Loss and re-adaptation of lumbar intervertebral disc water signal intensity after prolonged bedrest

The adaptation and re-adaptation process of the intervertebral disc (IVD) to prolonged bedrest is important for understanding IVD physiology and IVD herniations in astronauts. Little information is available on changes in IVD composition. In this study, 24 male subjects underwent 60-day bedrest and In/Out Phase magnetic resonance imaging sequences were performed to evaluate IVD shape and water signal intensity. Scanning was performed before bedrest (baseline), twice during bedrest, and three, six and twenty-four months after bedrest. Area, signal intensity, average height, and anteroposterior diameter of the lumbar L3/4 and L4/5 IVDs were measured. At the end of bedrest, disc height and area were significantly increased with no change in water signal intensity. After bedrest, we observed reduced IVD signal intensity three months (p=0.004 versus baseline), six months (p=0.003 versus baseline), but not twenty-four months (p=0.25 versus baseline) post-bedrest. At these same time points post-bedrest, IVD height and area remained increased. The reduced lumbar IVD water signal intensity in the first months after bedrest implies a reduction of glycosaminoglycans and/or free water in the IVD. Subsequently, at two years after bedrest, IVD hydration status returned towards pre-bedrest levels, suggesting a gradual, but slow, re-adaptation process of the IVD after prolonged bedrest.     

TNFα Transport Induced by Dynamic Loading Alters Biomechanics of Intact Intervertebral Discs

Objective

Intervertebral disc (IVD) degeneration is an important contributor to the development of back pain, and a key factor relating pain and degeneration are the presence of pro-inflammatory cytokines and IVD motion. There is surprisingly limited understanding of how mechanics and inflammation interact in the IVD. This study investigated interactions between mechanical loading and pro-inflammatory cytokines in a large animal organ culture model to address fundamental questions regarding (i.) how inflammatory mediators arise within the IVD, (ii.) how long inflammatory mediators persist, and (iii.) how inflammatory mediators influence IVD biomechanics.

Methods

Bovine caudal IVDs were cultured for 6 or 20-days under static & dynamic loading with or without exogenous TNFα in the culture medium, simulating a consequence of inflammation of the surrounding spinal tissues. TNFα transport within the IVD was assessed via immunohistochemistry. Changes in IVD structural integrity (dimensions, histology & aggrecan degradation), biomechanical behavior (Creep, Recovery & Dynamic stiffness) and pro-inflammatory cytokines in the culture medium (ELISA) were assessed.

Results

TNFα was able to penetrate intact IVDs when subjected to dynamic loading but not static loading. Once transported within the IVD, pro-inflammatory mediators persisted for 4–8 days after TNFα removal. TNFα exposure induced changes in IVD biomechanics (reduced diurnal displacements & increased dynamic stiffness).

Discussion

This study demonstrated that exposure to TNFα, as might occur from injured surrounding tissues, can penetrate healthy intact IVDs, induce expression of additional pro-inflammatory cytokines and alter IVD mechanical behavior. We conclude that exposure to pro-inflammatory cytokine may be an initiating event in the progression of IVD degeneration in addition to being a consequence of disease.     

