[Prediction of protection probability against Omicron BA.1, BA.4 and BA.5 variants in symptomatic infections with prototype strain based on neutralization antibody levels].

Objective: To predict the protection probability of different clinical outcomes after reinfection with Omicron variant in symptomatic and unvaccinated COVID-19 patients who infected with prototype strain. Methods: The data used in this study were derived from a systematic review and meta-analysis which systematically searched PubMed, Embase, Web of Science, and Europe PMC databases, included published and uploaded studies of dynamic changes of neutralizing antibodies in symptomatic COVID-19 patients from 1 January 2020 to 2 October 2022 and extracted the literature information, study design, serological experiment information and antibody results. According to the scatter distribution characteristics of antibody titer data, a generalized additive model based on Gaussian distribution was used to fit the titer value of neutralizing antibody based on logarithmic conversion and the dynamic change pattern of neutralizing antibody in symptomatic and unvaccinated COVID-19 patients infected with prototype strain over time was obtained. In this study, the fitted antibody titers of patients on the 28th, 51st, and 261st day after symptom onset was selected to predict the protection probability. Results: Neutralizing antibodies produced in symptomatic and unvaccinated patients infected with prototype strain could provide protection against Omicron reinfection, and the probability of protection gradually decreased with time. Neutralizing antibody level on day 28 after symptom onset provided protection probability of 30.3% (95%CI: 20.0%-45.5%) against reinfection, 51.5% (95%CI: 33.4%-75.9%) against symptomatic reinfection, and 91.2% (95%CI: 77.1%-97.7%) against severe reinfection caused by Omicron BA.5. The protection probability against Omicron BA.1, BA.4 and BA.5 reinfections decreased significantly 261 days after symptom onset, showing 9.6%-12.9%, 18.4%-23.9% and 63.1%-70.3% against three clinical outcomes, respectively. At the same time point and against the same clinical outcome, the protection probability of BA.1 was the highest, followed by BA.4 and BA.5. Conclusions: Neutralizing antibodies induced in symptomatic and unvaccinated COVID-19 patients previously infected with the prototype strain have limited protection probability against Omicron BA.5 reinfections and symptomatic reinfections. The protection probability against Omicron BA.5 reinfections is 30.3% 28 days after symptom onset and decreases to about 10% after 261 days. However, the protection probability against severe reinfections is considerable, with over 90% 28 days after symptom onset and still exceeding 60% after 261 days.

Relationship between Coffee Consumption and Osteoporosis Risk Determined by the ESR1 Polymorphism rs2982573.

BACKGROUND: The development of osteoporosis is partly explained by interactions between genetic and lifestyle or environmental factors. OBJECTIVES: In the current study, we determined the relationship between coffee consumption and the risk of osteoporosis among individuals with ESR1 rs2982573 in Taiwan. DESIGN, PARTICIPANTS AND SETTING: In this population-based cross-sectional study, we used genetic, demographic, and lifestyle data from participants recruited in Taiwan Biobank (TWB) between 2016 and 2019. We used multiple logistic regression analyses to determine the relationship between osteoporosis and variant rs2982573 genotypes (TT, TC, and CC). MAIN OUTCOME: The primary outcome was osteoporosis. RESULTS: Individuals with osteoporosis (n = 515) were older than those without the disease (mean age ±SE (year); 61.324±0.361 versus 53.068 ±0.130, p<0.001). There was no significant association between rs2982573 and osteoporosis (OR, 0.904; 95% CI, 0.706-1.157; p=0.422 for TC+CC when compared with the TT genotype). Coffee consumption was associated with a lower risk of osteoporosis (OR, 0.737; 95% CI, 0.592-0.918; p=0.006). The p-value for interaction between rs2982573 and coffee consumption was 0.0393. In our subgroup analyses, the adjusted ORs (95% CI) were 0.635 (0.410-0.985) in coffee drinking TC+CC individuals and 1.095 (0.809-1.482) in non-coffee drinking TC+CC individuals, respectively when compared with their TT genotype counterparts. CONCLUSION: According to our study, participants in the TWB with the TC+CC genotype of ESR1 rs2982573 who consumed at least three cups of coffee per week were less likely to have osteoporosis.

[Estimating the disease burden of seasonal influenza in China, 2006-2019].

