Atrial natriuretic hormone secretion in patients with renal failure

To study the effects of volume overload and renal failure on plasma levels of immunoreactive atrial natriuretic hormone (IR-ANH), we measured levels of this hormone in normal subjects, in patients with advanced chronic renal failure (CRF) with and without clinically evident volume overload, and in patients with end-stage renal disease (ESRD) treated with chronic hemodialysis. The levels were 13 +/- 2 pmol/l in normal volunteers, 77 +/- 24 pmol/l in patients with CRF without volume overload, and 219 +/- 50 pmol/l in patients with CRF and clinically evident volume overload (analysis of variance, p less than 0.001, alpha = 0.05 compared to normals). In patients with ESRD, the levels of IR-ANH were 145 +/- 46 pmol/l before dialysis and decreased to 87 +/- 31 after dialysis (p less than 0.025). No correlation was found between the decrease in IR-ANH levels and the decrease in weight during dialysis. A significant positive correlation was found between the IR-ANH levels and blood urea nitrogen in patients with CRF (r = 0.658, p less than 0.01). Volume overload appears to be the most important stimulatory factor for ANH secretion in renal failure patients but other mechanisms, especially a decrease in metabolic clearance, may also contribute to elevated plasma levels. The increased secretion of ANH in patients with renal failure may be an important adaptive response to volume overload and hypertension.     

Carbon content of the neritic scyphomedusa Chrysaora fuscescens

An analysis of the role of scyphomedusae in a planktonic ecosystemrequires that biomass or numerical abundance estimates be convertibleinto standard units for comparison with other components ofthe planktonic community. One species under investigation isthe brown sea nettle, Chrysaora fuscescens, which is very abundantin coastal waters of the west coast of North America (Shenker,1984). For this species, the carbon content of whole immatureanimals and the body components of sexually mature medusae weredetermined. Immature medusae contained a mean carbon content0.202% of the wet weight. The bell, oral arms and gonadal tissueof mature medusae had mean carbon levels 0.156, 0.554 and 0.576%of the wet weight, respectively. When the relative proportionsof these body tissues were calculated, the mean carbon contentof whole mature medusae was determined to be 0.280% of the wetweight.     

Trace metal modification of immunocompetence. I. Effect of trace metals in the cultures on in vitro transformation of B lymphocytes.

Lymphocytes obtained from the spleen of Balb/C mice have been subjected to transformation by LPS in the presence of varying concentrations of Pb²⁺, Cd²⁺, or Cr³⁺. Both DNA and protein turnover were followed. It was found that Pb²⁺ and Cr³⁺ are mitogenic over a broad range of concentrations, while Cd²⁺ is slightly mitogenic at very low (10^?6M) concentration and rapidly becomes inhibitory of both [³H]TdR and [³H]ALA uptake. Pb²⁺ appears to stimulate the action of LPS, while Cr³⁺ appears to inhibit. Each of the metals protects the lymphocytes from cell death arising from incubation with LPS. The mechanism of the observed changes is as yet obscure.     

A comprehensive biochemical characterization of settlement stage leptocephalus larvae of bonefish (Albula vulpes)

Little is known about early development of the near-threatened bonefish (Albula vulpes), a member of superorder Elopomorpha. Members of Elopomorpha are partially defined by their synapomorphic leptocephalus larval stage, for which the nutritional requirements are not well understood. Characterizing the nutritional profile, including major nutrients (such as lipids) used for energetic processes, can help to gain a better understanding of the nutritional requirements for leptocephalus larvae. A total of 24 settlement stage A. vulpes leptocephalus larvae were collected at Long Caye Island, Belize. Samples were used to determine various biochemical characteristics including lipid class, fatty acid and glycosaminoglycan compositions. Each of these biochemical components plays a role in early developmental processes such as cellular membrane formation and is crucial for healthy development. Biochemical characteristics of settlement stage A. vulpes leptocephalus are presented in this study for the first time. The dominant lipid classes and fatty acids detected in these samples were consistent with prior studies using closely related species like the Japanese eel, indicating possible similarities in diets at this stage. In the future, similar analyses can be applied to other species that share the leptocephalus life stage to determine if nutritional requirements at this stage of development are unique to this species. The findings in this study will also help to facilitate the establishment of adequate aquaculture systems for captive bonefish, ultimately leading to improved management strategies for wild bonefish habitats.     

The toxicity of the Aggregatibacter actinomycetemcomitans cytolethal distending toxin correlates with its phosphatidylinositol‐3,4,5‐triphosphate phosphatase activity

