Asymmetric aerobic oxidative NHC-catalysed synthesis of dihydropyranones utilising a system of electron transfer mediators.

In the context of green chemistry, the replacement of high molecular weight stoichiometric oxidants with O2 is most desirable but difficult. Here, we report the asymmetric aerobic oxidative synthesis of dihydropyranones. The oxidation is aided by a system of electron transfer mediators and is selective toward the homoenolate. The dihydropyranones can be isolated in high to excellent yields, with high ee (up to 95%).

Phlpp1 facilitates post-traumatic osteoarthritis and is induced by inflammation and promoter demethylation in human osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is the most common form of arthritis and a leading cause of disability. OA is characterized by articular chondrocyte deterioration, subchondral bone changes and debilitating pain. One strategy to promote cartilage regeneration and repair is to accelerate proliferation and matrix production of articular chondrocytes. We previously reported that the protein phosphatase Phlpp1 controls chondrocyte differentiation by regulating the activities of anabolic kinases. Here we examined the role of Phlpp1 in OA progression in a murine model. We also assessed PHLPP1 expression and promoter methylation. DESIGN: Knee joints of WT and Phlpp1(-/-) mice were surgically destabilized by transection of the medial meniscal ligament (DMM). Mice were assessed for signs of OA progression via radiographic and histological analyses, and pain assessment for mechanical hypersensitivity using the von Frey assay. Methylation of the PHLPP1 promoter and PHLPP1 expression were evaluated in human articular cartilage and chondrocyte cell lines. RESULTS: Following DMM surgeries, Phlpp1 deficient mice showed fewer signs of OA and cartilage degeneration. Mechanical allodynia associated with DMM surgeries was also attenuated in Phlpp1(-/-) mice. PHLPP1 was highly expressed in human articular cartilage from OA patients, but was undetectable in cartilage specimens from femoral neck fractures (FNFxs). Higher PHLPP1 levels correlated with less PHLPP1 promoter CpG methylation in cartilage from OA patients. Blocking cytosine methylation or treatment with inflammatory mediators enhanced PHLPP1 expression in human chondrocyte cell lines. CONCLUSION: Phlpp1 deficiency protects against OA progression while CpG demethylation and inflammatory cytokines promote PHLPP1 expression.

Clinical and immunochemical profiles of food challenge proven or anaphylactic shrimp allergy in tropical Singapore.

BACKGROUND: Shellfish allergy in Singapore is highly prevalent, and shrimp allergy is the most common. OBJECTIVE: This study aims to evaluate the clinical characteristics and immunological phenotype of shellfish allergy in this population. METHODS: Patients with self-reported shellfish allergy were recruited from outpatient clinics of three large hospitals and from a population survey. Open oral food challenges (OFC) to glass prawn (Litopenaeus vannamei) and tiger prawn (Penaeus monodon) were carried out on all patients except for those who had a history of severe anaphylaxis. Skin prick tests (SPT) and specific IgE to crude and recombinant allergens were carried out to evaluate shrimp and dust mite sensitization. Immunoblots were used to assess IgE-binding proteins. RESULTS: The 104 patients recruited were categorized into shellfish allergic (SA) when OFC was positive or had a history of severe anaphylaxis (n = 39), shellfish tolerant (ST) when OFC was negative (n = 27), and house dust mite positive controls (HDM(+) ) who were ST (n = 38). Oral symptoms (87.1%) were the predominant clinical manifestation. Positive challenge doses ranged from 2 to 80 g of cooked shrimp, with 25/52 patients reacting to either one or both shrimps challenged. The presence of specific IgE to shrimp either by SPT and/or ImmunoCAP(®) assay provided diagnostic test sensitivity of 82% and specificity of 22.2%. The inclusion of specific IgE to shrimp tropomyosin and IgE immunoblots with shrimp extracts did not improve the diagnostic proficiency substantially. CONCLUSIONS AND CLINICAL RELEVANCE: This study highlights the predominance of oral symptoms in shrimp allergy in tropical Asia and that a high provocation dose may be necessary to reveal shrimp allergy. Furthermore, specific IgE diagnostic tests and immunoblots were of limited use in this population.

Clinical relevance of cagPAI intactness in Helicobacter pylori isolates from Vietnam.

