Membrane diffusion- and capillary blood volume measurements are not useful as screening tools for pulmonary arterial hypertension in systemic sclerosis: a case control study

Background

Lung injury caused by both inhaled dusts and infectious agents depends on increased availability of iron and metal-catalyzed oxidative stress. Because inhaled particles, such as silica, and certain infections can cause secondary pulmonary alveolar proteinosis (PAP), we tested the hypothesis that idiopathic PAP is associated with an altered iron homeostasis in the human lung.

Methods

Healthy volunteers (n = 20) and patients with idiopathic PAP (n = 20) underwent bronchoalveolar lavage and measurements were made of total protein, iron, tranferrin, transferrin receptor, lactoferrin, and ferritin. Histochemical staining for iron and ferritin was done in the cell pellets from control subjects and PAP patients, and in lung specimens of patients without cardiopulmonary disease and with PAP. Lavage concentrations of urate, glutathione, and ascorbate were also measured as indices of oxidative stress.

Results

Lavage concentrations of iron, transferrin, transferrin receptor, lactoferrin, and ferritin were significantly elevated in PAP patients relative to healthy volunteers. The cells of PAP patients had accumulated significant iron and ferritin, as well as considerable amounts of extracellular ferritin. Immunohistochemistry for ferritin in lung tissue revealed comparable amounts of this metal-storage protein in the lower respiratory tract of PAP patients both intracellularly and extracellularly. Lavage concentrations of ascorbate, glutathione, and urate were significantly lower in the lavage fluid of the PAP patients.

Conclusion

Iron homeostasis is altered in the lungs of patients with idiopathic PAP, as large amounts of catalytically-active iron and low molecular weight anti-oxidant depletion are present. These findings suggest a metal-catalyzed oxidative stress in the maintenance of this disease.     

Allosteric regulation of BK channel gating by Ca(2+) and Mg(2+) through a nonselective, low affinity divalent cation site.

The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K(+) channels is enhanced by cytosolic calcium (Ca(2+)). Activation is sensitive to a range of [Ca(2+)] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 alpha subunits at [Ca(2+)] and [Mg(2+)] up to 100 mM. The half-activation voltage (V(0.5)) is steeply dependent on [Ca(2+)] in the micromolar range, but shows a tendency towards saturation over the range of 60-300 microM Ca(2+). As [Ca(2+)] is increased to millimolar levels, the V(0.5) is strongly shifted again to more negative potentials. When channels are activated by 300 microM Ca(2+), further addition of either mM Ca(2+) or mM Mg(2+) produces similar negative shifts in steady-state activation. Millimolar Mg(2+) also produces shifts of similar magnitude in the complete absence of Ca(2+). The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in [Ca(2+)] increase activation rates, addition of 10 mM Mg(2+) to 0 Ca(2+) produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg(2+) does not participate in Ca(2+)-dependent steps that influence current activation rate. We conclude that millimolar Mg(2+) and Ca(2+) concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca(2+)-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca(2+) binding steps, four voltage sensors, and four independent lower affinity Ca(2+)/Mg(2+) binding steps describes well the behavior of G-V curves over a range of Ca(2+) and Mg(2+). The ability of a broad range of [Ca(2+)] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.     

The mitotic checkpoint protein hBUB3 and the mRNA export factor hRAE1 interact with GLE2p-binding sequence (GLEBS)-containing proteins

The mRNA export factor RAE1 (also called GLE2) and the mitotic checkpoint protein BUB3 share extensive sequence homology in yeast as well as higher eukaryotes, although the biological relevance of their similarity is unclear. Previous work in HeLa cells has shown that human (h)RAE1 binds the nuclear pore complex protein hNUP98 via a short NUP98 motif called GLEBS (for GLE2p-binding sequence). Here we report that the two known binding partners of hBUB3, the mitotic checkpoint proteins hBUB1 and hBUBR1, both carry a region with remarkable similarity to the GLEBS motif of hNUP98. We show that the GLEBS-like motifs of mouse (m)BUB1 and mBUBR1 are sufficient for mBUB3 binding. mBUB3 lacks affinity for the hNUP98 GLEBS, demonstrating its binding specificity for GLEBS motifs of mitotic checkpoint proteins. Interestingly, mRAE1 does not exclusively bind to the GLEBS motif of hNUP98 and can cross-interact with the mBUB1 GLEBS. We show that full-length RAE1 and BUB1 proteins interact in mammalian cells and accumulate both at the kinetochores of prometaphase chromosomes. Our findings demonstrate that GLEBS motifs reside in mammalian nucleoporins and mitotic checkpoint proteins and apparently serve as specific binding sites for either BUB3, RAE1, or both.     

