Endothelium-specific sepiapterin reductase deficiency in DOCA-salt hypertension

The endothelial nitric oxide synthase (eNOS) requires tetrahydrobiopterin (H(4)B) as a cofactor and, in its absence, produces superoxide (O(2)(·-)) rather than nitric oxide (NO(·)), a condition referred to as eNOS uncoupling. DOCA-salt-induced hypertension is associated with H(4)B oxidation and uncoupling of eNOS. The present study investigated whether administration of sepiapterin or H(4)B recouples eNOS in DOCA-salt hypertension. Bioavailable NO(·) detected by electron spin resonance was markedly reduced in aortas of DOCA-salt hypertensive mice. Preincubation with sepiapterin (10 μmol/l for 30 min) failed to improve NO(·) bioavailability in hypertensive aortas while it augmented NO(·) production from control vessels, implicating a hypertension-associated deficiency in sepiapterin reductase (SPR), the rate-limiting enzyme for sepiapterin conversion to H(4)B. Indeed, a decreased SPR expression was observed in aortic endothelial cells, but not in endothelium-denuded aortic remains, implicating an endothelium-specific SPR deficiency. Administration of hypertensive aortas with H(4)B (10 μmol/l, 30 min) partially restored vascular NO(·) production. Combined administration of H(4)B and the NADPH oxidase inhibitor apocynin (100 μmol/l, 30 min) fully restored NO(·) bioavailability while reducing O(2)(·-) production. In angiotensin II-induced hypertension, however, aortic endothelial SPR expression was not affected. In summary, administration of sepiapterin is not effective in recoupling eNOS in DOCA-salt hypertension, due to an endothelium-specific loss in SPR, whereas coadministration of H(4)B and apocynin is highly efficient in recoupling eNOS. This is consistent with our previous observations that in angiotensin II hypertension, endothelial deficiency in dihydrofolate reductase is alternatively responsible for uncoupling of eNOS. Taken together, these data indicate that strategies specifically targeting at different H(4)B metabolic enzymes might be necessary in restoring eNOS function in different types of hypertension.     

Glutathione and p53 independently mediate responses against oxidative stress in ES cells.

We have investigated the roles of the antioxidant glutathione and p53 in the response of embryonic stem (ES) cells to oxidative stress. ES cells express gammaGCS, a critical enzyme in glutathione (GSH) biosynthesis. Treatment with the pro-oxidant menadione led to elevation of GSH, a strong apoptotic response and reduced clonogenic survival. Addition of BSO, a specific gammaGCS inhibitor depleted GSH pools and prevented the menadione-induced increase in GSH, sensitizing cells to oxidative insult. Although p53 status had no bearing on either the basal levels of GSH or the menadione-induced GSH response, the levels of menadione-induced apoptosis were reduced in the absence of p53. We conclude that the pathways involving p53 and GSH act independently to protect against the deleterious effects of oxidative damage. Furthermore, the presence of an intact p53 pathway confers a long-term growth advantage post oxidative stress. Thus, in the absence of p53 ES cells bearing genotoxic damage are less likely to be propagated, suggesting that p53-dependent apoptosis acts to limit the deleterious effects of oxidative stress during early development.     

Aging and glucose homeostasis in C57BL/6J male mice

Age-dependent changes in glucose homeostasis were assessed in specific pathogen-free C57BL/6J male mice. Increased islet size and pancreatic insulin content in old (21-25-month-old) mice were associated with lower nonfasting plasma glucose levels and improved clearance of either an oral or an i.p. administered glucose load in comparison with young, mature (4-5-month-old) males. The almost twofold increase in islet size correlated with a twofold increase of glucose-stimulated insulin secretion from perifused islets from 25-month-old males compared with 5-month-old males. These aging male mice did not become obese, and there were no fibrotic changes associated with the hyperplastic islets observed in the old males. Thus, the findings that glucose tolerance did not deteriorate with age, coupled with the lack of evidence for impaired beta cell responsiveness to glucose in old males, suggest that deterioration in glucose homeostasis is not an inevitable consequence of aging in the mouse.     