The purpose of this paper is to investigate the relationship between clinical outcome and the intactness of cagPAI in Helicobacter pylori strains from Vietnam. The presence or absence of 30 cagPAI genes was investigated by polymerase chain reaction (PCR) and dot-blotting. H. pylori-induced interleukin-8 secretion and hummingbird phenotype, and H. pylori adhesion to gastric epithelial cells were examined. The serum concentration of pepsinogen 1, pepsinogen 2, and gastrin was also measured in all patients. cagPAI was present in all 103 Vietnamese H. pylori isolates, of which 91 had intact cagPAI and 12 contained only a part of cagPAI. Infection with the partial cagPAI strains was less likely to be associated with peptic ulcer and chronic gastric mucosal inflammation than infection with strains possessing intact cagPAI. The partial cagPAI strains lacked almost all ability to induce interleukin-8 secretion and the hummingbird phenotype in gastric cells. Their adhesion to epithelial cells was significantly decreased in comparison with intact cagPAI strains. Moreover, for the first time, we found an association between cagPAI status and the serum concentration of pepsinogens 1 and 2 in infected patients. H. pylori strains with internal deletion within cagPAI are less virulent and, thus, less likely to be associated with severe clinical outcomes.

Protective mechanisms of inosine in platelet activation and cerebral ischemic damage.

OBJECTIVE: Inosine is a naturally occurring nucleoside degraded from adenosine. Recent studies have demonstrated that inosine has potent immunomodulatory and neuroprotective effects. In the present study, we further investigated the inhibitory effects of inosine on platelet activation in vitro and in vivo, as well as in attenuating middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. METHODS AND RESULTS: Inosine concentration-dependently (0.5 to 6.0 mmol/L) inhibited platelet aggregation stimulated by agonists. Inosine (1.5 and 3.0 mmol/L) inhibited phosphoinositide breakdown, [Ca+2]i, and TxA2 formation in human platelets stimulated by collagen (1 microg/mL). In addition, inosine (1.5 and 3.0 mmol/L) markedly increased levels of cyclic guanylate monophosphate (GMP) and cyclic GMP-induced vasodilator-stimulated phosphoprotein Ser157 phosphorylation. Rapid phosphorylation of a platelet protein of molecular weight 47,000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/mL). This phosphorylation was markedly inhibited by inosine (3.0 mmol/L). Inosine (1.5 and 3.0 mmol/L) markedly reduced hydroxyl radical in collagen (1 microg/mL)-activated platelets. In in vivo studies, inosine (400 mg/kg) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules of mice, and administration of 2 doses (100 mg/kg) or a single dose (150 mg/kg) of inosine significantly attenuated MCAO-induced focal cerebral ischemia in rats. CONCLUSIONS: Platelet aggregation contributes significantly to MCAO-induced focal cerebral ischemia. The most important findings of this study suggest that inosine markedly inhibited platelet activation in vitro and in vivo, as well as cerebral ischemia. Thus, inosine treatment may represent a novel approach to lowering the risk of or improving function in thromboembolic-related disorders and ischemia-reperfusion brain injury.

Expression of matrix metalloproteinase-9 in human platelets: regulation of platelet activation in in vitro and in vivo studies.

1. The aim of this study was to identify the presence of matrix metalloproteinase-9 (MMP-9) in human platelets and systematically examine its inhibitory mechanisms of platelet activation. 2. In this study, we report on an efficient method for the quantitative analysis of pro-MMP-9 in human platelets using capillary zone electrophoresis (CZE). To elucidate subcellular localization of MMP-9 in human platelets, we investigated intraplatelet MMP-9 by immunogold labeling and visualized it using electron microscopy. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium. 3. MMP-9-gold labeling was observed on the plasma membrane, alpha-granules, open canalicular system, and within the cytoplasma both in resting and activated platelets. Furthermore, activated MMP-9 concentration-dependently (15-90 ng ml(-1)) inhibited platelet aggregation stimulated by agonists. Activated MMP-9 (21 and 90 ng ml(-1)) inhibited phosphoinositide breakdown, intracellular Ca(2+) mobilization, and thromboxane A(2) formation in human platelets stimulated by collagen (1 microg ml(-1)). In addition, activated MMP-9 (21 and 90 ng ml(-1)) significantly increased the formation of nitric oxide/cyclic GMP. 4. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12, 13-dibutyrate (PDBu) (60 nm). This phosphorylation was markedly inhibited by activated MMP-9 (21 and 90 ng ml(-1)). Activated MMP-9 (1 microg g(-1)) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules. 5. These results indicate that the antiplatelet activity of activated MMP-9 may be involved in the following pathways. (1) Activated MMP-9 may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown, protein kinase C activation, and thromboxane A(2) formation, thereby leading to inhibition of intracellular Ca(2+) mobilization. (2) Activated MMP-9 also activated the formation of nitric oxide/cyclic GMP, resulting in inhibition of platelet aggregation. These results strongly indicate that MMP-9 is a potent inhibitor of aggregation. It may play an important role as a negative feedback regulator during platelet activation.

