Dietary sodium,glomerular filtration rate autoregulation,and glomerular size distribution profiles in domestic fowl (Gallus gallus)

Summary Mammalian glomerular filtration rate (GFR) autoregulation can be impaired by protocols that inhibit tubuloglomrular feedback, such as high sodium intake. Domestic fowl were fed diets containing either high sodium (0.39% Na: High-Na Group) or low sodium (0.03% Na: Low-Na Group). An arterial snare was used to reduce renal arterial perfusion pressure (RAPP) in a stepwise fashion to evaluate GFR autoregulation. Absolute sodium excretion, fractional sodium excretion (FE_Na), and ambient systemic arterial blood pressure were significantly elevated in the High-Na Group when compared with the Low-Na Group, and pressure natriuresis was abolished by the Low-Na diet. However, GFR autoregulatory profiles were identical in birds fed High-Na and Low-Na diets, suggesting that tubuloglomerular feed-back does not contribute significantly to avian GFR autoregulation. Filtering glomeruli were stained in vivo with alcian blue dye to determine if RAPP-induced reductions in GFR are associated with cessation of filtration (glomerular intermittency) by a portion of the nephron population. RAPP was held below the GFR autoregulatory range (experimental group) or was at ambient systemic arterial pressure (control group) during glomerular staining. Reducing RAPP below the autoregulatory range reduced GFR by 50%, but similar glomerular size distribution profiles were observed for experimental and control groups. These results indicate that sustained glomerular intermittency does not contribute to the decrease in GFR when RAPP is reduced below the autoregulatory range.Abbreviations BW body weight - C control - E excretion - FE fractional excretion - FF filtration fraction - GFR glomerular filtration rate - PAH p-amino hippuric acid - RAPP renal arterial perfusion pressure - RPF renal plasma flow - RT reptilian-type - SNGFR single nephron glomerular filtration rate - U _OSM urine osmolarity - UFR urine flow rate     

In situ activation of β-galactosidase of Kluyveromyces bulgaricus resting cells by sodium and potassium phosphates and chlorides

Summary Incubation of Kluyveromyces bulgaricus in sodium and potassium salts led to in vivo activation of -galactosidase. The activation reaction was relatively slow since, at 37°C, it took 30 min to come to completion. The reaction was irreversible and was favoured by high salt concentrations with chlorides proving to be more efficient than phosphates. After incubation in KCl, the final activity obtained was 1.49 U/mg dry yeast and this represented a 10-fold increase in activity compared with the control value measured in ammonium phosphate. Hydrolysis of onitrophenyl--galactoside (ONPG) was insensitive to inhibitors of the transport systems and energy metabolism. There results suggest that K. bulgaricus resting cells take up substrates and ions that readily influence -galactosidase activity.     

The small chemical vacuolin-1 alters the morphology of lysosomes without inhibiting Ca2+-regulated exocytosis

Ca2+-regulated exocytosis of lysosomes was previously shown to be required for the repair of plasma membrane wounds. The small chemical vacuolin-1 alters the morphology of lysosomes without affecting the ability of cells to reseal their plasma membrane after injury. On the basis of a failure to detect Ca2+-triggered lysosomal exocytosis in vacuolin-1-treated cells, a recent study proposed that lysosomes are dispensable for resealing. Here, we show that vacuolin-1, despite altering lysosome morphology, does not inhibit the exocytosis of lysosomes induced by exposure to a Ca2+ ionophore, or by plasma membrane wounding. Thus, lysosomes cannot be excluded as agents of membrane repair in vacuolin-1-treated cells.     

Inhibition of estrogen receptor alpha expression and function in MCF-7 cells by kaempferol

Estrogens are mitogenic for estrogen receptor (ER)-positive breast cancer cells. Current treatment of ER-positive breast tumors is directed towards interruption of estrogen activity. We report that treatment of ER-positive breast cancer cells with kaempferol resulted in a time- and dose-dependent decrease in cell number. The concentration required to produce 50% growth inhibition at 48 h was approximately 35.0 and 70.0 microM for ER-positive and ER-negative breast cancer cells, respectively. For MCF-7 cells, a reduction in the ER-alpha mRNA equivalent to 50, 12, 10% of controls was observed 24 h after treatment with 17.5, 35.0, and 70.0 microM of kaempferol, respectively. Concomitantly, these treatments led to a 58, 80, and 85% decrease in ER-alpha protein. The inhibitory effect of kaempferol on ER-alpha levels was seen as early as 6 h post-treatment. Kaempferol treatment also led in a dose-dependent decrease in the expression of progesterone receptor (PgR), cyclin D1, and insulin receptor substrate 1 (IRS-1). Immunocytochemical study revealed that ER-alpha protein in kaempferol-treated MCF-7 cells formed an aggregation in the nuclei. Kaempferol also induced degradation of ER-alpha by a different pathway than that were observed for the antiestrogen ICI 182,780 and estradiol. Estradiol-induced MCF-7 cell proliferation and expression of the estrogen-responsive-element-reporter gene activity were abolished in cells co-treated with kaempferol. These findings suggest that modulation of ER-alpha expression and function by kaempferol may be, in part, responsible for its anti-proliferative effects seen in in vitro.     

