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The aim of the study is to increase the efficiency of the operation of the facilities for the thermal processing of raw materials by exposure to electromagnetic radiation and the improvement of their main working organs that ensure the improvement of the quality of the product with the least operating costs. The work differs from existing studies by methodical generalization of the electrodynamic system with various design execution of the working chamber, ensuring the flow of the technological process of heat treatment and disinfection of raw materials due to repeated exposure to EMP. Because of the design features of the bulk resonators, the units have specificity in the principles of operation, in the methods of designing the nodes and all the elements of the energy transfer from the emitter to the raw material as a whole. In a constructive respect, the electrodynamic systems of microwave generators differ from existing ones by combining mobile resonator and beam chambers located in a toroidal shielding body. In microwave technology, this method of energy transfer constitutes an independent field of study. In this case, it is possible to detect the dynamics of the process taking into account the time variation in the dielectric and thermal physical parameters of the raw material, calculate the configuration and dimensions of the cavity resonator in accordance with the wavelength and the critical electric field strength, determine the number of sources and their location in the working chamber.

A facile deposition method of 3D photonic crystals made of yttrium-stabilized zirconia (YSZ) was developed. YSZ nanoparticles with primary particle size below 10 nm and cubic crystalline phase were synthesized by hydrothermal treatment of solutions of zirconyl nitrate, yttrium nitrate and acetylacetone. Before coassembly with polystyrene (PS) microspheres, a dispersant Dolapix CE64 was added to the dialyzed sol of YSZ nanoparticles to render their surface negatively charged. Vertical convective coassembly resulted in 3D ordered YSZ/PS hybrid films, which were inverted at 500 °C in air to produce inverse opals. The linear shrinkage of the coatings was in the range 15–20%, below previously reported values for YSZ. The obtained coatings demonstrated pronounced photonic properties and retained their ordered structure after annealing at 1000 °C for 2 h. Increasing the filling fraction of crystalline nanoparticles in the templates should enable production of fully functional 3D photonic crystals for applications in high-temperature photonics.

Für die vollständige Verwertung lignocellulosehaltiger Bio‐Reststoffe werden verschiedenste Verfahren genutzt, die sich jeweils beeinflussen. In dieser Arbeit werden nachhaltige, auf Wasser, CO2und Enzymen basierende Prozesse betrachtet. Der Vergleich der relevanten Prozessparameter Temperatur, Druck und Verweilzeit zeigt, dass sich die verschiedenen Verfahren in einem einzigen Reaktorkonzept zusammenführen lassen. Dies ermöglicht ein objektives und effizientes Screening unterschiedlicher Bio‐Reststoffe. Interaktionen zwischen den Biomassekomponenten werden betrachtet und daraus eine sinnvolle Prozessreihenfolge abgeleitet, die Abbauprodukte minimiert und zu möglichst hochwertigen Produkten führt.

Possible physical mechanisms of the formation of the spin flame front in deflagrating gas mixtures are discussed. Conditions for the observation of a new physical phenomenon -- the propagation of the spin flame front in a limiting propane-air mixture in an open narrow slot -- are identified. Experimental techniques for investigating the spin mode of flame propagation in a gas mixture with low Reynolds numbers are suggested. The conditions where transport can affect the formation of a spin front in a gas-air mixture are formulated and prospects for future research are outlined.

An overview of high pressure processes for producing chemicals from lignocellulosic biomass is presented. Special attention is given to the combination of different process steps and scale‐up possibilities for the apparatus involved. Based on literature and own experiments it is shown that, under the condition of an adapted choice of process parameters, high pressure biorefinery allows the connection and combination of different high efficient and selective unit operations to a single one‐stop process that can be realized in different scales. Such reactor design can be used for numerous lab scale experiments providing a basis for comparable data for different types of solid biomass. The advantage of such a screening unit is the possibility of cascading processes in the same extraction vessel. Thus, the knowledge on a convergent behavior of reaction and extraction rates can be gained, allowing to progress in modeling of solid biomass conversion under high pressure.

