Induced Stress on Red Blood Cell Promotes Red Blood Cell-Endothelial Adhesion

Cell and Tissue Biology - Besides disease condition, very few stress stimulants were determined to provoke red blood cell (RBC) adhesion to endothelial cells (EC). However, the possible role of...     

Trichosanthes tricuspidata Modulates Oxidative Toxicity in Brain Hippocampus Against Pilocarpine Induced Status Epilepticus in Mice

Epilepsy prevails to be a neurological disorder in anticipation of safer drugs with enhanced anticonvulsant efficacy as presently available drugs fails to offer adequate control of epileptic seizures in about one-third of patients. The objective of this study was to evaluate the effect of Trichosanthes tricuspidata methanolic extract (TTME) against epilepsy mediated oxidative stress in pilocarpine induced mice. Intraperitonial administration of pilocarpine (85 mg/kg) induced seizure in mice was assessed by behavior observations, which is significantly (p < 0.05) reduced by TTME (100 and 200 mg/kg; i.p) in a dose dependant manner, similar to diazepam. Seizure was accompanied by significant increase in lipid peroxidation and the hippocampal nitrite content in pilocarpine group when compared with control. Moreover, the antioxidant enzymes superoxide dismutase, catalase and glutathione levels were decreased in pilocarpine administered groups. TTME administration attenuated oxidative damage as evident by decreased lipid oxidative damage and nitrite–nitrate content and restored the level of enzymatic antioxidant defenses in hippocampus. Involvement of free radicals during epilepsy is further confirmed by histopathological analysis which showed the loss of neuronal cells in hippocampus CA1 and CA3 pyramidal region. Our findings strongly support the hypothesis that TTME has anticonvulsant activity accompanied with the strong antioxidant potential plays a crucial role in reducing the oxidative stress produced by seizure.     

The ligand specificity of yeast Rad53 FHA domains at the +3 position is determined by nonconserved residues

On the basis of the results from our laboratory and others, we recently suggested that the ligand specificity of forkhead-associated (FHA) domains is controlled by variations in three major factors: (i) residues interacting with pThr, (ii) residues recognizing the +1 to +3 residues from pThr, and (iii) an extended binding surface. While the first factor has been well established by several solution and crystal structures of FHA-phosphopeptide complexes, the structural bases of the second and third factors are not well understood and are likely to vary greatly between different FHA domains. In this work, we proposed and tested the hypothesis that nonconserved residues G133 and G135 of FHA1 and I681 and D683 of FHA2, located outside of the core FHA region of yeast Rad53 FHA domains, contribute to the specific recognition of the +3 position of different phosphopeptides. By rational mutagenesis of these residues, the specificity of FHA1 has been changed from predominantly pTXXD to be equally acceptable for pTXXD, pTXXL, and pYXL, which are similar to the specificities of the FHA2 domain of Rad53. Conversely, the +3 position specificity of FHA2 has been engineered to be more like FHA1 with the I681A mutation. These results were based on library screening as well as binding analyses of specific phosphopeptides. Furthermore, results of structural analyses by NMR indicate that some of these residues are also important for the structural integrity of the loops.     

Prognostic factors in canine appendicular osteosarcoma - a meta-analysis

ABSTRACT: BACKGROUND: Appendicular osteosarcoma is the most common malignant primary canine bone tumor. When treated by amputation or tumor removal alone, median survival times (MST) do not exceed 5 months, with the majority of dogs suffering from metastatic disease. This period can be extended with adequate local intervention and adjuvant chemotherapy, which has become common practice. Several prognostic factors have been reported in many different studies, e.g. age, breed, weight, sex, neuter status, location of tumor, serum alkaline phosphatase (SALP), bone alkaline phosphatase (BALP), infection, percentage of bone length affected, histological grade or histological subtype of tumor. Most of these factors are, however, only reported as confounding factors in larger studies. Insight in truly significant prognostic factors at time of diagnosis may contribute to tailoring adjuvant therapy for individual dogs suffering from osteosarcoma.The objective of this study was to systematically review the prognostic factors that are described for canine appendicular osteosarcoma and validate their scientific importance. RESULTS: A literature review was performed on selected studies and eligible data were extracted. Meta-analyses were done for two of the three selected possible prognostic factors (SALP and location), looking at both survival time (ST) and disease free interval (DFI). The third factor (age) was studied in a qualitative manner. Both elevated SALP level and the (proximal) humerus as location of the primary tumor are significant negative prognostic factors for both ST and DFI in dogs with appendicular osteosarcoma. Increasing age was associated with shorter ST and DFI, however, was not statistically significant because information of this factor was available in only a limited number of papers. CONCLUSIONS: Elevated SALP and proximal humeral location are significant negative prognosticators for canine osteosarcoma.     

