Interrelationship of renal vascular development and nephrogenesis

Within the cortex region of the neonatal rabbit kidney the developing microvasculature was investigated by means of two endothelium-detecting antibodies (EnPo 1 and EC1). Rows of antibody-labelled cells were found within tissue regions that had previously been described as avascular. We conclude that these vessel-like structures detected by EnPo 1 and EC1 are capillary precursors without lumina. Furthermore, beneath the fibrous capsule within the morphologically homogeneous mesenchyme two cell populations can be discriminated by use of differential antigen expression. The EnPo 1 antigen, which is abundant on endothelial cells and podocytes at different developmental stages, was detected on a subpopulation of mesenchymal cells. These cells were exclusively detected surrounding the tip of the collecting duct ampulla. Due to the unique specificity of EC1 and EnPo 1 the process of microvascular development can be readily followed on serial optical sections gained by laser scan microscopy. (1) Adjacent to EnPo 1-positive mesenchymal cell islets vessel-like structures are found that are in contact with the differentiated vasculature. (2) The renal vesicle is enclosed by a network of vessel-like structures establishing contact with differentiated vessels. (3) No guidance of invading capillary sprouts toward the developing glomerulus and nephron is required, since vascular elements already accompany the earliest detectable nephron stage.     

Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.

A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.     

Two-locus disease models with two marker loci: the power of affected-sib-pair tests.

Recently, Schork et al. found that two-trait-locus, two-marker-locus (parametric) linkage analysis can provide substantially more linkage information than can standard one-trait-locus, one-marker-locus methods. However, because of the increased burden of computation, Schork et al. do not expect that their approach will be applied in an initial genome scan. Further, the specification of a suitable two-locus segregation model can be crucial. Affected-sibpair tests are computationally simple and do not require an explicit specification of the disease model. In the past, however, these tests mainly have been applied to data with a single marker locus. Here, we consider sib-pair tests that make it possible to analyze simultaneously two marker loci. The power of these tests is investigated for different (epistatic and heterogeneous) two-trait-locus models, each trait locus being linked to one of the marker loci. We compare these tests both with the test that is optimal for a certain model and with the strategy that analyzes each marker locus separately. The results indicate that a straightforward extension of the well-known mean test for two marker loci can be much more powerful than single-marker-locus analysis and that is power is only slightly inferior to the power of the optimal test.     

Quantitative Resistance to Phytophthora Infestans in Potato: A Case Study for Qtl Mapping in an Allogamous Plant Species

Phytophthora infestans is the most important fungal pathogen in the cultivated potato (Solanum tuberosum). Dominant, race-specific resistance alleles and quantitative resistance-the latter being more important for potato breeding- are found in the germplasm of cultivated and wild potato species. Quantitative trait loci (QTLs) for resistance to two races of P. infestans have been mapped in an F(1) progeny of a cross between non-inbred diploid potato parents with multiple alleles. Interval mapping methods based on highly informative restriction fragment length polymorphism markers revealed 11 chromosome segments on 9 potato chromosomes showing significant contrasts between marker genotypic classes. Whereas phenotypically no difference in quantitative resistance response was observed between the two fungal races, QTL mapping identified at least one race specific QT locus. Two QT regions coincided with two small segments on chromosomes V and XII to which the dominant alleles R1, conferring race specific resistance to P. infestans, Rx1 and Rx2, both inducing extreme resistance to potato virus X, have been allocated in independent mapping experiments. Some minor QTLs were correlated with genetic loci for specific proteins related to pathogenesis, the expression of which is induced after infection with P. infestans.     

Ascorbic Acid Inhibits 125I-SCH 23982 Binding but Increases the Affinity of Dopamine for D1 Dopamine Receptors

Abstract: Effects of ascorbic acid (AA) on ¹²⁵I-SCH 23982 binding to D₁ dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µ M –0.33 m M inhibited 75% of specific binding of ¹²⁵I-SCH 23982 in a dose-dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 m M AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 m M AA, but reduced the inhibition by 3.3 m M AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 m M EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high-affinity ( K _h = 224.9 ± 48.9 n M ) and low-affinity ( K _l = 21,100 ± 2,400 n M ) binding sites. Although the maximum binding of ¹²⁵I-SCH 23982 decreased to 40% without affecting the K _D value in the presence of 1.67 m M AA, the value of the high-affinity site for dopamine was increased ( K _h = 23.3 ± 9.4 n M ) and that of the low-affinity site was decreased ( K _l = 136,800 ± 40,900 n M ). These results suggest that AA may affect D₁ dopamine receptor function by lipid peroxidation, competition with dopamine for low-affinity sites, and reduced oxidation of dopamine.     

