The effects of temperature on the filtration of diluted blood through 3 μm and 5 μm filters

The effect of temperature on the flow of diluted blood [Hct = 0.21], through 5 μm Nuclepore filters, is described by the Arrhenius equation with an energy of activation of 27.7 kJ/mol. Plasma, diluted with PBS, is almost three times less sensitive to temperature, with an energy of activation of 9.8 kJ/mol, while red cells are of intermediate sensitivity, with an energy of activation of 14.7 kJ/mol. The most sensitive elements to changes in temperature are leukocytes, with energies of activation of 31 kJ/mol and 35 kJ/mol for fast-flowing leukocytes (granulocytes and lymphocytes) and slow-flowing leukocytes (monocytes) respectively. Hence, the major determinants of the decline in filterability of blood through micropore filters are the leukocytes. This effect is compounded when blood is kept for 10 minutes or more at 10° C due to activation of granulocytes, which leads to permanent pore blocking when the affected blood is filtered at room temperature. The combination of increased passage time of leukocytes through peripheral areas at abnormally low temperatures and subsequent activation might influence the flow of blood in non-affected tissues. The effect of temperature on the filterability of red blood cells through 3 μm filters is not described by the Arrhenius equation and the deviations are seen as a gradual change of slope rather than a sharp break between two straight lines. The data are consistent with a gradual shift in rate limiting step away from the entry event into pores, which dominates at low temperature but becomes progressively less important at elevated temperatures. The changing parameter is probably the volume of the red cell, which is less important when flow is measured through 5 μm pores.     

Morphine Induces Hyperalgesia without Involvement of μ-Opioid Receptor or Morphine-3-glucuronide

Opioid-induced hyperalgesia (OIH) is a paradoxical increase in pain perception that may manifest during opioid treatment. For morphine, the metabolite morphine-3-glucuronide (M3G) is commonly believed to underlie this phenomenon. Here, in three separate studies, we empirically assess the role of M3G in morphine-induced hyperalgesia. In the first study, CD-1 mice injected with morphine (15 mg/kg subcutaneously) after pretreatment with the opioid receptor antagonist naltrexone (NTX) (15 mg/kg) showed tail withdrawal latency reductions indicative of hyperalgesia (2.5 ± 0.1 s at t = 30 min, P < 0.001 versus baseline). In these mice, the morphine/M3G concentration ratios versus effect showed a negative correlation (r_p = −0.65, P < 0.001), indicating that higher morphine relative to M3G concentrations are associated with increased OIH. In the second study, similar hyperalgesic responses were observed in mice lacking the multidrug resistance protein 3 (MRP3) transporter protein (Mrp3^−/− mice) in the liver and their wild-type controls (FVB mice; latency reductions: 3.1 ± 0.2 s at t = 30 min, P < 0.001 versus within-strain baseline). In the final study, the pharmacokinetics of morphine and M3G were measured in Mrp3^−/− and FVB mice. Mrp3^−/− mice displayed a significantly reduced capacity to export M3G into the systemic circulation, with plasma M3G concentrations just 7% of those observed in FVB controls. The data confirm previous literature that morphine causes hyperalgesia in the absence of opioid receptor activation but also indicate that this hyperalgesia may occur without a significant contribution of hepatic M3G. The relevance of these data to humans has yet to be demonstrated.     

Synergetic Effects of Caspase 3 and μ-Calpain in XIAP-Breakdown upon Focal Cerebral Ischemia

Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to neurodegenerative disorders. The recently discovered X-linked inhibitor of apoptosis protein (XIAP) is among the most potent inhibitors of apoptosis. This protein binds to and inhibits both initiator caspases and effector caspases such as caspase-3. The aim of this study was to investigate the relationships between XIAP-breakdown, caspase activation in the development of delayed infarct upon ischemia. We demonstrated that endogenous XIAP is cleaved at least into two fragments during reperfusion following the ischemic insult. The two fragments produced seem to be related to caspase-3 and μ-calpain activities, which are massively enhanced in tissues challenged by ischemia. Therefore, degradation of XIAP by μ-calpain in our system may decrease the activation threshold of caspase-3 normally held in check by the IAPs and/or lead to auto-activation of other caspases. Special issue in honor of Naren Banik.     