The Value of In Vitro Diagnostic Testing in Medical Practice: A Status Report

BackgroundIn vitro diagnostic (IVD) investigations are indispensable for routine patient management. Appropriate testing allows early-stage interventions, reducing late-stage healthcare expenditure (HCE).AimTo investigate HCE on IVDs in two developed markets and to assess the perceived value of IVDs on clinical decision-making. Physician-perceived HCE on IVD was evaluated, as well as desired features of new diagnostic markers.MethodsPast and current HCE on IVD was calculated for the US and Germany. A total of 79 US/German oncologists and cardiologists were interviewed to assess the number of cases where: physicians ask for IVDs; IVDs are used for initial diagnosis, treatment monitoring, or post-treatment; and decision-making is based on an IVD test result. A sample of 201 US and German oncologists and cardiologists was questioned regarding the proportion of HCE they believed to be attributable to IVD testing. After disclosing the actual IVD HCE, the physician’s perception of the appropriateness of the amount was captured. Finally, the association between physician-rated impact of IVD on decision-making and perceived contribution of IVD expenditure on overall HCE was assessed.ResultsIVD costs account for 2.3% and 1.4% of total HCE in the US and Germany. Most physicians (81%) believed that the actual HCE on IVDs was >5%; 19% rated the spending correctly (0–4%, p<0.001). When informed of the actual amount, 64% of physicians rated this as appropriate (p<0.0001); 66% of decision-making was based on IVD. Significantly, more physicians asked for either additional clinical or combined clinical/health economic data than for the product (test/platform) alone (p<0.0001).ConclusionsOur results indicate a poor awareness of actual HCE on IVD, but a high attributable value of diagnostic procedures for patient management. New markers should deliver actionable and medically relevant information, to guide decision-making and foster improved patient outcomes.     

Caloric Restriction Decreases Orthostatic Tolerance Independently from 6° Head-Down Bedrest

Astronauts consume fewer calories during spaceflight and return to earth with an increased risk of orthostatic intolerance. Whether a caloric deficiency modifies orthostatic responses is not understood. Thus, we determined the effects of a hypocaloric diet (25% caloric restriction) during 6° head down bedrest (an analog of spaceflight) on autonomic neural control during lower body negative pressure (LBNP). Nine healthy young men completed a randomized crossover bedrest study, consisting of four (2 weeks each) interventions (normocaloric bedrest, normocaloric ambulatory, hypocaloric bedrest, hypocaloric ambulatory), each separated by 5 months. Muscle sympathetic nerve activity (MSNA) was recorded at baseline following normocaloric and hypocaloric interventions. Heart rate (HR) and arterial pressure were recorded before, during, and after 3 consecutive stages (7 min each) of LBNP (-15, -30, -45 mmHg). Caloric and posture effects during LBNP were compared using two-way ANOVA with repeated measures. There was a strong trend toward reduced basal MSNA following caloric restriction alone (normcaloric vs. hypocaloric: 22±3 vs. 14±4 burst/min, p = 0.06). Compared to the normocaloric ambulatory, both bedrest and caloric restriction were associated with lower systolic blood pressure during LBNP (p<0.01); however, HR responses were directionally opposite (i.e., increase with bedrest, decrease with caloric restriction). Survival analysis revealed a significant reduction in orthostatic tolerance following caloric restriction (normocaloric finishers: 12/16; hypocaloric finishers: 6/16; χ2, p = 0.03). Caloric restriction modifies autonomic responses to LBNP, which may decrease orthostatic tolerance after spaceflight.     

Thyroid Hormone Signaling In Vivo Requires a Balance between Coactivators and Corepressors

Resistance to thyroid hormone (RTH), a human syndrome, is characterized by high thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels. Mice with mutations in the thyroid hormone receptor beta (TRβ) gene that cannot bind steroid receptor coactivator 1 (SRC-1) and Src-1^−/− mice both have phenotypes similar to that of RTH. Conversely, mice expressing a mutant nuclear corepressor 1 (Ncor1) allele that cannot interact with TRβ, termed NCoRΔID, have low TH levels and normal TSH. We hypothesized that Src-1^−/− mice have RTH due to unopposed corepressor action. To test this, we crossed NCoRΔID and Src-1^−/− mice to create mice deficient for coregulator action in all cell types. Remarkably, NCoR^ΔID/ΔID Src-1^−/− mice have normal TH and TSH levels and are triiodothryonine (T₃) sensitive at the level of the pituitary. Although absence of SRC-1 prevented T₃ activation of key hepatic gene targets, NCoR^ΔID/ΔID Src-1^−/− mice reacquired hepatic T₃ sensitivity. Using in vivo chromatin immunoprecipitation assays (ChIP) for the related coactivator SRC-2, we found enhanced SRC-2 recruitment to TR-binding regions of genes in NCoR^ΔID/ΔID Src-1^−/− mice, suggesting that SRC-2 is responsible for T₃ sensitivity in the absence of NCoR1 and SRC-1. Thus, T₃ targets require a critical balance between NCoR1 and SRC-1. Furthermore, replacement of NCoR1 with NCoRΔID corrects RTH in Src-1^−/− mice through increased SRC-2 recruitment to T₃ target genes.     

