are associated with the chromosomal regions containing K channel genes.