Objective: To estimate the health impact and economic burden of seasonal influenza in mainland China. Methods: From systematic literature reviews, we collected the influenza-associated excess influenza-like-illness (ILI) outpatient consultation rates, hospitalization rates of severe acute respiratory infections (SARI) and respiratory excess mortality, 2006-2017. Using these data, as well as demographic data (2019), the number of influenza-associated excess ILI outpatient consultations, SARI hospitalizations and respiratory excess deaths were estimated. Then using per capita economic burden of influenza-associated outpatient consultations and hospitalizations, as well as the productivity loss of influenza-related premature deaths, the annual influenza-associated total economic burden was estimated. All costs were adjusted to 2019 using the consumer price index. Results: The annual influenza-associated excess ILI outpatient consultations, SARI hospitalizations and excess respiratory deaths were 3 million, 2.34 million, 0.09 million, respectively. The total economic burden was 26.38 billion CNY, accounting for 0.266‰ GDP in 2019, of which the hospitalization-related economic burden accounted for the highest proportion (86.4%, 22.79 billion CNY), followed by the outpatient-related economic burden (11.3%, 2.97 billion CNY), and the indirect economic burden of productivity loss of premature deaths was the lowest (2.4%, 0.62 billion CNY). Largest economic burden was observed in East China (10.51 billion CNY) and smallest observed in Northeast China (0.38 billion CNY). Conclusion: The health burden of influenza-related outpatient visits and hospitalizations were substantial. The economic burden of influenza-related SARI hospitalization was higher than that of influenza-related outpatients and pre-mature deaths. The highest economic burden of influenza occurred in the East China.

Melt fluxing to elevate the forming ability of Al-based bulk metallic glasses.

Salt-fluxing treatment is an effective technique to improve the glass-forming ability (GFA) of bulk metallic glass (BMG)-forming melts, as demonstrated before in Pd- and Fe-based systems. However, it has been challenging to develop similar fluxing protocol for more reactive melts, such as Al-rich BMG-forming systems. Here we design new fluxing agents, from a thermodynamics perspective that takes into account combined effects of physical absorption and chemical absorption (reaction) between the fluxing agents and oxide inclusions. MgCl2-CaCl2 composite salts were selected, and their fluxing effects were systematically studied on an Al86Ni6.75Co2.25Y3.25La1.75 alloy, the best BMG-forming composition reported thus far for Al-rich alloy systems. The oxygen content was found to continuously decrease in the master alloy with increasing cycles of salt-fluxing treatment, with chlorate products on the surface suggesting concurrent physical absorption and chemical reaction. The fluxing treatment developed has enabled a record critical size (diameter) of 2.5 mm for Al-based BMGs. Our finding is thus an advance in developing highly desirable Al-based BMGs, and also provides guidance for designing processing protocol to produce larger-sized BMGs in other reactive systems.

Predicative values of serum microRNA-22 and microRNA-126 levels for non-small cell lung cancer development and metastasis: a case-control study.

Presented study aims to explore the predictive values of serum microRNA-22 (miR-22) and miR-126 levels for non-small cell lung cancer (NSCLC) development and metastasis.A total of 127 NSCLC patients who were admitted in the First People's Hospital of Yancheng City from May, 2013 to May, 2015 were selected as the case group, including 71 cases of adenocarcinoma and 56 cases of squamous cell carcinoma. There were 112 healthy individuals selected as the control group. The qRT-PCR was performed to testify the serum miR-22 and miR-126 levels. Logistic regression analysis was conducted to analyze independent factors influencing NSCLC metastasis and receiver operating characteristic (ROC) curve was drawn to analyze the sensitivity and specificity of serum miR-22 and miR-126 levels in predicting NSCLC developments and metastasis.The serum miR-22 level was significantly higher in the case group than that in the control group, while the serum miR-126 level was lower in the case group as compared with that in the control group. Compared with squamous cell carcinoma patients, serum miR-22 level significantly increased, while serum miR-126 level decreased in patients with adenocarcinoma. Patients at III + IV stage showed increased serum miR-22 level and relatively decreased serum miR-126 level as compared to patients at I + II stage. Serum miR-22 level elevated in patients with metastasis; in contrast serum miR-126 level reduced in comparison to those without metastasis. In patients with familial inheritance, serum miR-22 level increased but serum miR-126 level decreased as compared to those without familial inheritance. The specificity and sensitivity of serum miR-22 and miR-126 levels in predicting NSCLC development were 99.11%, 84.30%, 82.68% and 96.40%, respectively. The specificity and sensitivity of serum miR-22 and miR-126 levels in predicting NSCLC metastasis were 59.74%, 96.00%, 84.00% and 62.30%, respectively.Results indicated that serum miR-22 and miR-126 levels may be used as the predicative biomarkers for NSCLC development and metastasis.