Combining μX-ray fluorescence, μXANES and μXRD to shed light on Zn2+ cations in cartilage and meniscus calcifications

We aimed to examine the presence of Zn, a trace element, in osteoarthritis (OA) cartilage and meniscus from patients undergoing total knee joint replacement for primary OA. We mapped Ca²⁺ and Zn²⁺ at the mesoscopic scale by X-ray fluorescence microanalysis (μX-ray) to determine the spatial distribution of the 2 elements in cartilage, μX-ray absorption near edge structure spectroscopy to identify the Zn species, and μX-ray diffraction to determine the chemical nature of the calcification. Fourier transform infrared spectroscopy was used to determine the chemical composition of cartilage and meniscus. Ca²⁺ showed a heterogeneous spatial distribution corresponding to the calcifications within cartilage (or meniscus) or at their surface. At least 2 Zn²⁺ species were present: the first may correspond to Zn embedded in protein (different Zn metalloproteins are known to prevent calcification in biological tissues), and the second may be associated with a Zn trap in or at the surface of the calcification. Calcification present in OA cartilage may significantly modify the spatial distribution of Zn; part of the Zn may be trapped in the calcification and may alter the associated biological function of Zn metalloproteins.     

Connecting μ-fluidics to electron microscopy

A versatile methodology for electron microscopy (EM) grid preparation enabling total content sample analysis is presented. A microfluidic-dialysis conditioning module to desalt or mix samples with negative stain solution is used, combined with a robotic writing table to micro-pattern the EM grids. The method allows heterogeneous samples of minute volumes to be processed at physiological pH for structure and mass analysis, and allows the preparation characteristics to be finely tuned.     

Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex

Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14–19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.     

Proteolysis of Liver Plectin by μ-Calpain

Rat liver plectin was found to be mainly associated with plasma membrane fractions enriched in junctional complexes. The membrane-associated plectin has been partially isolated. Plectin co-purifies with a 200 kDa polypeptide which, on the basis of sequence homology, has been identified as a myosin like-protein. The interaction of μ-calpain with liver plectin has been investigated. Plectin is very sensitive to μ-calpain and is digested to give a fragment of 240 kDa.     

Theoretical description of the magnetic properties of μ3-hydroxo bridged trinuclear copper(II) complexes

A theoretical study of the magnetic properties, using density functional theory, of a family of trinuclear μ₃-OH copper(II) complexes reported in the literature is presented. The reported X-ray crystal structures of [Cu₃(μ₃-OH)(aat)₃(H₂O)₃](NO₃)₂·H₂O (HUKDUM), where aat: 3-acetylamine-1,2,4-triazole; [Cu₃(μ₃-OH)(aaat)₃(H₂SO₄)(HSO₄)(H₂O)] (HUKDOG), where aaat: 3-acetylamine-5-amine-1,2,4-triazole; [Cu₃(μ₃-OH)(PhPyCNO)₃(tchlphac)₂] (HOHQUR), where PhPyCNO: phenyl 2-pyridyl-ketoxime and tchlphac: acid 2,4,5-trichlorophenoxyacetic; [Cu₃(μ₃-OH)(PhPyCNO)₃(NO₃)₂(CH₃OH)] (ILEGEM); [Cu₃(μ₃-OH)(pz)₃(Hpz)₃(ClO₄)₂] (QOPJIP), where Hpz?=?pyrazole; [Cu₃(μ₃-OH)(pz)₃(Hpz)(Me₃CCOO)₂]?2Me₃CCOOH (DEFSEN) and [Cu₃(μ₃-OH)(8-amino-4-methyl-5-azaoct-3-en-2-one)₃][CuI₃] (RITXUO), were used in the calculations. The magnetic exchange constants were calculated using the broken-symmetry approach. The calculated J values are for HUKDUM J₁?=??68.6 cm^?1, J₂?=??69.9 cm^?1, J₃?=??70.4 cm^?1; for HUKDOG, J₁?=??73.5 cm^?1, J₂?=??58.9 cm^?1, J₃?=??62.1 cm^?1; for HOHQUR J₁?=??128.3 cm^?1, J₂?=??134.1 cm^?1, J₃?=??120.4 cm^?1; for ILEGEM J₁?=??151.6 cm^?1, J₂?=??173.9 cm^?1, J₃?=??186.9 cm^?1; for QOPJIP J₁?=??118.3 cm^?1, J₂?=??106.0 cm^?1, J₃?=??120.6 cm^?1; for DEFSEN J₁?=??74.9 cm^?1, J₂?=??64.0 cm^?1, J₃?=??57.7 cm^?1 and for RITXUO J₁?=??10.9 cm^?1, J₂?=?+14.3 cm^?1, J₃?=??35.4 cm^?1. The Kahn-Briat model was used to correlate the calculated magnetic properties with the overlap of the magnetic orbitals. Spin density surfaces show that the delocalization mechanism is predominant in all the studied compounds.

