Neutrophil depletion increases susceptibility to systemic and vaginal candidiasis in mice, and reveals differences between brain and kidney in mechanisms of host resistance

Infections caused by the yeast Candida albicans represent an increasing threat to debilitated and immunosuppressed patients, and neutropenia is an important risk factor. Monoclonal antibody depletion of neutrophils in mice was used to study the role of these cells in host resistance. Ablation of neutrophils increased susceptibility to both systemic and vaginal challenge. The fungal burden in the kidney increased threefold on day 1, and 100-fold on day 4, and infection was associated with extensive tissue destruction. However, a striking feature of the disseminated disease in neutrophil-depleted animals was the altered pattern of organ involvement. The brain, which is one of the primary target organs in normal mice, was little affected. There was a threefold increase in the number of organisms recovered from the brains of neutrophil-depleted mice on day 4 after infection, but detectable abscesses were rare. In contrast, the heart, which in normal mice shows only minor lesions, developed severe tissue damage following neutrophil depletion. Mice deficient in C5 demonstrated both qualitative and quantitative increases in the severity of infection after neutrophil depletion when compared with C5-sufficient strains. The results are interpreted as reflecting organ-specific differences in the mechanisms of host resistance.

Modulation of human cardiac function through 4 beta-adrenoceptor populations

In human heart there is now evidence for the involvement of four beta-adrenoceptor populations, three identical to the recombinant beta(1)-, beta(2)- and beta(3)-adrenoceptors, and a fourth as yet uncloned putative beta-adrenoceptor population, which we designate provisionally as the cardiac putative beta(4)-adrenoceptor. This review described novel features of beta-adrenoceptors as modulators of cardiac systolic and diastolic function. We also discuss evidence for modulation by unoccupied beta(1)- and beta(2)-adrenoceptors. Human cardiac and recombinant beta(1)- and beta(2)-adrenoceptors are both mainly coupled to adenylyl cyclase through Gs protein, the latter more tightly than the former. Activation of both human beta(1)- and beta(2)-adrenoceptors not only increases cardiac force during systole but also hastens relaxation through cyclic AMP-dependent phosphorylation of phospholamban and troponin I, thereby facilitating diastolic function. Furthermore, both beta(1) and beta(2)-adrenoceptors can mediate experimental arrhythmias in human cardiac preparations elicited by noradrenaline and adrenaline. Human ventricular beta(3)-adrenoceptors appear to be coupled to a pertussis toxin-sensitive protein (Gi?). beta(3)-Adrenoceptor-selective agonists shorten the action potential and cause cardiodepression, suggesting direct coupling of a Gi protein to a K+ channel. In a variety of species, including man, cardiac putative beta(4)-adrenoceptors mediate cardiostimulant effects of non-conventional partial agonists, i.e. high affinity beta(1)- and beta(2)-adrenoceptor blockers that cause agonist effects at concentrations considerably higher than those that block these receptors. Putative beta(4)-adrenoceptors appear to be coupled positively to a cyclic AMP-dependent cascade and can undergo some desensitisation.

Rapid increases in cytokinin concentration in lateral buds of chickpea (Cicer arietinum L) during release of apical dominance

We examined the role of cytokinins (CKs) in release of apical dominance in lateral buds of chickpea (Cicer arietinum L.). Shoot decapitation or application of CKs (benzyladenine, zeatin or dihydrozeatin) stimulated rapid bud growth. Time-lapse video recording revealed growth initiation within 2 h of application of 200 pmol benzyladenine or within 3 h of decapitation. Endogenous CK content in buds changed little in the first 2 h after shoot decapitation, but significantly increased by 6 h, somewhat later than the initiation of bud growth. The main elevated CK was zeatin riboside, whose content per bud increased 7-fold by 6 h and 25-fold by 24 h. Lesser changes were found in amounts of zeatin and isopentenyl adenine CKs. We have yet to distinguish whether these CKs are imported from the roots via the xylem stream or are synthesised in situ in the buds, but CKs may be part of an endogenous signal involved in lateral bud growth stimulation following shoot decapitation. To our knowledge, this is the first detailed report of CK levels in buds themselves during release of apical dominance.

Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions

Raf-1 activation is a complex process which involves plasma membrane recruitment, phosphorylation, protein-protein and lipid-protein interactions, We now show that PP1 and PP2A serine-threonine phosphatases also have a positive role in Ras dependent Raf-1 activation, General serine-threonine phosphatase inhibitors such sodium fluoride, or beta-glycerophosphate and sodium pyrophosphate, or specific PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I-1 or protein phosphatase inhibitor 2 all abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitors block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to serine phosphatase inhibitors. Sucrose gradient analysis demonstrates that serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and significantly alters the distribution of 14-3-3 and Raf-1 across different plasma membrane microdomains, These observations suggest that dephosphorylation of S259 is a critical early step in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement. Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated.

Epidermal growth factor sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia

Epidermal growth factor (EGF) has been reported to either sensitize or protect cells against ionizing radiation. We report here that EGF increases radiosensitivity in both human fibroblasts and lymphoblasts and downregulates both ATM (mutated in ataxia-telangiectasia (A-T)) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). No further radiosensitization was observed in A-T cells after pretreatment with EGF. The down-regulation of ATM occurs at the transcriptional level. Concomitant with the down-regulation of ATM, the DNA binding activity of the transcription factor Spl decreased. A causal relationship was established between these:observations by demonstrating that upregulation of Spl DNA binding activity by granulocyte/ macrophage colony-stimulating factor rapidly reversed the EGF-induced decrease in ATM protein and restored radiosensitivity to normal levels. Failure to radiosensitize EGF-treated cells to the same extent as observed for A-T cells ban be explained by induction of ATM protein and kinase activity with time post-irradiation, Although ionizing radiation damage to DNA rapidly activates ATM kinase and cell cycle checkpoints, we have provided evidence for the first time that alteration in the amount of ATM protein occurs in response to both EGF and radiation exposure. Taken together these data support complex control of ATM function that has important repercussions for targeting ATM to improve radiotherapeutic benefit.

Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites - In vivo assessment using phospho-specific antibodies

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1·Mre11·Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and UV; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after UV. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after UV) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530). However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1. Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.

Rapid radiation-induction of atm protein levels in situ

Ataxia-telangiectasia (A-T) is characterised by hypersensitivity to ionising radiation (IR), immunodeficiency, neurodegeneration and predisposition to malignancy. Mutations in the A-T gene (ATM) often result in reduced levels of ATM protein and/or compromise ATM function. IR induced DNA damage is known to rapidly upregulate ATM kinase activity/phosphorylation events in the control of cell cycle progression and other processes. Variable expression of ATM levels in different tissues and its upregulation during cellular proliferation indicate that the level of ATM is also regulated by mechanisms other than gene mutation. Here, we report on the IR induction of ATM protein levels within a number of different cell types and tissues. Induction had begun within 5 min and peaked within 2 h of exposure to 2 Gy of IR, suggesting a rapid post-translational mechanism. Low basal levels of ATM protein were more responsive to IR induction compared to high ATM levels in the same cell type. Irradiation of fresh skin biopsies led to an average three-fold increase in ATM levels while immunohistochemical analyses indicated low expressing cells within the basal layer with ten-fold increases in ATM levels following IR. ATM high expressing lymphoblastoid cell lines (LCLs) which were initially resistant to the radiation-induction of ATM levels also became responsive to IR after ATM antisense expression was used to reduce the basal levels of the protein. These results demonstrate that ATM is present in variable amounts in different tissue/cell types and where basal levels are low ATM levels can be rapidly induced by IR to saturable levels specific for different cell types. ATM radiation-induction is a sensitive and rapid radioprotective response that complements the IR mediated activation of ATM.

Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites - In vivo assessment using phospho-specific antibodies

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1.Mre11.Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and W; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after W. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after W) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530), However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1, Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.

Exposure to automotive pollution increases plasma susceptibility to oxidation

Low-density lipoprotein oxidation is implicated in the development of atherosclerosis. Plasma susceptibility to oxidation may be used as a marker of low-density lipoprotein oxidation and thus predict atherosclerotic risk. In this study the authors investigated the relationship between plasma susceptibility to oxidation and exposure to automotive pollution in a group of automobile mechanics (n = 16) exposed to high levels of automotive pollution, vs. matched controls (n = 13). The authors induced plasma oxidation by a free radical initiator and they determined susceptibility to oxidation by (1) change in absorbance at 234 nm, (2) lag time to conjugated diene formation, and (3) linear slope of the oxidation curve. Mechanics had significantly higher values (mean standard error) for change in absorbance (1.60 +/- 0.05 vs. 1.36 +/- 0.05; p < .002), and slope (1.6 x 10(-3) +/- 0.1 x 10(-3) vs. 1.3 x 10(-3) +/- 0.1 x 10(-3); p < .001), compared with controls. These results indicate that regular exposure to automotive pollutants increases plasma susceptibility to oxidation and may, in the long term, increase the risk of developing atherosclerosis.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

No evidence for inhibition of ENaC through CFTR-mediated release of ATP

Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl- secretion and inhibit amiloride-sensitive Na+ transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na+ channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl- channel blocker 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl- transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N-2,2'-O-dibutyrylguanosine 3',5-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl-. (C) 2002 Elsevier Science B.V. All rights reserved.

The role of putative phosphorylation sites in the targeting and shuttling of the aquaporin-2 water channel

In renal collecting ducts, a vasopressin-induced cAMP increase results in the phosphorylation of aquaporin-2 (AQP2) water channels at Ser-256 and its redistribution from intracellular vesicles to the apical membrane. Hormones that activate protein kinase C (PKC) proteins counteract this process. To determine the role of the putative kinase sites in the trafficking and hormonal regulation of human AQP2, three putative casein kinase II (Ser-148, Ser-229, Thr-244), one PKC (Ser-231), and one protein kinase A (Ser-256) site were altered to mimic a constitutively non-phosphorylated/phosphorylated state and were expressed in Madin-Darby canine kidney cells. Except for Ser-256 mutants, seven correctly folded AQP2 kinase mutants trafficked as wild-type AQP2 to the apical membrane via forskolin-sensitive intracellular vesicles. With or without forskolin, AQP2-Ser-256A was localized in intracellular vesicles, whereas AQP2-S256D was localized in the apical membrane. Phorbol 12-myristate 13-acetate-induced PKC activation following forskolin treatment resulted in vesicular distribution of all AQP2 kinase mutants, while all were still phosphorylated at Ser-256. Our data indicate that in collecting duct cells, AQP2 trafficking to vasopressin-sensitive vesicles is phosphorylation-independent, that phosphorylation of Ser-256 is necessary and sufficient for expression of AQP2 in the apical membrane, and that PMA-induced PKC-mediated endocytosis of AQP2 is independent of the AQP2 phosphorylation state.