[Association between ultrasound screening frequency and mortality in patients with hepatocellular carcinoma].

Objective: To discuss the association between ultrasound screening frequency and total mortality in patients with HCC before diagnosing HCC, and explore the optimal ultrasound screening frequency for HCC high-risk groups. Methods: Retrospectively collected clinical data of 615 cases of liver cirrhosis who developed to HCC from January 1, 2010 to December 31, 2015. Before diagnosing HCC, all patients were divided into five groups according to ultrasound screening frequency: 0-6, 7-12, 13-24, 25-36 months and not screened within 3 years (never screened). The chance to receive curative therapy, 5-year cumulative mortalities and independent factors of mortality in patients with HCC were analyzed. Results: Chances to receive curative therapy among the 0-6, 7-12, 13-24, 25-36 months and never screened groups were 38.2%, 27.2%, 25.4%, 23.8% and 19.7%, respectively (P<0.05). The 5-year overall mortality rates were 76.4%, 77.7%, 79.3%, 82.5% and 84.6%, respectively. Compared with 0-6 months, the adjusted OR of mortality for the other groups were 1.112, 1.235, 1.305 and 1.451, respectively (all P<0.05). Multivariate analysis showed that ultrasound screening frequency, curative treatment and Child-Pugh (class A/B) were the factors to affect long-term survival in patients with HCC (all P<0.05). Conclusion: For HCC high-risk groups, optimal ultrasound screening frequency is within 6 months, and high-frequency ultrasound screening can increase the chance of receiving curative treatment, reduce total mortality, and improve overall survival.

Structural basis of how stress-induced MDMX phosphorylation activates p53.

The tumor-suppressor protein p53 is tightly controlled in normal cells by its two negative regulators--the E3 ubiquitin ligase MDM2 and its homolog MDMX. Under stressed conditions such as DNA damage, p53 escapes MDM2- and MDMX-mediated functional inhibition and degradation, acting to prevent damaged cells from proliferating through induction of cell cycle arrest, DNA repair, senescence or apoptosis. Ample evidence suggests that stress signals induce phosphorylation of MDM2 and MDMX, leading to p53 activation. However, the structural basis of stress-induced p53 activation remains poorly understood because of the paucity of technical means to produce site-specifically phosphorylated MDM2 and MDMX proteins for biochemical and biophysical studies. Herein, we report total chemical synthesis, via native chemical ligation, and functional characterization of (24-108)MDMX and its Tyr99-phosphorylated analog with respect to their ability to interact with a panel of p53-derived peptide ligands and PMI, a p53-mimicking but more potent peptide antagonist of MDMX, using FP and surface plasmon resonance techniques. Phosphorylation of MDMX at Tyr99 weakens peptide binding by approximately two orders of magnitude. Comparative X-ray crystallographic analyses of MDMX and of pTyr99 MDMX in complex with PMI as well as modeling studies reveal that the phosphate group of pTyr99 imposes extensive steric clashes with the C-terminus of PMI or p53 peptide and induces a significant lateral shift of the peptide ligand, contributing to the dramatic decrease in the binding affinity of MDMX for p53. Because DNA damage activates c-Abl tyrosine kinase that phosphorylates MDMX at Tyr99, our findings afford a rare glimpse at the structural level of how stress-induced MDMX phosphorylation dislodges p53 from the inhibitory complex and activates it in response to DNA damage.

Quality of life in cervical cancer treated with systematic nerve-sparing and modified radical hysterectomies.

The aim of this study was to compare the quality of life (QoL) between cervical cancer patients treated with systematic nerve-sparing radical hysterectomy (SNSRH) and modified radical hysterectomy (MRH). A total of 127 patients with early cervical cancer treated with radical hysterectomy (RH) were included in the study. The patients were divided into two groups: MRH group (n = ) and SNSRH group (n = ). The patients' QoL scores were assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 questionnaire (Chinese version). The overall QoL scores were no different between the SNSRH and MRH groups in preoperative period (P > 0.05). In postoperative period, the overall QoL score in SNSRH group was slightly lower than that in MRH group at different follow-up time, but there was no difference between two groups (P > 0.05). Patients with early cervical cancer subjected to SNSRH or MRH are satisfied with their overall QoL scores. QoL may be negatively impacted by the cancer itself, surgery and adjuvant therapy.