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Introduction

The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.

Methods

Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.

Results

LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.

Conclusions

Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.     

Simultaneous Imaging of CBF Change and BOLD with Saturation-Recovery-T1 Method

A neuroimaging technique based on the saturation-recovery (SR)-T₁ MRI method was applied for simultaneously imaging blood oxygenation level dependence (BOLD) contrast and cerebral blood flow change (ΔCBF), which is determined by CBF-sensitive T₁ relaxation rate change (ΔR₁ ^CBF). This technique was validated by quantitatively examining the relationships among ΔR₁ ^CBF, ΔCBF, BOLD and relative CBF change (rCBF), which was simultaneously measured by laser Doppler flowmetry under global ischemia and hypercapnia conditions, respectively, in the rat brain. It was found that during ischemia, BOLD decreased 23.1±2.8% in the cortical area; ΔR₁ ^CBF decreased 0.020±0.004s^-1 corresponding to a ΔCBF decrease of 1.07±0.24 ml/g/min and 89.5±1.8% CBF reduction (n=5), resulting in a baseline CBF value (=1.18 ml/g/min) consistent with the literature reports. The CBF change quantification based on temperature corrected ΔR₁ ^CBF had a better accuracy than apparent R₁ change (ΔR₁ ^app); nevertheless, ΔR₁ ^app without temperature correction still provides a good approximation for quantifying CBF change since perfusion dominates the evolution of the longitudinal relaxation rate (R₁ ^app). In contrast to the excellent consistency between ΔCBF and rCBF measured during and after ischemia, the BOLD change during the post-ischemia period was temporally disassociated with ΔCBF, indicating distinct CBF and BOLD responses. Similar results were also observed for the hypercapnia study. The overall results demonstrate that the SR-T₁ MRI method is effective for noninvasive and quantitative imaging of both ΔCBF and BOLD associated with physiological and/or pathological changes.     

Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

Germination of Bacillus spores with a high pressure (HP) of ∼150 MPa is via activation of spores'' germinant receptors (GRs). The HP germination of multiple individual Bacillus subtilis spores in a diamond anvil cell (DAC) was monitored with phase-contrast microscopy. Major conclusions were that (i) >95% of wild-type spores germinated in 40 min in a DAC at ∼150 MPa and 37°C but individual spores'' germination kinetics were heterogeneous; (ii) individual spores'' HP germination kinetic parameters were similar to those of nutrient-triggered germination with a variable lag time (T_lag) prior to a period of the rapid release (ΔT_release) of the spores'' dipicolinic acid in a 1:1 chelate with Ca²⁺ (CaDPA); (iii) spore germination at 50 MPa had longer average T_lag values than that at ∼150 MPa, but the ΔT_release values at the two pressures were identical and HPs of <10 MPa did not induce germination; (iv) B. subtilis spores that lacked the cortex-lytic enzyme CwlJ and that were germinated with an HP of 150 MPa exhibited average ΔT_release values ∼15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average T_lag values; and (v) the germination of wild-type spores given a ≥30-s 140-MPa HP pulse followed by a constant pressure of 1 MPa was the same as that of spores exposed to a constant pressure of 140 MPa that was continued for ≥35 min; (vi) however, after short 150-MPa HP pulses and incubation at 0.1 MPa (ambient pressure), spore germination stopped 5 to 10 min after the HP was released. These results suggest that an HP of ∼150 MPa for ≤30 s is sufficient to fully activate spores'' GRs, which remain activated at 1 MPa but can deactivate at ambient pressure.     