Nitric oxide in experimental joint inflammation. Benefit or detriment?

The host response to infection or injury initiates a cascade of events involving recruitment of leukocytes and the release of multiple inflammatory mediators. One of these mediators, nitric oxide (NO), not only represents an important microbicidal agent in host defense, but also functions as a biological signaling and effector molecule in inflammation and immunity. However, overproduction of NO can be autotoxic and contribute to tissue damage and has been implicated in pathogenesis of tumors, and infectious, autoimmune and chronic degenerative diseases. NO is generated via constitutive and inducible nitric oxide synthases (iNOS) which catalyze the oxidation of a guanidino nitrogen associated with L-arginine. Whereas endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively expressed, iNOS is transcriptionally induced by bacterial constituents and inflammatory mediators, including TNF alpha and IL-1. In an experimental model of bacterial component-induced joint inflammation and tissue degradation, functionally distinct roles of the constitutive NOS and iNOS were demonstrated. Following systemic delivery of an arthritogenic dose of streptococcal cell walls (SCW), these bacterial peptidoglycan-polysaccharide complexes disseminate and target the peripheral joints, liver and spleen of the treated animals. Following deposition of the SCW in the peripheral joints, an initial innate inflammatory response to the bacterial components progresses into an adaptive immune response with the recruitment and activation of mononuclear phagocytes and T lymphocytes. With the release of cytokines and inflammatory mediators, there is an upregulation of gene expression for iNOS, but not the constitutive nNOS or eNOS. Nonetheless, the constitutive NOS isoforms, regulated by calcium fluxes and interaction with calmodulin, may also enhance NO production. Increased release of NO was detected not only in the synovium, but also in the circulation, and plasma levels of nitrate plus nitrite, the stable products of NO reactions, correlated with disease progression. Following inhibition of NO production with nonspecific NOS inhibitors, such as N(G)-monomethyl-L-arginine, which target all three isoforms, there is a striking therapeutic benefit with reduced signs and symptoms of erosive arthritis. In contrast, selective targeting of iNOS with N-iminoethyl-L-lysine resulted in exacerbation of the synovial inflammation and degradation of joint structures. Based on these data, it appears that the constitutive isoforms of NOS contribute to the pathophysiology of the arthropathy, and that induced NOS and NO may function, in part, in a protective pathway. Moreover, the suppression of NO following treatment with TNF alpha antagonists results in reduced inflammation and the associated synovial pathology. Collectively, these data implicate discrete roles for the NOS isoforms in the emergence of local tissue pathology and underscore the need to define the specific pathways that are being targeted for interventional strategies.

Mechanisms involved in the antiplatelet activity of midazolam in human platelets.

BACKGROUND: Midazolam is widely used as a sedative and anesthetic induction agent. The aim of this study was to systematically examine the inhibitory mechanisms of midazolam in platelet aggregation. METHODS: The inhibitory mechanisms of midazolam in platelet aggregation were explored by means of analysis of the platelet glycoprotein IIb-IIIa complex, phosphoinositide breakdown, intracellular Ca+2 mobilization, measurement of membrane fluidity, thromboxane B2 formation, and protein kinase C activity. RESULTS: In this study, midazolam dose-dependently (6-26 microm) inhibited platelet aggregation in human platelets stimulated by agonists. Midazolam also dose-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. Midazolam (6-26 mum) significantly inhibited thromboxane A2 formation stimulated by collagen in human platelets. Moreover, midazolam (15 and 26 mum) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by collagen (2 microg/ml). This phosphorylation was markedly inhibited by midazolam (26 microm). CONCLUSIONS: These results indicate that the antiplatelet activity of midazolam may be involved in the following pathways: the effects of midazolam may initially be caused by induction of conformational changes in platelet membrane, leading to a change in the activity of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca+2 mobilization and phosphorylation of P47 protein.