Effects of furosemide on allergic asthmatic responses in mice.

BACKGROUND: The syndrome of allergic asthma features reversible bronchoconstriction, airway inflammation and hyperresponsiveness as well as airway remodelling, including goblet cell hyperplasia. Managing severe asthma is still a clinical challenge. Numerous studies report that furosemide, an inhibitor of Na(+)-K(+)-Cl(-) cotransporter (NKCC) reduces airway hyperresponsiveness (AHR) in asthmatic patients. However, the mechanism by which furosemide exerts anti-asthmatic action remains unclear. OBJECTIVE: This study sought to investigate the cellular profile of NKCC1 expression in the lung and examine the effects of furosemide on several outcome measurements in a mouse model of allergic asthma. METHODS: Mice were sensitized and challenged with ovalbumin (OVA). Before challenge, the OVA-sensitized mice were treated with furosemide (4.0 mg/kg/day, via daily intraperitoneal injection for 5 days). Outcome measurements in naïve, OVA-exposure, furosemide-treated naïve and furosemide-treated OVA-exposed mice included the slope of the relationship between inhaled methacholine (MCh) concentration and respiratory system resistance (Slope·R(RS)), bronchoalveolar lavage (BAL) cell counts and immunohistochemical and immunoblotting assays of lung tissues. RESULTS: NKCC1 immunoreactivity was observed in airway epithelial cells (AECs) and alveolar type II (ATII) cells of the control mice. OVA exposure enhanced the expression of NKCC1 in AECs and ATII cells, and increased the infiltration of NKCC1-expressing T lymphocytes in the lung. NKCC1 immunoreactivity was not detected in the airway smooth muscle (ASM) cells. Furosemide treatment reduced the Slope·R(RS) in both naïve and OVA-exposed mice by about 50%. Furosemide treatment also increased T lymphocyte infiltration to the lung in OVA-exposed mice by approximately 53%, but had no effect on pulmonary goblet cell hyperplasia. CONCLUSIONS AND CLINICAL RELEVANCE: Furosemide decreases basal airway responsiveness, thereby reducing the extent of allergen-induced AHR. However, the same treatment also increases T lymphocytes infiltration in the course of allergic asthma. Further studies are necessary to address the usefulness of furosemide in the clinical treatment of asthma.

The role of interleukin-4Ralpha in the induction of glutamic acid decarboxylase in airway epithelium following acute house dust mite exposure.

Background Asthma is a disease characterized by airway inflammation, remodelling and dysfunction. Airway inflammation contributes to remodelling, a term that is used to describe structural changes including goblet cell metaplasia (GCM), matrix deposition, and smooth muscle hyperplasia/hypertrophy. GCM has been implicated in asthma mortality by contributing to mucus plugs and leading to asphyxiation. In animal models, this process is highly dependent on IL-13. Recently, we have described an IL-13-dependent up-regulation of a GABAergic signalling system in airway epithelium that contributes to GCM. The mechanism by which IL-13 up-regulates GABA signalling in airway epithelium is unknown. Objectives To test the hypothesis that IL-4Ralpha signalling is required for allergen induced up-regulation of GABAergic signalling and GCM. Methods BALB/c mice were exposed to an acute house dust mite (HDM) protocol and received vehicle, anti-IL-4Ralpha-monoclonal antibody, or control antibody. Outcomes included airway responses to inhaled methacholine (MCh), histology for eosinophilia and GCM, phosphorylated STAT6 levels using immunohistochemistry and immunoblot, and glutamic acid decarboxylase (GAD) 65/67 and GABA(A)beta(2/3) receptor subunit expression using confocal microscopy. Results Acute HDM exposure resulted in increased airway responses to MCh, lung eosinophilia, STAT6 phosphorylation, elevations in GAD65/67 and GABA(A)beta(2/3) receptor expression, and GCM that were inhibited with anti-IL-4Ralpha-monoclonal treatment. Control antibody had no effect. Conclusion The IL-4Ralpha is required for allergen-induced up-regulation of a GABAergic system in airway epithelium implicated in GCM following acute HDM exposure.

Gamma-aminobutyric acid nurtures allergic asthma.