Slow Leakage of Ca-Dipicolinic Acid from Individual Bacillus Spores during Initiation of Spore Germination

When exposed to nutrient or nonnutrient germinants, individual Bacillus spores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ∼20% of spore DPA) from individual spores that takes place prior to the lag time, T_lag, when spores begin rapid release of remaining DPA. Major conclusions from this work with Bacillus subtilis spores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous time T₁; (ii) the period of slow DPA leakage (ΔT_leakage = T_lag − T₁) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreased T₁ times but increased values of ΔT_leakage; (iv) upon germination with l-valine for 10 min followed by addition of d-alanine to block further germination, all germinated spores had T₁ times of less than 10 min, suggesting that T₁ is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreased T₁ and T_lag times but not the amount of DPA released in ΔT_leakage; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ΔT_leakage times in which more DPA was released; and (vii) there was slow DPA leakage early in germination of B. subtilis spores by the nonnutrients CaDPA and dodecylamine and in nutrient germination of Bacillus cereus and Bacillus megaterium spores. Overall, these findings have identified and characterized a new early event in Bacillus spore germination.     

Random Coil to Globular Thermal Response of a Protein (H3.1) with Three Knowledge-Based Coarse-Grained Potentials

The effect of temperature on the conformation of a histone (H3.1) is studied by a coarse-grained Monte Carlo simulation based on three knowledge-based contact potentials (MJ, BT, BFKV). Despite unique energy and mobility profiles of its residues, the histone H3.1 undergoes a systematic (possibly continuous) structural transition from a random coil to a globular conformation on reducing the temperature. The range over which such a systematic response in variation of the radius of gyration (R_g) with the temperature (T) occurs, however, depends on the potential, i.e. ΔT_MJ ≈ 0.013–0.020, ΔT_BT ≈ 0.018–0.026, and ΔT_BFKV ≈ 0.006–0.013 (in reduced unit). Unlike MJ and BT potentials, results from the BFKV potential show an anomaly where the magnitude of R_g decreases on raising the temperature in a range ΔT_A ≈ 0.015–0.018 before reaching its steady-state random coil configuration. Scaling of the structure factor, S(q) ∝ q^−1/ν, with the wave vector, q = 2π/λ, and the wavelength, λ, reveals a systematic change in the effective dimension (D_e∼1/ν) of the histone with all potentials (MJ, BT, BFKV): D_e∼3 in the globular structure with D_e∼2 for the random coil. Reproducibility of the general yet unique (monotonic) structural transition of the protein H3.1 with the temperature (in contrast to non-monotonic structural response of a similar but different protein H2AX) with three interaction sets shows that the knowledge-based contact potential is viable tool to investigate structural response of proteins. Caution should be exercise with the quantitative comparisons due to differences in transition regimes with these interactions.     

The First Identification of Carbohydrate Binding Modules Specific to Chitosan

Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (T_m) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)₆. The T_m elevation (ΔT_m) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)₆ and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔT_m = 13.6 °C), when (GlcN)₆ was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)_n; ΔG_r° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)_n with the lower affinities (ΔG_r° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)₆ for individual DD1 and DD2 sites (ΔG_r° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)_n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.     

Accelerated Recovery of Mitochondrial Membrane Potential by GSK-3β Inactivation Affords Cardiomyocytes Protection from Oxidant-Induced Necrosis