Natural course of penicillamine nephropathy: a long term study of 33 patients

To elucidate the natural course of the nephropathy associated with penicillamine and thereby facilitate its clinical management 33 patients with rheumatoid arthritis who developed proteinuria during treatment with oral penicillamine were studied in detail throughout their renal illness. Renal biopsies were performed, and creatinine clearance and proteinuria were measured serially for 74 months (range 16-148 months). Fourteen patients developed proteinuria within six months after the start of treatment and 27 within 12 months. When treatment was stopped the proteinuria reached a median peak of 4·2 g/24 h (range 0·3-15·0 g/24 h) at one month (range 0-7 months) before resolving spontaneously by six months (12 patients), 12 months (21), or 18 months (29). In all patients but one, who developed carcinoma of the renal pelvis, proteinuria resolved by 21 months and its median duration was eight months. The median first and last measurements of creatinine clearance showed no appreciable change (80 ml/min and 78 ml/min), and no patient died from or needed treatment for renal failure. The HLA-B8 or HLA-DR3 alloantigen, or both, were identified in 10 patients. Renal biopsy specimens showed membranous glomerulonephritis in 29 patients, minimal change nephropathy in two, and electron dense deposits in the mesangial regions in two.In all the patients whose nephropathy was due solely to treatment with penicillamine the proteinuria resolved completely when the drug was withdrawn; renal function did not deteriorate, and corticosteroids were unnecessary.     

Environmental and Seasonal Factors Affecting the Frost-induced Stage of Cold Acclimation in Cornus stolonifera Michx

Stem tissues of red-osier dogwood (Cornus stolonifera Michx.) acclimated from −3 C to −40 or −50 C in 8 to 10 weeks under a short photoperiod (9 hours) and controlled temperature conditions. During the summer months plants did not acclimate as well as at other times. The sequence of day/night temperature regimes which induced maximum acclimation was 20/15 C for 5 to 6 weeks; 15/5 C for 2 to 3 weeks; 15/5 C plus 1 hour of frost per day for 1 week. The duration of exposure to each temperature regime influenced the rate and intensity of frost-induced acclimation. Less than 5 weeks of warm temperature preconditioning at 20/15 C reduced subsequent frost-induced acclimation. The inductive influence of frost on cold acclimation was additive over 5 days of repeated exposure, but its effects after the first exposure(s) were not immediate—requiring 1 to 4 days of 15/5 C following the frost treatments for the expression of the frost-induced acclimation to be manifest. There was a 75% increase in rRNA following 3 days of frost exposure and plants in an O₂-free atmosphere during frost exposure failed to acclimate. The results suggest that seasonal acclimation behavior was due to endogenous rhythms rather than developmental stage, and that the frost-induced phase of acclimation involves aerobic metabolic processes.     

Genetic and structural analysis of a virulence determinant in polyomavirus VP1.

The LID strain of polyomavirus differs from other laboratory strains in causing a rapidly lethal infection of newborn C3H/Bi mice. This virulent behavior of LID was attenuated by dilution, yet at sublethal doses LID was able to induce tumors at a high frequency, like its parent virus PTA. By constructing and assaying LID-PTA recombinant viruses and by DNA sequencing, the determinant of virulence in LID was mapped to the major viral capsid protein, VP1. The VP1s of LID and PTA differed at two positions: at 185, LID has phenylalanine and PTA has tyrosine, and at 296, LID has alanine and PTA has valine. Results obtained with viruses constructed by site-directed mutagenesis showed that alanine at position 296 is sufficient to confer a fully virulent phenotype regardless of which amino acid is at position 185. However, with valine at position 296, an effect of phenylalanine at position 185 is apparent, as this virus possesses an intermediate level of virulence. A crystal structure of polyomavirus complexed with 3'-sialyl lactose previously indicated van der Waals contacts between the side chain of valine 296 and the sialic acid ring (T. Stehle, Y. Yan, T. L. Benjamin, and S. C. Harrison, Nature [London] 369:160-163, 1994). When this interaction was modeled with alanine, these contacts were greatly reduced. Direct confirmation that the substitutions in VP1 affected receptor binding was obtained by studying virus hemagglutination behavior. The ensemble of results are discussed in terms of the idea that a lower affinity of the virus for its receptor can result in more rapid spread and increased pathogenicity.     