Asthma often occurs as a result of immune-based inflammatory responses, which consequently cause pathological changes in airway structural cells. However, the underlying mechanisms of airway pathology in asthma are still not fully understood. Our recent studies revealed a critical role of gamma-aminobutyric acid (GABA) signalling pathway in the airway epithelium of allergic asthma through its ability to stimulate mucus production. This review briefly describes the GABAergic signalling system and its role in the regulation of mucus protein production in bronchial airway epithelial cells.

Nonsteroidal anti-inflammatory drugs upregulate function of wild-type and mutant CFTR.

Small-scale clinical trials show that treatment of cystic fibrosis (CF) patients with ibuprofen, a nonsteroidal anti-inflammatory drug, improves the symptoms of CF and slows down the decline of lung function. Paradoxically, ibuprofen inhibits ligand-stimulated CF transmembrance conductance regulator (CFTR) activity. The aim of the present study was to investigate the effects of ibuprofen on CFTR function under different conditions. Patch-clamp recordings were performed in two lines of human airway epithelial cells: IB3-8-3-7 cells, which express wild-type CFTR; and IB3-1 cells, which express the variant CFTR with deletion of phenylalanine 580 (DeltaF580CFTR). Addition of ibuprofen to the extracellular solution caused a rapid inhibition of CFTR activity in IB3-8-3-7 cells in the presence of a high intracellular concentration of cAMP, whereas ibuprofen enhanced the CFTR conductance at low levels of cAMP. Introducing ibuprofen into the interior of cells occluded the enhancing effect of ibuprofen. Notably, the variant CFTR-mediated conductance was detected in IB3-1 cells treated with myoinositol and was enhanced by ibuprofen at endogenous levels of cAMP. In summary, nonsteroidal anti-inflammatory drugs increase the function of both wild-type cystic fibrosis transmembrane conductance regulator and the phenylalanine 580 deletion in cultured human airway epithelial cells at endogenous levels of cAMP.

Non-steroidal anti-inflammatory drugs increase insulin release from beta cells by inhibiting ATP-sensitive potassium channels.

BACKGROUND AND PURPOSE: Some non-steroidal anti-inflammatory drugs (NSAIDs) incidentally induce hypoglycemia, which is often seen in diabetic patients receiving sulphonylureas. NSAIDs influence various ion channel activities, thus they may cause hypoglycemia by affecting ion channel functions in insulin secreting beta cells. This study investigated the effects of the NSAID meclofenamic acid (MFA) on the electrical excitability and the secretion of insulin from pancreatic beta cells. EXPERIMENTAL APPROACH: Using patch clamp techniques and insulin secretion assays, the effects of MFA on the membrane potential and transmembrane current of INS-1 cells, and insulin secretion were studied. KEY RESULTS: Under perforated patch recordings, MFA induced a rapid depolarization in INS-1 cells bathed in low (2.8 mM), but not high (28 mM) glucose solutions. MFA, as well as acetylsalicylic acid (ASA) and flufenamic acid (FFA), excited the cells by inhibiting ATP-sensitive potassium channels (K(ATP)). In whole cell recordings, K(ATP) conductance consistently appeared when intracellular ATP was diluted. Intracellular glibenclamide prevented the development of K(ATP) activity, whereas intracellular MFA had no effect. At low glibenclamide concentrations, MFA induced additional inhibition of the K(ATP) current. Live cell Ca(2+) imaging displayed that MFA elevated intracellular Ca(2+) at low glucose concentrations. Furthermore, MFA dose-dependently increased insulin release under low, but not high, glucose conditions. CONCLUSIONS AND IMPLICATIONS: MFA blocked K(ATP) through an extracellular mechanism and thus increased insulin secretion. As some NSAIDs synergistically inhibit K(ATP) activity together with sulphonylureas, the risk of NSAID-induced hypoglycemia should be considered when glucose-lowering compounds are administered.

Gamma-aminobutyric acid up- and downregulates insulin secretion from beta cells in concert with changes in glucose concentration.