Loss of mitochondrial membrane potential (ΔΨ_m) is known to be closely linked to cell death by various insults. However, whether acceleration of the ΔΨ_m recovery process prevents cell necrosis remains unclear. Here we examined the hypothesis that facilitated recovery of ΔΨ_m contributes to cytoprotection afforded by activation of the mitochondrial ATP-sensitive K⁺ (mK_ATP) channel or inactivation of glycogen synthase kinase-3β (GSK-3β). ΔΨ_m of H9c2 cells was determined by tetramethylrhodamine ethyl ester (TMRE) before or after 1-h exposure to antimycin A (AA), an inducer of reactive oxygen species (ROS) production at complex III. Opening of the mitochondrial permeability transition pore (mPTP) was determined by mitochondrial loading of calcein. AA reduced ΔΨ_m to 15±1% of the baseline and induced calcein leak from mitochondria. ΔΨ_m was recovered to 51±3% of the baseline and calcein-loadable mitochondria was 6±1% of the control at 1 h after washout of AA. mK_ATP channel openers improved the ΔΨ_m recovery and mitochondrial calcein to 73±2% and 30±7%, respectively, without change in ΔΨ_m during AA treatment. Activation of the mK_ATP channel induced inhibitory phosphorylation of GSK-3β and suppressed ROS production, LDH release and apoptosis after AA washout. Knockdown of GSK-3β and pharmacological inhibition of GSK-3β mimicked the effects of mK_ATP channel activation. ROS scavengers administered at the time of AA removal also improved recovery of ΔΨ_m. These results indicate that inactivation of GSK-3β directly or indirectly by mK_ATP channel activation facilitates recovery of ΔΨ_m by suppressing ROS production and mPTP opening, leading to cytoprotection from oxidant stress-induced cell death.     

Reciprocally Coupled Residues Crucial for Protein Kinase Pak2 Activity Calculated by Statistical Coupling Analysis

Regulation of Pak2 activity involves at least two mechanisms: (i) phosphorylation of the conserved Thr⁴⁰² in the activation loop and (ii) interaction of the autoinhibitory domain (AID) with the catalytic domain. We collected 482 human protein kinase sequences from the kinome database and globally mapped the evolutionary interactions of the residues in the catalytic domain with Thr⁴⁰² by sequence-based statistical coupling analysis (SCA). Perturbation of Thr⁴⁰² (34.6%) suggests a communication pathway between Thr⁴⁰² in the activation loop, and Phe³⁸⁷ (ΔΔE_387F,402T = 2.80) in the magnesium positioning loop, Trp⁴²⁷ (ΔΔE_427W,402T = 3.12) in the F-helix, and Val⁴⁰⁴ (ΔΔE_404V,402T = 4.43) and Gly⁴⁰⁵ (ΔΔE_405G,402T = 2.95) in the peptide positioning loop. When compared to the cAMP-dependent protein kinase (PKA) and Src, the perturbation pattern of threonine phosphorylation in the activation loop of Pak2 is similar to that of PKA, and different from the tyrosine phosphorylation pattern of Src. Reciprocal coupling analysis by SCA showed the residues perturbed by Thr⁴⁰² and the reciprocal coupling pairs formed a network centered at Trp⁴²⁷ in the F-helix. Nine pairs of reciprocal coupling residues crucial for enzymatic activity and structural stabilization were identified. Pak2, PKA and Src share four pairs. Reciprocal coupling residues exposed to the solvent line up as an activation groove. This is the inhibitor (PKI) binding region in PKA and the activation groove for Pak2. This indicates these evolutionary conserved residues are crucial for the catalytic activity of PKA and Pak2.     

Deletion of Vacuolar Proton-translocating ATPase Voa Isoforms Clarifies the Role of Vacuolar pH as a Determinant of Virulence-associated Traits in Candida albicans