Characterization of MOCS1A, an oxygen-sensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis

The human proteins MOCS1A and MOCS1B catalyze the conversion of a guanosine derivative to precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine (AdoMet)-dependent radical enzymes, which catalyze the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus. MOCS1A was heterologously expressed in Escherichia coli and purified under aerobic and anaerobic conditions. Individual mutations of the conserved cysteines to serine revealed that all are essential for synthesis of precursor Z in vivo. The type and properties of the iron-sulfur (FeS) clusters were investigated using a combination of UV-visible absorption, variable temperature magnetic circular dichroism, resonance Raman, M?ssbauer, and EPR spectroscopies coupled with iron and acid-labile sulfide analyses. The results indicated that anaerobically purified MOCS1A is a monomeric protein containing two oxygen-sensitive FeS clusters, each coordinated by only three cysteine residues. A redox-active [4Fe-4S](2+,+) cluster is ligated by an N-terminal CX(3)CX(2)C motif as is the case with all other AdoMet-dependent radical enzymes investigated thus far. A C-terminal CX(2)CX(13)C motif that is unique to MOCS1A and its orthologs primarily ligates a [3Fe-4S](0) cluster. However, MOCS1A could be reconstituted in vitro under anaerobic conditions to yield a form containing two [4Fe-4S](2+) clusters. The N-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen via a semistable [2Fe-2S](2+) cluster intermediate, and the C-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen to yield a semistable [3Fe-4S](0) cluster intermediate.     

The minimal essential core of a cysteine-based protein-tyrosine phosphatase revealed by a novel 16-kDa VH1-like phosphatase, VHZ

The smallest active protein-tyrosine phosphatase yet (only 16 kDa) is described here and given the name VHZ for VH1-like member Z because it belongs to the group of small Vaccinia virus VH1-related dual specific phosphatases exemplified by VHR, VHX, and VHY. Human VHZ is remarkably well conserved through evolution as it has species orthologs in frogs, fish, fly, and Archaea. The gene for VHZ, which we designate as DUSP25, is located on human chromosome 1q23.1 and consists of only two coding exons. VHZ is broadly expressed in tissues and cells, including resting blood lymphocytes, Jurkat T cells, HL-60, and RAMOS. In transfected cells, VHZ was located in the cytosol and in other cells also in the nucleoli. Endogenous VHZ showed a similar but more granular distribution. We show that VHZ is an active phosphatase and analyze its structure by computer modeling, which shows that in comparison with the 185-amino acid residue VHR, the 150-residue VHZ is a shortened version of VHR and contains the minimal set of secondary structure elements conserved in all known phosphatases from this class. The surface charge distribution of VHZ differs from that of VHR and is therefore unlikely to dephosphorylate mitogen-activated protein kinases. The remarkably high degree of conservation of VHZ through evolution may indicate a role in some ancient and fundamental physiological process.     

Activation of IP prostanoid receptors prevents cardiomyocyte hypertrophy via cAMP-dependent signaling

The antihypertrophic action of angiotensin-converting enzyme inhibitors in the heart results partly from local potentiation of bradykinin. We have demonstrated that the antihypertrophic action of bradykinin is mediated by the release of nitric oxide from endothelium and elevation of cardiomyocyte cGMP. Whether other paracrine factors derived from the coronary endothelium, such as prostacyclin (PGI2), may act to prevent hypertrophy has not been explored. In the vasculature, activation by PGI2 of IP and EP1 prostanoid receptors elicits vasodilatation (via cAMP-dependent signaling) and vasoconstriction, respectively. The present objective was to determine whether IP prostanoid receptor activation has antihypertrophic actions in adult rat cardiomyocytes (ARCM). The selective IP agonist cicaprost (1 microM) virtually abolished the increase in [3H]phenylalanine incorporation (a marker of hypertrophy) induced either by endothelin-1 (ET-1; 60 nM, n = 10, P < 0.005) or by angiotensin II (1 microM, n = 6, P < 0.005). Cicaprost also inhibited ET-1 induction of c-fos mRNA expression, an additional marker of hypertrophy in ARCM (n = 5, P < 0.005). In the absence of hypertrophic stimuli, cicaprost alone did not significantly influence either marker. The antihypertrophic actions of cicaprost were mimicked by the dual IP/EP1 agonist iloprost (1 microM) in the presence of the EP1 antagonist AH-6809 (3 microM). Furthermore, cicaprost modestly but significantly increased cardiomyocyte cAMP content by 13 +/- 6% (P < 0.05, n = 4), and the antihypertrophic effect of cicaprost was lost in the presence of the cAMP-dependent protein kinase inhibitor H-89 (1 microM, n = 5, P < 0.05). However, ET-1 also induced increases in the activity of the intracellular growth signals ERK1 (by 3-fold) and ERK2 (by 5-fold) in ARCM, and these were not inhibited by cicaprost (P < 0.01, n = 5). Activation of IP receptors thus represents a novel approach to prevention of hypertrophy, and this effect is linked to cAMP-dependent signaling.     

Mass spectrometry provides accurate characterization of two genetic marker types in Bacillus anthracis

Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) could be combined on a single analysis platform. Here we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B. anthracis. The results obtained with ESI-FTICR-MS were consistent with independent results obtained from traditional approaches using electrophoretic detection of fluorescent products. However, ESI-FTICR-MS improves on the traditional approaches because it does not require fluorescent labeling of PCR products, minimizes post-PCR processing, obviates electrophoresis, and provides unambiguous base composition of both SNP and VNTR PCR products. In addition, ESI-FTICR-MS allows both marker types to be examined simultaneously and at a rate of approximately 1 sample per min. This technology represents a significant advance in our ability to rapidly characterize B. anthracis isolates using VNTR and SNP loci.