Engagement of CD14 mediates the inflammatory potential of monosodium urate crystals

Phagocyte ingestion of monosodium urate (MSU) crystals can induce proinflammatory responses and trigger acute gouty inflammation. Alternatively, the uptake of MSU crystals by mature macrophages can be noninflammatory and promote resolution of gouty inflammation. Macrophage activation by extracellular MSU crystals involves apparent recognition and ingestion mediated by TLR2 and TLR4, with subsequent intracellular recognition linked to caspase-1 activation and IL-1beta processing driven by the NACHT-LRR-PYD-containing protein-3 inflammasome. In this study, we examined the potential role in gouty inflammation of CD14, a phagocyte-expressed pattern recognition receptor that functionally interacts with both TLR2 and TLR4. MSU crystals, but not latex beads, directly bound recombinant soluble (s) CD14 in vitro. CD14(-/-) bone marrow-derived macrophages (BMDMs) demonstrated unimpaired phagocytosis of MSU crystals but reduced p38 phosphorylation and approximately 90% less IL-1beta and CXCL1 release. Attenuated MSU crystal-induced IL-1beta release in CD14(-/-) BMDMs was mediated by decreased pro-IL-1beta protein expression and additionally by decreased caspase-1 activation and IL-1beta processing consistent with diminished NACHT-LRR-PYD-containing protein-3 inflammasome activation. Coating of MSU crystals with sCD14, but not sTLR2 or sTLR4, restored IL-1beta and CXCL1 production in CD14(-/-) BMDMs in vitro. Gain of function of CD14 directly enhanced TLR4-mediated signaling in response to MSU crystals in transfected Chinese hamster ovary cells in vitro. Last, MSU crystal-induced leukocyte influx at 6 h was reduced by approximately 75%, and local induction of IL-1beta decreased by >80% in CD14(-/-) mouse s.c. air pouches in vivo. We conclude that engagement of CD14 is a central determinant of the inflammatory potential of MSU crystals.     

The Isomerase Active Site of Cyclophilin A Is Critical for Hepatitis C Virus Replication

