Mutational analysis of gap junction formation.

The paired oocyte cell-cell channel assay was used to investigate the mechanisms involved in the process of formation of gap junction channels. Single oocytes, injected with connexin-specific mRNAs, accumulate a pool of precursors from which cell-cell channels can form rapidly upon pairing. Several lines of evidence, including immunohistochemistry and surface labeling, indicate that part of this precursor pool is located in the cell membrane, probably in the form of closed hemichannels. The homophilic binding of hemichannels to each other can be mimicked by synthetic peptides representing the extracellular loop sequences of connexin32. The peptides specifically suppress channel formation. A crucial role is established for the six cysteines in the extracellular domains that are conserved in all vertebrate gap junction proteins. Change of any of these cysteines into serines results in absolute loss of function of the mutant connexin. The effects of thiol-specific reagents on channel formation suggest that docking and/or opening of channels involves disulfide exchange. Several of the variable amino acids in the extracellular loop sequences were found to determine specificity of connexin-connexin interactions.     

Human dihydropteridine reductase: characterisation of a cDNA clone and its use in analysis of patients with dihydropteridine reductase deficiency.

Deficiency of human dihydropteridine reductase (hDHPR) causes malignant hyperphenylalaninemia. We report the isolation of a cDNA clone for hDHPR that spans the complete coding region, and present the nucleotide sequence and the predicted amino acid sequence. The hDHPR protein does not share extensive homology with the enzymatically related protein human dihydrofolate reductase. Patients with hDHPR deficiency were analysed for the presence of hDHPR cross-reacting protein, mRNA encoding hDHPR, and chromosomal DNA rearrangements. The results show that this inherited error of metabolism can result from a variety of mutations. However, no major rearrangements were seen in 11 patients analysed by Southern blotting. Three RFLPs were found with the restriction endonucleases AvaII and MspI. These RFLPs are useful for prenatal diagnosis of hDHPR deficiency.     

Characterization of ischemia-induced loss of epithelial polarity

Summary Total renal ischemia for various time intervals (0–50) min) resulted in the rapid and duration-dependent redistribution of polarized membrane lipids and proteins in renal proximal tubule cells. Following only 15 min of ischemia, apical membrane enrichment of NaK-ATPase, normally a basolateral membrane (BLM) enzyme, had increased (1.6±0.6vs. 2.9±1.2,P<0.01). In vivo histochemical localization of NaK-ATPase showed reaction product throughout the apical microvillar region. PTH-stimulatable adenylate cyclase, another BLM protein, was also found in ischemic but not control apical membrane fractions. One dimensional SDS-PAGE showed four bands, present in control BLM and ischemic apical membranes, which could not be found in control apical membrane fractions. Immunohistochemical localization of leucine aminopeptidase (LAP) showed the enzyme was limited to the apical domain in control cells. Following ischemic injury (50 min), LAP staining could be seen within the cell and along the BLM. Following 24 hr of reperfusion, the BLM distribution of LAP was further enhanced. With cellular recovery from ischemic injury (5 days), LAP was again only visualized in the apical membrane. Duration-dependent alterations in apical and BLM lipids were also observed. Apical sphingomyelin and phosphatidylserine and the cholesterol-tophospholipid ratio decreased rapidly while apical phosphatidylcholine and phosphatidylinositol increased. Taken together, these results indicate renal ischemia causes rapid duration-dependent reversible loss of surface membrane polarity in proximal tubule cells.     

Dynorphin (1-13): analgesia, hypothermia, cross-tolerance with morphine and beta-endorphin

Intracerebroventricular administration of 20, 40 and 60 nmol of dynorphin (1-13) produced analgesia, as assessed by flinch/jump response to footshock, and hypothermia in the rat. Rats developed tolerance to both the analgesic and thermic effects of the 20 nmol dose of dynorphin. Dynorphin and beta-endorphin showed cross-tolerance with respect to their analgesic but not their thermic effects. Dynorphin and morphine also produced cross-tolerant analgesic effects. Naloxone (10 mg/kg, IP) completely blocked the barrel rolling produced by 20 nmol dynorphin but did not alter its analgesic or thermic effects.     