Phosphorylation of centrin during the cell cycle and its role in centriole separation preceding centrosome duplication

Once during each cell cycle, mitotic spindle poles arise by separation of newly duplicated centrosomes. We report here the involvement of phosphorylation of the centrosomal protein centrin in this process. We show that centrin is phosphorylated at serine residue 170 during the G(2)/M phase of the cell cycle. Indirect immunofluorescence staining of HeLa cells using a phosphocentrin-specific antibody reveals intense labeling of mitotic spindle poles during prophase and metaphase of the cell division cycle, with diminished staining of anaphase and no staining of telophase and interphase centrosomes. Cultured cells undergo a dramatic increase in centrin phosphorylation following the experimental elevation of PKA activity, suggesting that this kinase can phosphorylate centrin in vivo. Surprisingly, elevated PKA activity also resulted intense phosphocentrin antibody labeling of interphase centrosomes and in the concurrent movement of individual centrioles apart from one another. Taken together, these results suggest that centrin phosphorylation signals the separation of centrosomes at prophase and implicates centrin phosphorylation in centriole separation that normally precedes centrosome duplication.     

Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.     

New Pedal Remains of Megaladapis and Their Functional Significance. Am J Phys Anthropol 100:115–139

Erratum: Wunderlich RE, Simons EL, Jungers WL. 1996. New Pedal Remains of Megaladapis and Their Functional Significance. Am J Phys Anthropol 100:115–139.     

Microtubule nucleating capacity of centrosomes in tissue sections.

We used a novel adaptation of methods for microtubule polymerization in vitro to assess the MTOC activity of centrosomes in frozen-sectioned tissues. Remarkably, centrosomes of tissue sections retain the ability to nucleate microtubules even after several years of storage as frozen tissue blocks. Adaptations of these methods allow accurate counts of microtubules from individual cells and the quantitative estimation the MTOC activity of the intact tissue. These methods can be utilized to characterize MTOC activity in normal and diseased tissues and in particular tissues at different stages of development. (J Histochem Cytochem 47:1265-1273, 1999)     

Visualization of bioluminescence as a marker of gene expression in rhizobium-infected soybean root nodules

The linked structural genes lux A and lux B, encoding bacterial luciferase of a marine bacterium Vibrio harveyi, were fused with the nitrogenase nifD promoter from Bradyrhizobium japonicum and with the P1 promoter of pBR322. Both fusions were integrated into the B. japonicum chromosome by site-specific recombination. Soybean roots infected with the two types of rhizobium transconjugants formed nitrogen-fixing nodules that produced bright blue-green light. Cells containing the P1 promoter/lux AB fusion resulted in continuously expressed bioluminescence in both free-living rhizobium and in nodule bacteriods. However, when under control of the nifD promoter, luciferase activity was found only in introgen-fixing nodules. Light emission from bacteroids allowed us to visualize and to photograph nodules expressing this marker gene fusion in vivo at various levels of resolution, including within single, living plant cells. Localization of host cells containing nitrogen-fixing bacteroids within nodule tissue was accomplished using low-light video microscopy aided by realtime image processing techniques developed specifically to enhance extreme low-level luminescent images.     

Control of centrin stability by Aurora A

Aurora A is an oncogenic serine/threonine kinase which can cause cell transformation and centrosome amplification when over-expressed. Human breast tumors show excess Aurora A and phospho-centrin in amplified centrosomes. Here, we show that Aurora A mediates the phosphorylation of and localizes with centrin at the centrosome, with both proteins reaching maximum abundance from prophase through metaphase, followed by their precipitous loss in late stages of mitosis. Over-expression of Aurora A results in excess phospho-centrin and centrosome amplification. In contrast, centrosome amplification is not seen in cells over-expressing Aurora A in the presence of a recombinant centrin mutant lacking the serine phosphorylation site at residue 170. Expression of a kinase dead Aurora A results in a decrease in mitotic index and abrogation of centrin phosphorylation. Finally, a recombinant centrin mutation that mimics centrin phosphorylation increases centrin's stability against APC/C-mediated proteasomal degradation. Taken together, these results suggest that the stability of centrin is regulated in part by Aurora A, and that excess phosphorylated centrin may promote centrosome amplification in cancer.