AIMS/HYPOTHESIS: The role of gamma-aminobutyric acid (GABA) and A-type GABA receptors (GABA(A)Rs) in modulating islet endocrine function has been actively investigated since the identification of GABA and GABA(A)Rs in the pancreatic islets. However, the reported effects of GABA(A)R activation on insulin secretion from islet beta cells have been controversial. METHODS: This study examined the hypothesis that the effect of GABA on beta cell insulin secretion is dependent on glucose concentration. RESULTS: Perforated patch-clamp recordings in INS-1 cells demonstrated that GABA, at concentrations ranging from 1 to 1,000 micromol/l, induced a transmembrane current (I(GABA)) which was sensitive to the GABA(A)R antagonist bicuculline. The current-voltage relationship revealed that I(GABA) reversed at -42+/-2.2 mV, independently of glucose concentration. Nevertheless, the glucose concentration critically controlled the membrane potential (V (M)), i.e., at low glucose (0 or 2.8 mmol/l) the endogenous V (M) of INS-1 cells was below the I(GABA) reversal potential and at high glucose (16.7 or 28 mmol/l), the endogenous V (M) of INS-1 cells was above the I(GABA) reversal potential. Therefore, GABA dose-dependently induced membrane depolarisation at a low glucose concentration, but hyperpolarisation at a high glucose concentration. Consistent with electrophysiological findings, insulin secretion assays demonstrated that at 2.8 mmol/l glucose, GABA increased insulin secretion in a dose-dependent fashion (p<0.05, n=7). This enhancement was blocked by bicuculline (p<0.05, n=4). In contrast, in the presence of 28 mmol/l glucose, GABA suppressed the secretion of insulin (p<0.05, n=5). CONCLUSIONS/INTERPRETATION: These findings indicate that activation of GABA(A)Rs in beta cells regulates insulin secretion in concert with changes in glucose levels.

Role of barium nitrate on the sulfur fixation of calcium oxide.

In this paper, the effect of Ba(NO3)2 on the efficiency of sulfur fixation of calcium oxide during coal combustion was studied. The results showed that addition of barium nitrate to the CaO can enhance the sulfur removal rate of CaO significantly. The X-ray diffraction spectrum of residual ash of coal added some sulfur fixative expressed that Ba2+ can form a compound of Ba-Al-Si-O which encloses the CaSO4 to prevent it's decomposition, so Ba2+ can improve the action of sulfur fixation of CaO. The combustion character of the original coal and original coal added sulfur fixative was researched with thermal-gravity analyzer and the results expressed that adding some sulfur fixative to the coal will make the combustion character of coal change little.

Convergence of PKC-dependent kinase signal cascades on NMDA receptors.

Synaptic plasticity, or long-term potentiation (LTP), of excitatory synapses in the hippocampus contributes to learning and the establishment of spatial memories. In the CA1 region, induction of LTP enhances the function of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) because of the Ca2+-calmodulin kinase II (CaMKII)-dependent phosphorylation of this subtype of glutamate receptor. Entry of Ca2+, via N-methyl-D-aspartate receptors (NMDARs), during strong synaptic stimulation provides the stimulus to trigger phosphorylation of AMPARs. However, this induction also requires activation of a protein kinase C (PKC)-dependent tyrosine kinase signal cascade and a concomitant upregulation of NMDARs. This review focuses upon NMDARs as potential targets of PKC and/or of the PKC-dependent tyrosine kinase cascade. PKC, acting via the CAKbeta/Src tyrosine kinase cascade, enhances NMDAR activation and may increase the number of receptors expressed in synapses. In contrast, direct phosphorylation of NMDARs by PKC increases the sensitivity of NMDA channel inactivation to intracellular Ca2+. In CAI neurons, PKC provides a point of convergence of control of NMDARs and synaptic plasticity for a wide variety of G-protein coupled and growth factor receptors.

In CA1 pyramidal neurons of the hippocampus protein kinase C regulates calcium-dependent inactivation of NMDA receptors.