SYM-2081 a kainate receptor antagonist reduces allodynia and hyperalgesia in a freeze injury model of neuropathic pain.

Cold-freeze injury at -4 degrees C to the rat sciatic nerve produces mechanical allodynia and thermal hyperalgesia [M.A. Kleive, P.S. Jungbluth, J.A. Uhlenkamp, K.C. Kajander, Cold injury to rat sciatic nerve induces thermal hyperalgesia or analgesia, 8th World Congress on Pain, Vancouver, BC, Canada, August 1996 (Abstract).]. The NMDA receptor, an excitatory amino acid (EAA) receptor, appears to be involved in the development of allodynia and hyperalgesia following nerve injury. The role, if any, of the kainate receptor, another EAA receptor, remains unknown. In the current study, we evaluated whether (2S,4R)-4-methylglutamic acid (SYM-2081), a recently developed kainate receptor antagonist, attenuates increased responsiveness following cold injury to the sciatic nerve. During baseline testing, Sprague-Dawley rats were evaluated for frequency of withdrawal from von Frey filaments and latency of withdrawal from a radiant thermal source. Animals were then anesthetized, the left sciatic nerve was exposed, and the nerve was cooled to -4 degrees C for 15 min (n=24). For control rats (n=24), all procedures were identical except that the nerve was maintained at 37 degrees C. Testing resumed on the third day following surgery. On the fifth post-operative day, SYM-2081 (150 or 100 mg/kg), fentanyl citrate (0. 04 mg/kg) or vehicle was injected intraperitoneally. Injury to the rat sciatic nerve induced a significant increase in withdrawal frequency and a significant decrease in withdrawal latency (ANOVA, p<0.05). SYM-2081 and fentanyl significantly reduced these responses (p<0.05). These results suggest that kainate and opioid receptors are involved in the mechanical allodynia and thermal hyperalgesia that develop following cold injury to the sciatic nerve.

Critical incident analysis: nursing a dying man.

When a nursing student cares for a terminally ill person for the first time, that student faces many challenges. Here, a third year student describes and analyses her thoughts, feelings and actions, reflecting on her perceptions of the patient's (Mr A's) needs; the mutual benefits of a close relationship between nurse and patient; and the personal and professional growth which resulted from writing about this challenging experience. The memory of Mr A is an inspirational legacy.

Dorsal raphe stimulation modulates responses of substantia nigra, pars reticulata neurons to painful stimulation.

There are but few data that substantia nigra neurons are involved in the pain-processing mechanisms in the CNS. In contrast, a great deal of evidence suggests a participation of the dorsal raphe nucleus (RD) in these processes. The purpose of the present study was to examine the effects of painful stimulation (suprathreshold electrical stimulation of the peroneal nerve--NP) on the extracellular activity of neurons of substantia nigra, pars reticulata (SNR) in cats, and to determine if a train of stimuli applied to RD could affect the responses of SNR neurons to this type of painful stimulation. The majority of SNR neurons (83.3%) responded to NP-stimulation either with an increase or with a decrease in the firing rate. The RD-stimulation when applied simultaneously or just before the NP-stimulation reduced or even eliminated the increase in the firing rate caused by pain irrespective of the ability of RD-stimulation applied proviously alone to reduce or not the spontaneous activity of the SNR neurons. In cells which responded with inhibition to NP- and RD-stimulations when applied alone, RD-stimulation applied just before NP-stimulation slightly prolonged the NP-induced inhibition. The results indicate that in addition to the descending pain modulation pathway from the dorsal raphe nucleus to the spinal cord, which pathway is well established, there may be an ascending one to some supraspinal structures, the substantia nigra being one of them.