Interferon gamma (IFNγ) has been demonstrated to inhibit tumor growth in vivo as well as proliferation of multiple types of cultured transformed cells. In this study, we showed that IFNγ promoted progressive death in A431 cells, overexpressing EGF receptor (EGFR). Based on the data provided by evaluating cell morphology, MTT assay, FACS analysis, and cleaved caspase-3 staining we concluded that the major cause of IFNγ-induced A431 cell growth inhibition was not cell cycle arrest, but apoptosis. We investigated a role for the EGFR and ERK1/2 MAPK signaling pathways in IFNγ-induced apoptosis of A431 cells. IFNγ-induced cell death was accompanied by both an increase of the ERK1/2 MAPK activation and a simultaneous reduction of the EGFR activation. Activation of ERK1/2 was crucial for IFNγ-induced cell death because MEK1/2 inhibitors, PD0325901 and U0126 efficiently protected cells from apoptosis by suppressing caspase-3 activation. Even though EGFR tyrosine kinase inhibitor AG1478 also rescued A431 cells from IFNγ-induced apoptosis, unlike MEK1/2 inhibitors, it initiated G1arrest. Together, these results suggest that sustained inhibition of both EGFR and ERK1/2 leads to significant protection of the cells from IFNγ-induced apoptosis, indicating important roles for the EGFR tyrosine kinase and ERK1/2 MAP-kinases in regulating A431 cell death.

Bei der Nutzung lignocellulosehaltiger Biomasse führen hohe Feststoffbeladungen im Rührkessel noch zu Problemen bei der enzymatischen Hydrolyse. Die Integration von thermischer Vorbehandlung und enzymatischer Hydrolyse der Biomasse in einem Festbettreaktor wird daher in dieser Arbeit untersucht. Es wird gezeigt, dass der Einfluss von Pumpen auf die Enzymaktivität für eine spätere Anwendung vernachlässigbar ist, starke Druckabfälle sich jedoch negativ auf die Enzyme auswirken. Eine mechanische Vorbehandlung vor der thermischen Hydrolyse bietet deutliche Vorteile bei der Enzymreaktion (> 95 % Umsatz nach 48 h für pelletiertes Stroh). Desweiteren wird die enzymatische Hydrolyse im Festbett bei verschiedenen Feststoff‐Konzentrationen (7 – 20 %) gezeigt.

The existence of morphologically distinct populations of islets in the pancreas was described over 60 years ago. Unfortunately, little attention has been paid to possible functional differences between islet subpopulations until recently. We demonstrated that one population, the small islets, were superior to large islets in a number of functional aspects. However, that work did not determine whether these differences were inherent, or whether they arose because of the challenge of isolation procedures. Nor, were there data to explain the differences in insulin secretion. We utilized immunohistochemistry, immunofluorescence, ELISA, and transmission electron microscopy to compare the unique characteristics found in isolated rat islet populations in situ and after isolation. Insulin secretion of small isolated islets was significantly higher compared to large islets, which correlated with higher insulin content/area in small islets (in situ), a higher density of insulin secretory granules, and greater insulin content/volume in isolated islets. Specifically, the core b-cells of the large islets contained less insulin/cell with a lower insulin granule density than peripheral b-cells. When insulin secretion was normalized for total insulin content, large and small islets released the same percentage of total insulin. Small islets had a higher density of cells/area than large islets in vitro and in situ. The data provide a possible explanation for the inferior insulin secretion from large islets, as they have a lower total cell density and the b-cells of the core contain less insulin/cell.

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter > 150 μm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter < 100 μm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 μm/min in small islets and 2.8 μm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150μm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.