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol‐3,4,5‐triphosphate (PIP3) phosphatase activity, leading us to propose that Cdt toxicity is the result of PIP3 depletion and perturbation of phosphatidylinositol‐3‐kinase (PI‐3K)/PIP3/Akt signalling. To further explore this relationship, we have focused our analysis on identifying residues that comprise the catalytic pocket and are critical to substrate binding rather than catalysis. In this context, we have generated several CdtB mutants and demonstrate that, in each instance, the ability of the toxin to induce cell cycle arrest correlates with retention of phosphatase activity. We have also assessed the effect of Cdt on downstream components of the PI‐3K signalling pathway. In addition to depletion of intracellular concentrations of PIP3, toxin‐treated lymphocytes exhibit decreases in pAkt and pGSK3β. Further analysis indicates that toxin‐treated cells exhibit a concomitant loss in Akt activity and increase in GSK3β kinase activity consistent with observed changes in their phosphorylation status. We demonstrate that cell susceptibility to Cdt is dependent upon dephosphorylation and concomitant activation of GSK3β. Finally, we demonstrate that, in addition to lymphocytes, HeLa cells exposed to a CdtB mutant that retains phosphatase activity and not DNase activity undergo G2 arrest in the absence of H2AX phosphorylation. Our results provide further insight into the mode of action by which Cdt may function as an immunotoxin and induce cell cycle arrest in target cells such as lymphocytes.     

Comprehensive chemical profiling of gramineous plant root exudates using high-resolution NMR and MS

Root exudates released into soil have important functions in mobilizing metal micronutrients and for causing selective enrichment of plant beneficial soil micro-organisms that colonize the rhizosphere. Analysis of plant root exudates typically has involved chromatographic methods that rely on a priori knowledge of which compounds might be present. In the research reported here, the combination of multinuclear and 2-D NMR with GC-MS and high-resolution MS provided de novo identification of a number of components directly in crude root exudates of different plant types. This approach was applied to examine the role of exudate metal ion ligands (MIL) in the acquisition of Cd and transition metals by barley and wheat. The exudation of mugineic acids and malate was enhanced by Fe deficiency. which in turn led to an increase in the tissue content of Cu, Mn, and Zn. The presence of elevated Cd maintained at a free activity pCd of 8.8 (10(-8.8) M), resulted in reduced phytosiderophore production by Fe deficient plants. The buffer morpholinoethane sulfonate (MES), which is commonly used in chelator-buffering nutrient solutions, was detected in the root exudate mixture, suggesting uptake and re-secretion of this compound by the roots. The ability to detect this compound in complex mixtures containing organic acids, amino acids, and other substances suggests that the analytical methods used here provide an unbiased method for simultaneous detection of all major components contained in root exudates.     

Expression of the cytolethal distending toxin (Cdt) operon in Actinobacillus actinomycetemcomitans: evidence that the CdtB protein is responsible for G2 arrest of the cell cycle in human T cells

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several gram-negative bacteria. In this study, we report that the cdt locus in A. actinomycetemcomitans is composed of five open reading frames, designated orf1, orf2, cdtA, cdtB, and cdtC. The deduced amino acid sequences of the five open reading frames are highly conserved among A. actinomycetemcomitans strains 652, Y4, 29522, and HK1651. There is also strong homology with the Cdt proteins of Haemophilus ducreyi (87-91%), but only partial homology with that of Campylobacter jejuni and Escherichia coli (29-48%). Analysis of A. actinomycetemcomitans mRNA by RT-PCR suggests that the two small open reading frames upstream of cdtA are coexpressed with cdtA, cdtB, and cdtC. We next utilized a series of plasmids that express various combinations of the cdt genes to determine their requirement for expression of immunoinhibitory activity. Cell extracts of E. coli transformed with each of the plasmids were tested for their capacity to induce G2 arrest in the cell cycle of PHA-activated human T cells. These experiments suggest that expression of cdtB alone is sufficient to induce G2 arrest in human T cells, but do not exclude the possibility that cdtC also contributes to cell cycle arrest. The implications of our results with respect to the function of the individual Cdt proteins are discussed.     

Plasma levels of immunoreactive atrial natriuretic hormone in patients with diabetes mellitus

In order to determine whether atrial natriuretic hormone (ANH) secretion is altered in diabetic patients with autonomic neuropathy, plasma immunoreactive ANH (IR-ANH) levels were measured in 23 patients with insulin-dependent diabetes mellitus, 12 of whom had definite cardiac autonomic neuropathy determined by noninvasive maneuvers. Levels were also measured in 31 healthy control subjects. Whereas only one of the 11 diabetics without cardiac autonomic neuropathy had elevated IR-ANH levels, four of the 12 diabetics with cardiac autonomic neuropathy had elevated IR-ANH levels (P = 0.03 compared to control subjects). 24-h urinary sodium excetion was not different among the groups. There was no significant correlation between IR-ANH levels and diabetes control and any of the parameters of autonomic nervous system activity nor between IR-ANH levels and plasma norepinephrine or epinephrine levels. Furthermore, no relationship was observed in the diabetic subjects between IR-ANH levels and left ventricular ejection fraction determined by radionuclide ventriculography. Thus, elevated IR-ANH levels occur with greater frequency in diabetic patients with autonomic neuropathy. These elevations do not appear to be due to alterations in dietary sodium intake or left ventricular dysfunction.     

A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5-triphosphate phosphatase activity

The Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) is a potent immunotoxin that induces G(2) arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P(3) to PI-3,4-P(2) and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G(2) arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P(3) in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P(3) levels were more sensitive to the toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P(3). Finally, reduction of Jurkat cell PI-3,4,5-P(3) synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P(3) phosphatase activity, but also that toxicity in lymphocytes is related to this activity.