Elaborations of the basal surface of the cells of the Malpighian tubules of an insect

Electrical measurements of membrane potential and resistance using intracellular microelectrodes showed that the fluid-secreting parts of the Malpighian tubules of Rhodnius have superficial regions different in electrical properties from the main body of the cell. The membrane potential in these superficial regions was smaller by 30-40 mV, but showed a standard depolarization on changing the potassium concentration of the bathing medium. The response to changes in the external chloride concentration also differed in the two regions, a finding that was reinforced by different responses to the drug, furosemide. Electron microscopy of the basal regions of the cells revealed many long cellular projections that run parallel to the cell surface and interdigitate with similar projections from neighbouring cells. The degree of interdigitation was examined by marking individual cells with alcian blue or by horseradish peroxidase injection. A survey of the published micrographs of insect Malpighian tubules shows that most have similar projections on their basal surfaces and not the simple basal infoldings previously supposed.     

Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity.

A family of peptide antimycotics, termed pseudomycins, has been isolated from liquid cultures of Pseudomonas syringae, a plant-associated bacterium. These compounds were purified using Amberlite XAD-2 and reverse-phase liquid chromatography. Pseudomycin A, the predominant peptide in a family of four, showed selective phytotoxicity, and had impressive activity against the human pathogen Candida albicans. Amino acid, mass spectroscopic, and comparative electrophoretic and chromatographic analyses revealed that the pseudomycins are different from previously described antimycotics from P. syringae, including syringomycin, syringotoxin and syringostatins. Pseudomycins A-C contain hydroxyaspartic acid, aspartic acid, serine, arginine, lysine and diaminobutyric acid. The molecular masses of pseudomycins A-C, as determined by plasma desorption mass spectrometry, are 1224, 1208 and 1252 Da, respectively. Pseudomycin D, on the other hand, has a molecular mass of 2401 Da and is more complex than pseudomycins A-C.     

Classically activated macrophages use stable microtubules for matrix metalloproteinase-9 (MMP-9) secretion

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages up-regulate MMP-9 expression and secretion upon immunological challenge and require its activity for migration during the inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution as well as the mechanisms responsible for its trafficking are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and protein-disulfide isomerase in activated RAW 264.7 macrophages. We demonstrated vesicular organelles of MMP-9 aligned along stable subsets of MTs and showed that selective modulation of MT dynamics contributes to the enhanced trafficking of MMP-9 extracellularly. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. Finally, we implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly.     

The mitogen-activated protein kinase kinase kinase kinase GCKR positively regulates canonical and noncanonical Wnt signaling in B lymphocytes

Wnt ligands bind receptors of the Frizzled (Fz) family to control cell fate, proliferation, and polarity. Canonical Wnt/Fz signaling stabilizes beta-catenin by inactivating GSK3beta, leading to the translocation of beta-catenin to the nucleus and the activation of Wnt target genes. Noncanonical Wnt/Fz signaling activates RhoA and Rac, and the latter triggers the activation of c-Jun N-terminal kinase (JNK). Here, we show that exposure of B-lymphocytes to Wnt3a-conditioned media activates JNK and raises cytosolic beta-catenin levels. Both the Rac guanine nucleotide exchange factor Asef and the mitogen-activated protein kinase kinase kinase kinase germinal center kinase-related enzyme (GCKR) are required for Wnt-mediated JNK activation in B cells. In addition, we show that GCKR positively affects the beta-catenin pathway in B cells. Reduction of GCKR expression inhibits Wnt3a-induced phosphorylation of GSK3beta at serine 9 and decreases the accumulation of cytosolic beta-catenin. Furthermore, Wnt signaling induces an interaction between GCKR and GSK3beta. Our findings demonstrate that GCKR facilitates both canonical and noncanonical Wnt signaling in B lymphocytes.