ABSTRACTMouse-derived macrophages have the unique ability to restrict or permit Legionella pneumophilaintracellular growth. The common inbred mouse strain C57BL/6J (B6) restricts L. pneumophilagrowth, whereas macrophages derived from A/J mice allow >103-fold bacterial growth within three days. This phenotypic difference was mapped to the mouse Naip5allele. The B6 restrictive Naip5allele is dominant, and six amino acid changes in its product were predicted to control permissiveness. By using the wild-derived mouse strain MOLF/Ei, we found that MOLF/Ei-derived macrophages also restrict L. pneumophilagrowth, yet the Naip5 protein is identical to the A/J Naip5 at the six-amino-acid signature. The MOLF/Ei restrictive trait, unlike that of B6-derived macrophages, was not dominant over the A/J trait. In spite of this phenotypic difference, the L. pneumophilagrowth restriction in MOLF/Ei macrophages was mapped to the Naip5region as well, indicating that the originally predicted change in the A/J Naip5allele may not be critical for restriction. In the product of the A/J Naip5permissive allele, there are four unique amino acid changes that map to a NACHT-like domain. Similar misregulating mutations have been identified in the NACHT domains of Nod-like receptor (NLR) proteins. Therefore, one of these mutations may be critical for restriction of L. pneumophilaintracellular growth, and this parallels results found with human NLR variants with defects in the innate immune response.

Mouse-derived macrophages have the unique ability to restrict or permit Legionella pneumophila intracellular growth. The common inbred mouse strain C57BL/6J (B6) restricts L. pneumophila growth, whereas macrophages derived from A/J mice allow >103-fold bacterial growth within three days. This phenotypic difference was mapped to the mouse Naip5 allele. The B6 restrictive Naip5 allele is dominant, and six amino acid changes in its product were predicted to control permissiveness. By using the wild-derived mouse strain MOLF/Ei, we found that MOLF/Ei-derived macrophages also restrict L. pneumophila growth, yet the Naip5 protein is identical to the A/J Naip5 at the six-amino-acid signature. The MOLF/Ei restrictive trait, unlike that of B6-derived macrophages, was not dominant over the A/J trait. In spite of this phenotypic difference, the L. pneumophila growth restriction in MOLF/Ei macrophages was mapped to the Naip5 region as well, indicating that the originally predicted change in the A/J Naip5 allele may not be critical for restriction. In the product of the A/J Naip5 permissive allele, there are four unique amino acid changes that map to a NACHT-like domain. Similar misregulating mutations have been identified in the NACHT domains of Nod-like receptor (NLR) proteins. Therefore, one of these mutations may be critical for restriction of L. pneumophila intracellular growth, and this parallels results found with human NLR variants with defects in the innate immune response.

The program BRIZ has been developed for displaying Brillouin zones and Fermi sphere configurations. The underlying physics relates to the nearly-free-electron model for simple sp-metals. A Brillouin zone boundary near the Fermi level provides formation of the energy gap and leads to the lowering of the structure energy. Consideration of the Brillouin zone with respect to a free-electron Fermi sphere and zone filling by electron states provides useful information for phase structure stability and physical properties.

The oncogenic potential of hepatitis C virus (HCV) core protein has been demonstrated, but the precise mechanism of cell transformation triggered by HCV core is still unclear. This study shows that constitutive expression of HCV core protein (core) in NIH 3T3 murine fibroblasts triggers malignant transformation. At the preneoplastic stage, clones that expressed HCV core constitutively demonstrated genomic instability seen as disruption of the mitotic spindle cell checkpoint leading to increased ploidy. Transformation was completed by the loss of DNA and resistance to apoptosis induced by serum starvation. Simultaneously, cells acquired a capacity for anchorage independent growth and absence of contact inhibition. Inoculation of these transformed cells into severe combined immune deficiency (SCID) mice led to formation of solid core-expressing tumors. Transformation and tumorigenicity of core-expressing cell lines coincided with a 5- to 10-fold repression of endogenous p53 transactivation. Thus, long-term HCV core expression alone is sufficient for complete transformation of immortal fibroblasts that can then induce tumors in a susceptible host. This data suggests that malignant transformation by HCV core may occur through primary stress, induction of genomic instability, and further HCV core-induced rescue of surviving mutated cells.

