Differences of Ca2+ regulation in skin fibroblasts from blacks and whites

Black people have a higher propensity than caucasians toward essential hypertension. To explore the possibility that this racial difference relates to cellular Ca2+ metabolism, we measured 45Ca2+ washout and uptake and cytosolic free concentration of Ca2+ [Ca2+]i in serially passed skin fibroblasts from normotensive black and white males. Depending on the experimental conditions, 45Ca2+ washout in these cells was described by either two or three exponential functions, whereas 45Ca2+ uptake was described only by a two-exponent function. There were no racial differences in 45Ca2+ uptake and washout of unstimulated fibroblasts. However, stimulation by human serum resulted in an increase in the 45Ca2+ washout that was higher in fibroblasts from blacks than from whites. The racial differences were expressed primarily by higher values of the apparent washout rate constant (k1) of 45Ca2+ from the largest and most rapidly exchangeable cellular pool. The effect of human serum was not related to its origin (blacks vs. whites). In 2 mM Ca2+ medium and 10% serum from blacks, the respective k1 (mean +/- SEM; x 10(-2)/min) values for fibroblasts from blacks and whites were 89.68 +/- 5.23 and 73.29 +/- 4.0; in the presence of 10% serum from whites, the k1 values for cells from blacks and whites were 84.14 +/- 2.80 and 76.36 +/- 3.23 (overall significance of P less than .01). In Ca2+-deficient medium in the presence of 10% human serum, the k1 for fibroblasts from blacks and whites were 115.57 +/- 3.76 and 102.15 +/- 3.30 (P less than .05). Serum substantially increased the 45Ca2+ uptake in fibroblasts from both blacks and whites; however, racial differences were not observed. Basal levels of [Ca2+]i were not different in fibroblasts of blacks vs. whites (46.8 +/- 6.8 and 43.2 +/- 7.1 nM for blacks and whites, respectively). However, the peak response of Cai2+ transients for cell stimulated by 5% human serum was significantly higher in blacks than whites (blacks = 963 +/- 213, whites = 481 +/- 162 nM; P = .0286). We conclude that Ca2+ regulation is different in serum-stimulated fibroblasts from blacks and whites and that, at least in part, this difference may relate to a greater agonist-induced mobilization of Ca2+ in fibroblasts from blacks.     

Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium

A chimaeric gene has been constructed that expresses -D-glucuronidase (GUS) in transformed plant tissues, but not in bacterial cells. This gene has proved extremely useful for monitoring transformation during the period immediately following gene transfer from Agrobacterium tumefaciens. GUS expression was detectable 2 days after inoculation, peaked at 3–4 days and then declined; if selection was imposed expression increased again after 10–14 days. The extent of transient expression after 4 days correlated well with stable integration as measured by kanamycin resistance, hormone independence, and gall formation. Histochemical staining of inoculated leaf discs confirmed the transient peak of GUS expression 3–4 days after inoculation. The most surprising result was that the blue staining was concentrated in localized zones on the circumference of the disc; within these zones, essentially all the cells appeared to be expressing GUS. We suggest that the frequency of gene transfer from Agrobacterium is extremely high within localized regions of leaf explants, but that the frequency of stable integration is several orders of magnitude lower.     

Genetic Analysis of the Bacteriophage λ Attl Nucleoprotein Complex

Site-specific recombination in bacteriophage λ involves interactions among proteins required for integration and excision of DNA molecules. We have analyzed the elements required to form an in vivo nucleoprotein complex of integrase (Int) and integration host factor (IHF). Interaction of Int with the core (the site of strand exchange) is stabilized by the flanking arm region of attL. IHF, in addition to Int, is required for efficient Int-core binding. We used the in vivo attL binding assay to characterize several Int variants for their abilities to form stable attL complexes. Substitution of Int active site tyrosine 342 by phenylalanine had no effect on the ability of the protein to form attL complexes. Three other amino acids that are completely conserved in the integrase family of recombinases (arginine 212, histidine 308, and arginine 311) were separately substituted by glutamine, leucine, and histidine, respectively. In each case, the mutant protein was altered in its ability to form attL complexes while retaining its ability to bind to the λ arm-type sites. We propose that, in addition to their role in catalysis, this triad of amino acids helps the Int protein to interact with the λ core sites.     