Predicting the intensity of the birch pollen season

We present a model for the prediction of the magnitude ofBetula flowering and pollen dispersal which may be used in the management of birch pollinosis and in the planning of clinical trials. The pollen sum during the flowering season is regressed on the temperature sum from May 1st to July 20th during the initiation year, the pollen sum of the initiation year, and the temperature sum during the main pollen season in the flowering year. We suggest that the fluctuating flowering pattern inBetula alba-species is primarily determined by the availability of assimilation products during inflorescence initiation and development during the spring one year before anthesis. When inflorescences, which are initiated during the previous year, elongate in the beginning of anthesis, they act as strong sinks to stored carbohydrates, and thus compete with developing leaves and shoots. The result is an initially reduced photosynthetic capacity in years with intense flowering, and a limited potential for the initiation of new inflorescences for the following year. The ambient temperature during catkin initiation affects assimilation efficiency and is a determinant of about equal importance to flowering intensity as is the magnitude of the flowering in the initiation year. The amount of pollen dispersed is also dependent on the weather during anthesis, which is not possible to predict until about one month in advance. The two other independent variables are available during the previous summer, making it possible to give a sufficiently valid prediction to allergologists about the magnitude of the next birch pollen season, according to its botanical determinants. We suggest that the varying reproductive output inBetula alba should not be described as true masting. A more parsimonious explanation to the flowering pattern is that an individual continually maximizes reproductive effort, according to what is possible, but that reproduction is often constrained by the environment.     

Neuroglial ATP release through innexin channels controls microglial cell movement to a nerve injury

Microglia, the immune cells of the central nervous system, are attracted to sites of injury. The injury releases adenosine triphosphate (ATP) into the extracellular space, activating the microglia, but the full mechanism of release is not known. In glial cells, a family of physiologically regulated unpaired gap junction channels called innexons (invertebrates) or pannexons (vertebrates) located in the cell membrane is permeable to ATP. Innexons, but not pannexons, also pair to make gap junctions. Glial calcium waves, triggered by injury or mechanical stimulation, open pannexon/innexon channels and cause the release of ATP. It has been hypothesized that a glial calcium wave that triggers the release of ATP causes rapid microglial migration to distant lesions. In the present study in the leech, in which a single giant glial cell ensheathes each connective, hydrolysis of ATP with 10 U/ml apyrase or block of innexons with 10 µM carbenoxolone (CBX), which decreased injury-induced ATP release, reduced both movement of microglia and their accumulation at lesions. Directed movement and accumulation were restored in CBX by adding ATP, consistent with separate actions of ATP and nitric oxide, which is required for directed movement but does not activate glia. Injection of glia with innexin2 (Hminx2) RNAi inhibited release of carboxyfluorescein dye and microglial migration, whereas injection of innexin1 (Hminx1) RNAi did not when measured 2 days after injection, indicating that glial cells’ ATP release through innexons was required for microglial migration after nerve injury. Focal stimulation either mechanically or with ATP generated a calcium wave in the glial cell; injury caused a large, persistent intracellular calcium response. Neither the calcium wave nor the persistent response required ATP or its release. Thus, in the leech, innexin membrane channels releasing ATP from glia are required for migration and accumulation of microglia after nerve injury.     

A eutherian X-linked gene, PDHA1, is autosomal in marsupials:A model for the evolution of a second, testis-specific variant in eutherian mammals

We report the cloning and mapping of a gene (PDHA)for the pyruvate dehydrogenase E1α subunit in marsupials. In situ hybridization and Southern blot analysis show that PDHA is autosomal in marsupials, mapping to chromosome 3q in Sminthopsis macroura and 5p in Macropus eugenii. Since these locations represent a region that was translocated to the p arm of the human X chromosome following marsupial/eutherian divergence, we suggest that the marsupial PDHA gene is homologous to PDHA1, the somatic eutherian isoform located on human Xp and mouse X. Only one copy of PDHA is found in marsupials, whereas a second, testis-specific, intronless form is observed in eutherian mammals. We also suggest that translocation of PDHA to the eutherian X chromosome, which is inactivated during spermatogenesis, led to the evolution of a second testis-specific locus by retroposition to an autosome.     