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies.Hepatitis C virus (HCV)² is the main contributing agent of acute and chronic liver diseases worldwide (1). Primary infection is often asymptomatic or associated with mild symptoms. However, persistently infected individuals develop high risks for chronic liver diseases such as hepatocellular carcinoma and liver cirrhosis (1). The combination of IFNα and ribavirin that serves as current therapy for chronically HCV-infected patients not only has a low success rate (about 50%) (2) but is often associated with serious side effects (2). There is thus an urgent need for the development of novel anti-HCV treatments (2).The immunosuppressive drug cyclosporine A (CsA) was reported to be clinically effective against HCV (3). Controlled trials showed that a combination of CsA with IFNα is more effective than IFNα alone, especially in patients with a high viral load (4, 5). Moreover, recent in vitro studies provided evidence that CsA prevents both HCV RNA replication and HCV protein production in an IFNα-independent manner (6–10). CsA exerts this anti-HCV activity independently of its immunosuppressive activity because the nonimmunosuppressive Cyp inhibitors such as Debio 025, NIM811, and SCY-635 also block HCV RNA and protein production (9, 11–14). Unlike CsA, these molecules do not display calcineurin affinity and specifically inhibit the peptidyl-prolyl cis-trans-isomerase (PPIase) Cyps. Most importantly, recent clinical data demonstrated that Debio 025 dramatically decreased HCV viral load (3.6 log decrease) in patients coinfected with HCV and HIV (15). This 14-day Debio 025 treatment (1200 mg orally administered twice daily) was effective against the three genotypes (genotypes 1, 3, and 4) represented in the study. More recently, the anti HCV effect of Debio 025 in combination with peginterferon α 2a (peg-IFNα2a) was investigated in treatment-inexperienced patients with chronic hepatitis C. Debio 025 (600 mg administered once daily) in combination with peg-IFNα2a (180 μg/week) for 4 weeks induced a continuous decay in viral load that reached −4.61 ± 1.88 IU/ml in patients with genotypes 1 and 4 and −5.91 ± 1.11 IU/ml in patients with genotypes 2 and 3 at week 4 (16). The Debio 025 findings are critical because they suggest that Cyp inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. The fact that several recent studies using small RNA interference knockdown approaches suggest that Cyps are critical for the HCV life cycle (9, 17, 18) strongly implies that there is a direct or indirect link between the CsA- and CsA derivative-mediated inhibitory effect on HCV replication and host Cyps.The discovery 20 years ago of the first cellular protein showing PPIase activity (19) was entirely unrelated to the discovery of CypA as an intracellular protein possessing a high affinity for CsA (20). It is only a few years later that Fischer et al. (21) demonstrated that the 18-kDa protein with PPIase activity and CypA represent a single unique protein. All Cyps contain a common domain of 109 amino acids, called the Cyp-like domain, which is surrounded by domains specific to each Cyp members and which dictates their cellular compartmentalization and function (22). Bacteria, fungi, insects, plants, and mammals contain Cyps, which all have PPIase activity and are structurally conserved (22). To date, 16 Cyp members have been identified, and 7 of them are found in humans: CypA, CypB, CypC, CypD, CypE, Cyp40, and CypNK (22).Although there is a growing body of evidence that Cyps control HCV replication in human hepatocytes, a major disagreement currently exists on the respective roles of Cyp members in HCV replication. One study suggests that CypB, but not CypA, is critical for HCV replication (17), another suggests that CypA, but not CypB and CypC, is critical for HCV replication (18), and a third study suggests that three Cyps, CypA, B, and C, are all required for HCV replication (9). Thus, although it becomes evident that Cyps serve as HCV co-factors, their respective contributions and roles in the HCV life cycle remain to be determined. An understanding of the mechanisms that control the Cyp inhibitor-mediated anti-HCV effect is imperative because it will provide new alternate anti-HCV therapies and shed light on the still poorly understood early and late steps of the HCV life cycle.     

Mdt1, a novel Rad53 FHA1 domain-interacting protein, modulates DNA damage tolerance and G(2)/M cell cycle progression in Saccharomyces cerevisiae

The Rad53 kinase plays a central role in yeast DNA damage checkpoints. Rad53 contains two FHA phosphothreonine-binding domains that are required for Rad53 activation and possibly downstream signaling. Here we show that the N-terminal Rad53 FHA1 domain interacts with the RNA recognition motif, coiled-coil, and SQ/TQ cluster domain-containing protein Mdt1 (YBl051C). The interaction of Rad53 and Mdt1 depends on the structural integrity of the FHA1 phosphothreonine-binding site as well as threonine-305 of Mdt1. Mdt1 is constitutively threonine phosphorylated and hyperphosphorylated in response to DNA damage in vivo. DNA damage-dependent Mdt1 hyperphosphorylation depends on the Mec1 and Tel1 checkpoint kinases, and Mec1 can directly phosphorylate a recombinant Mdt1 SQ/TQ domain fragment. MDT1 overexpression is synthetically lethal with a rad53 deletion, whereas mdt1 deletion partially suppresses the DNA damage hypersensitivity of checkpoint-compromised strains and generally improves DNA damage tolerance. In the absence of DNA damage, mdt1 deletion leads to delayed anaphase completion, with an elongated cell morphology reminiscent of that of G(2)/M cell cycle mutants. mdt1-dependent and DNA damage-dependent cell cycle delays are not additive, suggesting that they act in the same pathway. The data indicate that Mdt1 is involved in normal G(2)/M cell cycle progression and is a novel target of checkpoint-dependent cell cycle arrest pathways.