Temperature Sensitivity of Agonist High-Affinity Binding Sites of Solubilized and Reconstituted D1 Dopamine Receptors

Abstract: Solubilization of rat striatal membranes with sodium cholate, followed by reconstitution into phospholipid vesicles, leads to a 6.5-fold increase in the agonist high-affinity binding sites of the D₁ dopamine receptor. These high-affinity binding sites display differential sensitivity toward temperature. When reconstituted receptors were preincubated for 1 h at 0–4°C (on ice) or at 22°C (room temperature) followed by radioligand binding assays with dopamine, neither the high-affinity values of the receptor for dopamine nor the percent receptors in the high-affinity state (31–39%) were changed from control reconstituted receptors, which were not subject to any preincubations. At 30°C, there was a partial loss in the number of high-affinity D₁ receptors with only 25% of the total receptor population in the high-affinity state; there was no change in the affinity values of the high-affinity binding sites. At 37°C, there was a 40% loss in total number of D₁ receptor binding sites. All the high-affinity binding sites were lost and the remaining 60% of binding activity represented the low-affinity binding state of the receptor. These results indicate that the high-affinity binding sites of the reconstituted D₁ dopamine receptors are uniquely sensitive to higher temperatures.     

The biodegradation of piperazine and structurally-related linear and cyclic amines

The biodegradability of a range of linear and cyclic amines was assessed. All proved to be biodegradable but there were interesting differences in their susceptibility. The least degradable was piperazine although piperazine-degrading microorganisms were of widespread occurrence in samples of water and activated sludge and, to a lesser extent, soils. Piperazine degraders are only present in very small numbers — on averageca. 0.8/ml of river water. Of six isolates capable of using piperazine as a sole source of carbon, nitrogen and energy in pure culture five were identified asMycobacterium spp. and one asArthrobacter sp., all strains were capable only of slow growth (mean generation time ofca. 30 to 40 hours) on this substrate. Piperidine, pyrrolidine, ethanolamine and diethanolamine were all readily biodegradable. The relationship between structure and degradability of amines is discussed as are the possible reasons for the relative recalcitrance of piperazine.     

Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation

Summary The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 × 10^–7) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 × 10^–7). No colonies were seen on control plates (frequency < 0.96 × 10^–9). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.     

Biology of the bamboo Chusquea culeou (Poaceae: Bambusoideae) in southern Argentina

Over a period of 7 years the biology and phenotypic variability of Chusquea culeou were studied at 5 locations in cool temperate forests of southern Argentina. Excavated rhizomes had an average of 1.1 successful rhizome buds, and an average of 2.1 years elapsed between successive generations of rhizomes. Rhizome buds usually develop within the first four years after a rhizome forms. Height, volume and weight of a culm can be calculated from its diameter 1 m above the ground. Culm size, length of foliage leaf blades, and pattern of secondary branching differed among study sites. Dead culms were numerous and commonly remained erect for more than 7 years after dying. New culm shoots appear in spring and reach full size within a few months. Shoots can grow more than 9 cm/day. Less than half of the shoots survived a year; most were killed by moth larvae. Multiple primary branch buds emerge through the culm leaf sheaths in the second spring. The mean number of branch buds at mid-culm nodes varied between 34.8 and 81.5, and the mean number of primary branches was between 22.8 and 40.8. Number and length of branches, and number and length of foliage leaf blades at each node is related to the position of the node on a culm. Most branches grow about 3 cm and produce 1 to 3 foliage leaves annually. Foliage leaf blades generally live 2 years or more; few survive 6 years. Relative lengths of foliage leaf blades and their spacing along a branch permit recognition of annual cohorts.Both gregarious and sporadic flowering have been reported, and every year a few isolated plants flower and die. Length of the life cycle is unknown. Seedlings require up to 15 years to produce culms of mature size. Foliage branches may live more than 23 years, and culms may survive 33 years. Extensive loss of new shoots to predation suggests that gregarious flowering may be driven by a need to escape parasitism. C. culeou clumps expand slowly. Average annual rate of increase of the number of live culms in a clump was 4.6%. Methods of seed dispersal are undocumented. A dense stand of Chusquea culeou had an estimated phytomass of 179 tons/hectare (dry weight), 28% of which was underground. Net annual production was about 16 t/ha dry weight.