Spatial distribution and speciation of copper in root tips of cucumber revealed by μ-XRF and μ-XANES

The localization, biotransformation, and chemical speciation of copper in root tips of cucumber (Cucumis sativus) were investigated using synchrotron-based micro X-ray fluorescence (μ-XRF) and micro X-ray absorption near edge structure (μ-XANES). The highest content of Cu was found in root cap and meristematic zone whereas low Cu content in elongation and maturation zone. There was a dramatic increase of Cu content in root cap and meristematic zone after treatment with 100 μM CuSO₄ for 72 h. The μ-XANES analysis revealed that most of Cu in root tip was bound with alginate, citrate, and cysteine-like ligands whereas rarely deposited in form of CuO. From root cap to maturation zone, the proportion of Cu bound with alginate-like ligands increased whereas that bound with citrate-like ligands decreased. The proportion of Cu bound with cysteine-like ligands increased from root cap to elongation zone but sharply declined in maturation zone. The results suggested that Cu was chelated by S ligands in the cell walls which protect protoplasm against possible damage caused by Cu excess.     

Potentiation of μ-opioid receptor-mediated signaling by ketamine

Ketamine, a clinically relevant drug, has been shown to enhance opioid-induced analgesia and prevent hyperalgesia. However, the molecular mechanisms involved are not clearly understood. As previous studies found that activation of opioid receptors leads to the phosphorylation of mitogen-activated protein kinases, we investigated whether ketamine could modulate μ-opioid receptor (μOR)-mediated ERK1/2 phosphorylation. We find that acute treatment with ketamine enhances (～2- to 3-fold) the levels of opioid-induced ERK1/2 phosphorylation in recombinant as well as cells endogenously expressing μOR. Interestingly, we find that in the absence of ketamine ERK1/2 signaling is desensitized 10 min after opioid exposure whereas in its presence significant levels (～3-fold over basal) are detected. In addition, ketamine increases the rate of resensitization of opioid-mediated ERK1/2 signaling (15 min in its presence vs. 30 min in its absence). These results suggest that ketamine increases the effectiveness of opiate-induced signaling by affecting multiple mechanisms. In addition, these effects are observed in heterologous cells expressing μOR suggesting a non-NMDA receptor-mediated action of ketamine. Together this could, in part, account for the observed effects of ketamine on the enhancement of the analgesic effects of opiates as well as in the duration of opiate-induced analgesia.     

Scaling VOI size in 3D μCT studies of trabecular bone: a test of the over-sampling hypothesis

For comparative 3D microCT studies of trabecular bone, the use of a volume of interest (VOI) scaled to body size may avoid over-sampling the trabecular mass in smaller versus larger-bodied taxa and comparison of regions that are not functionally homologous (Fajardo and Müller: Am J Phys Anthropol 115 (2001) 327-336), though the influence on quantitative analyses using scaled versus nonscaled VOIs remains poorly characterized. We compare trabecular architectural properties reflecting mass, organization, and orientation from three volumes of interest (large, scaled, and small) obtained from the distal first metacarpal in a sample of Homo (n = 10) and Pan (n = 12). We test the null hypotheses that neither absolute VOI size, nor scaling of the VOI to metacarpal size as a proxy for body size, biases intraspecific analyses nor impacts the detection of interspecific differences. These hypotheses were only partially supported. While certain properties (e.g., bone volume fraction or trabecular thickness) were not affected by varying VOI size within taxa, others were significantly impacted (e.g., intersection surface, connectivity, and structure). In comparing large versus scaled VOIs, we found that the large VOI inflated the number and/or magnitude of significant differences between Homo and Pan. In summary, our results support the use of scaled VOIs in studies of trabecular architecture.     