Haplotype diversity and population structure in cultivated and wild barley evaluated for Fusarium head blight responses

Fusarium head blight (FHB) is a threat to barley (Hordeum vulgare L.) production in many parts of the world. A number of barley accessions with partial resistance have been reported and used in mapping experiments to identify quantitative trait loci (QTL) associated with FHB resistance. Here, we present a set of barley germplasm that exhibits FHB resistance identified through screening a global collection of 23,255 wild (Hordeum vulgare ssp. spontaneum) and cultivated (Hordeum vulgare ssp. vulgare) accessions. Seventy-eight accessions were classified as resistant or moderately resistant. The collection of FHB resistant accessions consists of 5, 27, 46 of winter, wild and spring barley, respectively. The population structure and genetic relationships of the germplasm were investigated with 1,727 Diversity Array Technology (DArT) markers. Multiple clustering analyses suggest the presence of four subpopulations. Within cultivated barley, substructure is largely centered on spike morphology and growth habit. Analysis of molecular variance indicated highly significant genetic variance among clusters and within clusters, suggesting that the FHB resistant sources have broad genetic diversity. The haplotype diversity was characterized with DArT markers associated with the four FHB QTLs on chromosome 2H bin8, 10 and 13 and 6H bin7. In general, the wild barley accessions had distinct haplotypes from those of cultivated barley. The haplotype of the resistant source Chevron was the most prevalent in all four QTL regions, followed by those of the resistant sources Fredrickson and CIho4196. These resistant QTL haplotypes were rare in the susceptible cultivars and accessions grown in the upper Midwest USA. Some two- and six-rowed accessions were identified with high FHB resistance, but contained distinct haplotypes at FHB QTLs from known resistance sources. These germplasm warrant further genetic studies and possible incorporation into barley breeding programs.     

Detection of biotinylated DNA probes by using Eu-labeled streptavidin and time-resolved fluorometry

Europium has been used as a label in immunoassays as it can be measured with high sensitivity by means of time-resolved fluorometry. Here we have used streptavidin labeled with europium chelates in the detection of adenovirus type 2 DNA bound to microtiter wells after hybridization with a biotinylated probe. The method gave quantitative results and a sensitivity of about 10 pg of the specific DNA.     

Evolution of mammalian X-linked and autosomal Pgk and Pdh E1 alpha subunit genes

The phylogeny and substitution rates of the mammalian X chromosome- located and autosomal phosphoglycerate kinase and pyruvate dehydrogenase genes were investigated. Compatibility analysis was used to show reticulate evolution in these genes. Analysis of the marsupial, mouse, and human phosphoglycerate kinase genes suggests that at least two recombination events have taken place, one occurring about the time of the placental-marsupial split involving exons 1-5 and the other before the primate-rodent split involving exons 9-10. Similar analysis of the pyruvate dehydrogenase genes indicates a recombination event involving exons 2-3 at a time before the primate-rodent split and a gene conversion between exons 3-4 in the human somatic and testis- specific pyruvate dehydrogenase genes after the primate-rodent split. This demonstrates that genetic exchange can occur between paralogous genes at widely separated chromosomal locations. Estimation of nucleotide substitution rates in these genes confirmed a higher substitution rate in the pyruvate dehydrogenase genes. In the phosphoglycerate kinase genes, there is no difference between the substitution rates in mice and humans and between the X chromosome- and autosome-located genes. A greater substitution rate was noted in the mouse autosomal pyruvate dehydrogenase gene when compared with the other mouse and human genes. This may be a result of either directional natural selection or a relaxation of functional constraint at this specific gene.