The NMDA subtype of the glutamate-gated channel exhibits a high permeability to Ca(2+). The influx of Ca(2+) through NMDA channels is limited by a rapid and Ca(2+)/calmodulin (CaM)-dependent inactivation that results from a competitive displacement of cytoskeleton-binding proteins from the NR1 subunit of the receptor by Ca(2+)/CaM (Zhang et al., 1998; Krupp et al., 1999). The C terminal of this subunit can be phosphorylated by protein kinase C (PKC) (Tingley et al., 1993). The present study sought to investigate whether PKC regulates Ca(2+)-dependent inactivation of the NMDA channel in hippocampal neurons. Activation of endogenous PKC by 4beta-phorbol 12-myristate 13-acetate enhanced peak (I(p)) and depressed steady-state (I(ss)) NMDA-evoked currents, resulting in a reduction in the ratio of these currents (I(ss)/I(p)). We demonstrated previously that PKC activity enhances I(P) via a sequential activation of the focal adhesion kinase cell adhesion kinase beta/proline-rich tyrosine kinase 2 (CAKbeta/Pyk2) and the nonreceptor tyrosine kinase Src (Huang et al., 1999; Lu et al., 1999). Here, we report that the PKC-induced depression of I(ss) is unrelated to the PKC/CAKbeta/Src-signaling pathway but depends on the concentration of extracellular Ca(2+). Intracellular applications of CaM reduced I(ss)/I(p) and occluded the Ca(2+)-dependent effect of phorbol esters on I(ss.) Moreover, increasing the concentration of intracellular Ca(2+) buffer or intracellular application of the inhibitory CaM-binding peptide (KY9) greatly reduced the phorbol ester-induced depression of I(ss). Taken together, these results suggest that PKC enhances Ca(2+)/CaM-dependent inactivation of the NMDA channel, most likely because of a phosphorylation-dependent regulation of interactions between receptor subunits, CaM, and other postsynaptic density proteins.

Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis.

Eglin c from the leech Hirudo medicinalis is a potent protein inhibitor of many serine proteinases including chymotrypsin and subtilisins. Unlike most small protein inhibitors whose solvent-exposed enzyme-binding loop is stabilized primarily by disulfide bridges flanking the reactive-site peptide bond, eglin c possesses an enzyme-binding loop supported predominantly by extensive electrostatic/H-bonding interactions involving three Arg residues (Arg48, Arg51, and Arg53) projecting from the scaffold of the inhibitor. As an adjacent residue, the C-terminal Gly70 participates in these interactions via its alpha-carboxyl group interacting with the side chain of Arg51 and the main chain of Arg48. In addition, the amide NH group of Gly70 donates an H-bond to the carbonyl C=O groups of Arg48 and Arg51. To understand the structural and functional relevance of the electrostatic/H-bonding network, we chemically synthesized wild-type eglin c and three analogues in which Gly70 was either deleted or replaced by glycine amide (NH(2)CH(2)CONH(2)) or by alpha-hydroxylacetamide (HOCH(2)CONH(2)). NMR analysis indicated that the core structure of eglin c was maintained in the analogues, but that the binding loop was significantly perturbed. It was found that deletion or replacement of Gly70 destabilized eglin c by an average of 2.7 kcal/mol or 20 degrees C in melting temperature. As a result, these inhibitors become substrates for their target enzymes. Binding assays on these analogues with a catalytically incompetent subtilisin BPN' mutant indicated that loss or weakening of the interactions involving the carboxylate of Gly70 caused a decrease in binding by approximately 2 orders of magnitude. Notably, for all four synthetic inhibitors, the relative free energy changes (DeltaDeltaG) associated with protein destabilization are strongly correlated (slope = 0.94, r(2) = 0. 9996) with the DeltaDeltaG values derived from a decreased binding to the enzyme.

Distinct roles of synaptic and extrasynaptic NMDA receptors in excitotoxicity.

Excitatory synaptic activity governs excitotoxicity and modulates the distribution of NMDA receptors (NMDARs) among synaptic and extrasynaptic sites of central neurons. We investigated whether NMDAR localization was functionally linked to excitotoxicity by perturbing F-actin, a cytoskeletal protein that participates in targeting synaptic NMDARs in dendritic spines. Depolymerizing F-actin did not affect NMDA-evoked whole-cell currents. However, the number of dendritic NMDAR clusters and the NMDAR-mediated component of miniature spontaneous EPSCs were reduced, whereas the number of AMPA receptor clusters and AMPA receptor-mediated component of EPSCs was unchanged. This selective perturbation of synaptically activated NMDARs had no effect on neuronal death or the accumulation of (45)Ca(2+) evoked by applying exogenous NMDA or L-glutamate, which reach both synaptic and extrasynaptic receptors. However, it increased survival and decreased (45)Ca(2+) accumulation in neurons exposed to oxygen glucose deprivation, which causes excitotoxicity by glutamate release at synapses. Thus, synaptically and extrasynaptically activated NMDARs are equally capable of excitotoxicity. However, their relative contributions vary with the location of extracellular excitotoxin accumulation, a factor governed by the mechanism of extracellular neurotransmitter accumulation, not the synaptic activation of NMDARs.