Structural basis for μ-opioid receptor binding and activation

Opioids that stimulate the μ-opioid receptor (MOR1) are the most frequently prescribed and effective analgesics. Here we present a structural model of MOR1. Molecular dynamics simulations show a ligand-dependent increase in the conformational flexibility of the third intracellular loop that couples with the G protein complex. These simulations likewise identified residues that form frequent contacts with ligands. We validated the binding residues using site-directed mutagenesis coupled with radioligand binding and functional assays. The model was used to blindly screen a library of ～1.2 million compounds. From the 34 compounds predicted to be strong binders, the top three candidates were examined using biochemical assays. One compound showed high efficacy and potency. Post hoc testing revealed this compound to be nalmefene, a potent clinically used antagonist, thus further validating the model. In summary, the MOR1 model provides a tool for elucidating the structural mechanism of ligand-initiated cell signaling and for screening novel analgesics.     

Functional analyses of mammalian reovirus nonstructural protein μNS

Genome replication of reovirus occurs in cytoplasmic inclusion bodies called viral factories or viroplasms. The viral nonstructural protein μNS, encoded by genome segment M3, is not a component of mature virions, but is expressed to high levels in infected cells and is concentrated in the infected cell factory matrix. Recent studies have demonstrated that μNS plays a central role in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly.     

Oxo-bridged Ta(+3) dimers, (TaCl2L2)2(μ-O)(μ-SR2), revisited. Structural differences between isoelectronic μ-O and μ-OH complexes

The compounds Ta₂Cl₄(dmpe)₂(μ-Me₂S)(μ-O) (1) and Ta₂Cl₄(py)₄(μ-THT)(μ-O) (2) where dmpe = Me₂PCH₂CH₂PMe₂ and THT = tetrahydrothiophene, have been prepared and structurally characterized. They are authentic examples of μ-O bridged Ta^IIITa^III edge-sharing bioctahedral complexes. Their structures are virtually identical with respect to all of the bonds that they have in common. However, the structure of 1 differs significantly in its TaTa, TaO and one type of TaCl distance from the previously reported Ta₂Cl₄(dmpe)₂(μ-Me₂S)(μ-O)·HCl (3). These differences show that in 3 there is a μ-OH group hydrogen bonded to a Cl⁻ ion. The structural differences attendant upon the μ-OH ⇌ μ-O change are of general interest and are discussed. The crystallographic data for the new compounds are as follows: 1: monoclinic (P2₁/c) with a = 10.412(2), b = 14.749(2), c = 22.177(3) Å, β = 99.25(1)°, V = 3361(1) ų and Z = 4. 2: monoclinic (P2₁/a) with a = 18.238(4), b = 10.402(3), c = 19.070(2) Å, β = 95.37(2), V = 3602(2) ų and Z = 4.     

Etonitazene-induced antinociception in μ1 opioid receptor deficient CXBK mice: Evidence for a role for μ2 receptors in supraspinal antinociception

The prevailing view is that supraspinal μ opioid-mediated antinociception in mice is mediated via the μ₁ subtype. The purpose of the present study was to determine if the highly μ-selective compound etonitazene could produce supraspinal (intracerebroventricular; i.c.v.) antinociception in CXBK mice, which are deficient in brain μ₁, but not μ₂, opioid receptors. CXBK or normal Crl:CD-1 ^®(ICR)BR mice were administered graded doses of etonitazene i.c.v. and 15 min later antinociception was assessed by a standard radiant-heat or 55°C water tail-flick test. Etonitazene produced dose-related antinociception that was blocked by naloxone and by β-FNA (demonstrating a μ opioid mechanism), but not by either ICI-174,864 or naltrindole (demonstrating the lack of involvement of δ opioid receptors). These findings suggest that μ₂ opioid receptors are important contributors to opioid-induced supraspinal antinociception in mice.     

New μ-Oxo-heterobinuclear complex containing the FeOSi linkage: Synthesis and structural characterization of [FeSALEN] OSi(CH3)3

The reaction of hexamethyldisilazane with the μ-oxo iron SALEN dimer in toluene under N₂ gives the μ-heterobinuclear title complex, FeC₁₉H₂₃N₂0₃Si. Crystals of the compound are monoclinic, space group P2₁/c with a = 6.087(2), b = 10.497(3), c = 28.567(7) Å, β = 97.78(2) and Z = 4. Solution of the structure by direct methods led to a final weighted and unweighted R factors of 6.2 and 5.6%, respectively. The coordination geometry of the iron center is square-pyramidal with the iron displaced 0.56 Å from the best least-squares plane of the coordinating oxygens and nitrogens of the SALEN ligand. The FeOSi angle is bent at 142.7°. The FeOSi linkage does not hydrolyze under neutral pH conditions.