Vacuolar proton-translocating ATPase (V-ATPase) is a central regulator of cellular pH homeostasis, and inactivation of all V-ATPase function has been shown to prevent infectivity in Candida albicans. V-ATPase subunit a of the V_o domain (V_oa) is present as two fungal isoforms: Stv1p (Golgi) and Vph1p (vacuole). To delineate the individual contribution of Stv1p and Vph1p to C. albicans physiology, we created stv1Δ/Δ and vph1Δ/Δ mutants and compared them to the corresponding reintegrant strains (stv1Δ/ΔR and vph1Δ/ΔR). V-ATPase activity, vacuolar physiology, and in vitro virulence-related phenotypes were unaffected in the stv1Δ/Δ mutant. The vph1Δ/Δ mutant exhibited defective V₁V_o assembly and a 90% reduction in concanamycin A-sensitive ATPase activity and proton transport in purified vacuolar membranes, suggesting that the Vph1p isoform is essential for vacuolar V-ATPase activity in C. albicans. The vph1Δ/Δ cells also had abnormal endocytosis and vacuolar morphology and an alkalinized vacuolar lumen (pH_vph1Δ_/Δ = 6.8 versus pH_vph1Δ_/Δ_R = 5.8) in both yeast cells and hyphae. Secreted protease and lipase activities were significantly reduced, and M199-induced filamentation was impaired in the vph1Δ/Δ mutant. However, the vph1Δ/Δ cells remained competent for filamentation induced by Spider media and YPD, 10% FCS, and biofilm formation and macrophage killing were unaffected in vitro. These studies suggest that different virulence mechanisms differentially rely on acidified vacuoles and that the loss of both vacuolar (Vph1p) and non-vacuolar (Stv1p) V-ATPase activity is necessary to affect in vitro virulence-related phenotypes. As a determinant of C. albicans pathogenesis, vacuolar pH alone may prove less critical than originally assumed.     

Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells

IntroductionNutrient deprivation is a likely contributor to intervertebral disc (IVD) degeneration. Silent mating type information regulator 2 homolog 1 (SIRT1) protects cells against limited nutrition by modulation of apoptosis and autophagy. However, little evidence exists regarding the extent to which SIRT1 affects IVD cells. Therefore, we conducted an in vitro study using human IVD nucleus pulposus (NP) cells.MethodsThirty-two IVD specimens were obtained from patients who underwent surgical intervention and were categorized based on Pfirrmann IVD degeneration grades. Cells were isolated from the NP and cultured in the presence of recombinant human SIRT1 (rhSIRT1) under different serum conditions, including 10 % (v/v) fetal bovine serum (FBS) as normal nutrition (N) and 1 % (v/v) FBS as low nutrition (LN). 3-Methyladenine (3-MA) was used to inhibit autophagy. Autophagic activity was assessed by measuring the absorbance of monodansylcadaverine and immunostaining and Western blotting for light chain 3 and p62/SQSTM1. Apoptosis and pathway analyses were performed by flow cytometry and Western blotting.ResultsCells cultured under LN conditions decreased in number and exhibited enhanced autophagy compared with the N condition. Medium supplementation with rhSIRT1 inhibited this decrease in cell number and induced an additional increase in autophagic activity (P < 0.05), whereas the combined use of rhSIRT1 and 3-MA resulted in drastic decreases in cell number and autophagy (P < 0.05). The incidence of apoptotic cell death increased under the LN condition, which was decreased by rhSIRT1 (P < 0.05) but increased further by a combination of rhSIRT1 and 3-MA (P < 0.05). Under LN conditions, NP cells showed a decrease in antiapoptotic Bcl-2 and an increase in proapoptotic Bax, cleaved caspase 3, and cleaved caspase 9, indicating apoptosis induction via the mitochondrial pathway. These changes were suppressed by rhSIRT1 but elevated further by rhSIRT1 with 3-MA, suggesting an effect of rhSIRT1-induced autophagy on apoptosis inhibition. Furthermore, the observed autophagy and apoptosis were more remarkable in cells from IVDs of Pfirrmann grade IV than in those from IVDs of Pfirrmann grade II.ConclusionsSIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human IVD NP cells, suggesting that rhSIRT1 may be a potent treatment agent for human degenerative IVD disease.     

Comparison of Apparent Diffusion Coefficient and T2 Relaxation Time Variation Patterns in Assessment of Age and Disc Level Related Intervertebral Disc Changes

Purpose

To compare the variation patterns of ADC and T2 values in different age and intervertebral disc (IVD) levels, thus to identify their sensitivities in assessing age and disc level related IVDs changes.

Materials and Methods

The T2 and ADC values were recorded from 345 IVDs of 69 volunteers. Kendall''s correlation analysis was used to identify the relationship between age and T2/ADC mean values respectively. The one-way analysis of variance (ANOVA) with post hoc analysis was then applied to test the differences of T2 and ADC values among different IVD levels and age groups, followed by linear regression analysis between age (<45 and >45 years) and T2/ADC mean values. This study was approved by the Ethics Committee of the Chinese Academy of Medical Sciences and the Peking Union Medical College Hospital.