Phytochrome control of short-day-induced bud set in black cottonwood

In trees and other woody perennial plants, short days (SDs) typically induce growth cessation, the initiation of cold acclimation, the formation of a terminal bud and bud dormancy. Phytochrome control of SD-induced bud set was investigated in two northern clones of black cottonwood (Populus trichocarpa Torr. & Gray) by using night breaks with red light (R) and far-red light (FR). For both clones (BC-1 and BC-2), SD-induced bud set was prevented when R night breaks as short as 2 min were given in the middle of the night. When night breaks with 2 min of R were immediately followed by 2 min of FR, substantial reversibility of bud set was observed for BC-1 but not for BC-2. By comparing the effects of the R night breaks on bud set and the length of specific internodes, we determined that the R night breaks influenced internode elongation in two opposing ways. First, the addition of a R night break to the SD treatment prevented the cessation of internode elongation that is associated with bud set. Those internodes that would not have elongated under SDs (and would have been found within the terminal bud) elongated in the R treatment. Second, the R night breaks decreased internode length relative to the long-day (LD) control. In contrast to the clonal differences in reversibility that we observed for bud set, the decrease in internode length (i.e. the second effect of R) was R/FR reversible in both clones. Based on these results, we conclude that internode elongation is influenced by two distinct types of phytochrome-mediated response. The first response is a typical response to photpperiod, whereas the second response is a typical “end-of-day” response to light quality. Our results demonstrate that SD-induced bud set in black cottonwood is controlled by phytochrome but that clonal differences have an important influence on the R/FR reversibility of this response. The availability of an experimental system in which SD-induced bud set is R/FR reversible will be valuable for studying the physiological genetics of photoperiodism in trees.     

The status and ecology of a Juniperus excelsa subsp. polycarpos woodland in the northern mountains of Oman

Juniperus excelsa subsp. polycarpos (K. Koch) Takhtajan is found in mountain areas from Turkey through to India and as an isolated population on Jebel Akhadar in the northern mountains of Oman. Juniperus is one of the dominant plant species in these mountains and a major landscape feature of several proposed National Nature and Scenic Reserves and of Hayl Juwari, a wooded valley at 2250 m altitude proposed as a Botanical Site of Special Interest. Above 2400 m altitude the Juniperus woodlands generally appear to be regenerating and in good condition, both on exposed slopes and in wadis and sheltered gullies, whereas below 2400 m most stands are in poor condition and exhibit few signs of regeneration. If the apparently poor condition of the lower altitude woodlands is due to any long term change in climatic conditions, both tree status and regeneration would be poorer in relatively more xeric habitats. To test this prediction we have carried out a detailed survey of the status and ecology of a 32 ha area of Hayl Juwari, and analysed differences in tree status and regeneration between wadis (relatively more nesic sites) and non-wadi areas (relatively more xeric). Approximately one third of the trees are dead, and an analysis of the height, condition, regeneration, female cone production, preferred germination sites and spatial distribution of trees indicates the importance of topography, hydrology and microclimate for growth. However, although there are relatively greater numbers of dead and poor-condition trees in the more xeric non-wadi habitat, there is no unequivocal evidence that the present distribution of small, sexually immature trees in both habitats could not form a pattern of larger, sexually mature trees similar to that seen today. We speculate, however, that the climate at this altitude may be marginal for the survival of a J. excelsa subsp. polycarpos woodland and that even small increases in climatic stress could imperil the woodland's present status.     

B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP.

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.     

Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins,osteocalcin, and matrix Gla protein in vitro

The role of the vitamin K dependent proteins, osteocalcin which is bone specific and matrix Gla protein (MGP) found in many tissues, has been studied by inhibition of synthesis of their characteristic amino acid, γ-carboxyglutamic acid (Gla) with the anticoagulant sodium warfarin. The effect of sodium warfarin on expression of these proteins, and other phenotypic markers of bone and cartilage during cellular differentiation and development of tissue extracellular matrix, was examined in several model systems. Parameters assayed include cell growth (reflected by histone gene expression) and collagen types I and II, osteopontin, alkaline phosphatase, and mineralization. Studies were carried out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 and 25/1. In normal diploid cells, warfarin consistently stimulated cell proliferation (twofold). In osteoblast cultures, MGP mRNA levels were generally increased (three to tenfold). Notably, MGP mRNA levels were not affected in chondrocyte cultures, either with chronic or acute warfarin treatments. Osteocalcin mRNA levels and synthesis were decreased up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 μg/ml sodium warfarin) rat osteoblast cultures after 22 days. Early stages of osteoblast phenotype development from the proliferation period to initial tissue formation (nodules) appeared unaffected; while after day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin-treated cultures. In summary, warfarin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regulated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on endoplasmic reticulum associated enzymes. © 1994 Wiley-Liss, Inc.     

The evolutionary origin of the 35 kb circular DNA of Plasmodium falciparum: new evidence supports a possible rhodophyte ancestry

In common with other Apicomplexan parasites, Plasmodium falciparum carries two extrachromosomal DNAs, one of which, the 6 kb element, is undoubtedly mitochondrial. The second, generally referred to as the 35 kb circle, is of unknown provenance, but the nature and organization of its genetic content makes a mitochondrial association unlikely and the molecule has features reminiscent of plastid genomes. We now report the occurrence on the circle of an open reading frame specifying a predicted 470 amino acid protein that shares more than 50% identity with a gene currently known only on the plastome of red algae. This high degree of conservation confirms the 35 kb circle's plastid ancestry, and we speculate that it may have originated from the rhodoplast of an ancient red algal endosymbiont in the progenitor of the Apicomplexa.