Results

Significant negative correlation was observed between age and T2/ADC mean values. The T2 and ADC values showed significant differences among IVD levels and among age groups except for T2 values in age group 1 (25–34 years) and group 2 (35–44 years), and for ADC values at L1–2 level. Both T2 and ADC values showed significant differences between young (age<45 years) and elderly group (age>45 years) at each IVD level. A linear relationship was observed between age and T2/ADC mean values in the elderly group as well as in the young group for the ADC mean values, while no such tendency was identified in the young group for the T2 mean values.

Conclusions

ADC values may be a more sensitive parameter than T2 in assessing age and disc level related intervertebral disc changes.     

The Effect of Proteolytic Enzymes on E. coli Phages and on Native Proteins

Lambda coli phage is not inactivated by chymotrypsin, trypsin, or ficin. T₂ phage is slowly inactivated by high concentrations of (α-, β-, γ-, or Δ-chymotrypsin, but not by trypsin or ficin. P₁ phage is slowly inactivated by α-, β-, or γ-chymotrypsin, or ficin, more rapidly by Δ-chymotrypsin, and much more rapidly by trypsin. Crystalline egg albumin, crystalline serum albumin, E. coli nucleoprotein, and yeast nucleoprotein are hydrolyzed slowly by α-chymotrypsin. Yeast nucleoprotein, like P₁ phage, is hydrolyzed more rapidly by Δ-chymotrypsin than by α-chymotrypsin, but not by trypsin or ficin. Neither phages nor native proteins were attacked by papain, carboxypeptidase, deoxyribonuclease, or ribonuclease.     

Cold denaturation of monoclonal antibodies

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔG_u, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔG_u versus temperature data from fluorescence gave a ΔC_p of 8.0 kcal mol⁻¹ K⁻¹, a maximum apparent stability of 23.7 kcal mol⁻¹ at 18°C, and an apparent cold denaturation temperature (T_CD) of −23°C. ΔG_u values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T_CD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.Key words: monoclonal antibodies, thermodynamic stability, cold denaturation, free energy, fluorescence     

Replacing with whole grains and legumes reduces Lp-PLA2 activities in plasma and PBMCs in patients with prediabetes or T2D

To determine dietary effects on circulating lipoprotein-associated phospholipase A₂ (Lp-PLA₂) activity and enzyme activity in peripheral blood mononuclear cells (PBMCs), 99 patients with impaired fasting glucose, impaired glucose tolerance, or newly-diagnosed T2D were randomly assigned to either a control group (usual diet with refined rice) or the whole grain and legume group. Substitution of whole grains and legumes for refined rice was associated with the replacement of 7% of energy from carbohydrates with energy from protein (about 4%) and fat. After 12 weeks, the whole grain and legume group showed a significant decrease in fasting glucose, insulin, homeostasis model assessment-insulin resistance, hemoglobin A_1c, malondialdehyde, plasma Lp-PLA₂ activity, and oxidized LDL (ox-LDL), and an increase in LDL particle size. The changes (Δs) in these variables in the whole grain and legume group were significantly different from those in controls after adjustment for the baseline levels. When all subjects were considered, Δ plasma Lp-PLA₂ positively correlated with Δ glucose, Δ PBMC Lp-PLA₂, Δ ox-LDL, and Δ urinary 8-epi-prostaglandin F_2α after being adjusted for confounding factors. The Δ PBMC Lp-PLA₂ correlated positively with Δ glucose and Δ ox-LDL, and negatively with Δ LDL particle size and baseline PBMC Lp-PLA₂. The substitution of whole grains and legumes for refined rice resulted in a reduction in Lp-PLA₂ activities in plasma and PBMCs partly through improved glycemic control, increased consumption of protein relative to carbohydrate, and reduced lipid peroxides.