ABSTRACTSpontaneous Ca2+oscillations in vascular smooth muscle cells have been modeled using a single Ca2+pool. This report describes spontaneous Ca2+oscillations dependent on two separate Ca2+sources for the nuclear versus cytoplasmic compartments. Changes in free intracellular Ca2+were monitored with ratiometric Ca2+- fluorophores using confocal microscopy. On average, spontaneous oscillations developed in 79% of rat aortic smooth muscle cells that were synchronous between the cytoplasm and nucleus. Reduction of extracellular Ca2+(< 1 μM) decreased the frequency and amplitude of the cytoplasmic oscillations with 48% of the oscillations asynchronous between the nuclear and cytoplasmic compartments. Similar results were obtained with the Ca2+channel blockers, nimodipine and diltiazem. Arg-vasopressin (AVP) induced a rapid release of intracellular Ca2+stores that was greater in the nuclear compartment (4.20 ± 0.23 ratio units, n = 56) than cytoplasm (2.54 ± 0.28) in cells that had spontaneously developed prior oscillations. Conversely, cells in the same conditions lacking oscillations had a greater AVP-induced Ca2+transient in the cytoplasm (4.99 ± 0.66, n = 17) than in the nucleus (2.67 ± 0.29). Pre-treatment with Ca2+channel blockers depressed the AVP responses in both compartments with the cytoplasmic Ca2+most diminished. Depletion of internal Ca2+stores prior to AVP exposure blunted the nuclear response, mimicking the response of cells that lacked prior oscillations. Spontaneous oscillating cells had a greater sarcoplasmic reticulum network than cells that did not oscillate. We propose that spontaneous nuclear oscillations rely on perinuclear sarcoplasmic reticulum stores, while the cytoplasmic oscillations rely on Ca2+influx.

ABSTRACTSpontaneous Ca2+ oscillations in vascular smooth muscle cells have been modeled using a single Ca2+ pool. This report describes spontaneous Ca2+ oscillations dependent on two separate Ca2+ sources for the nuclear versus cytoplasmic compartments. Changes in free intracellular Ca2+ were monitored with ratiometric Ca2+- fluorophores using confocal microscopy. On average, spontaneous oscillations developed in 79% of rat aortic smooth muscle cells that were synchronous between the cytoplasm and nucleus. Reduction of extracellular Ca2+ ( 1 μM) decreased the frequency and amplitude of the cytoplasmic oscillations with 48% of the oscillations asynchronous between the nuclear and cytoplasmic compartments. Similar results were obtained with the Ca2+ channel blockers, nimodipine and diltiazem. Arg-vasopressin (AVP) induced a rapid release of intracellular Ca2+ stores that was greater in the nuclear compartment (4.20 ± 0.23 ratio units, n = 56) than cytoplasm (2.54 ± 0.28) in cells that had spontaneously developed prior oscillations. Conversely, cells in the same conditions lacking oscillations had a greater AVP-induced Ca2+ transient in the cytoplasm (4.99 ± 0.66, n = 17) than in the nucleus (2.67 ± 0.29). Pre-treatment with Ca2+ channel blockers depressed the AVP responses in both compartments with the cytoplasmic Ca2+ most diminished. Depletion of internal Ca2+ stores prior to AVP exposure blunted the nuclear response, mimicking the response of cells that lacked prior oscillations. Spontaneous oscillating cells had a greater sarcoplasmic reticulum network than cells that did not oscillate. We propose that spontaneous nuclear oscillations rely on perinuclear sarcoplasmic reticulum stores, while the cytoplasmic oscillations rely on Ca2+ influx.Cell Research (2004) 14, 379–388. doi:10.1038/sj.cr.7290238

The synthesis of silica aerogels was modified by addition of supercritical CO2during the sol-gel process. It was shown, that CO2acts as a catalyst and accelerates the gelation significantly. This effect was studied under a multitude of experimental conditions. The influence of the precursor concentration, temperature and the nature of the catalysts and solvent on the gel formation in presence of CO2was studied. Several gels obtained by this method were dried and transparent silica aerogels were produced.

The cells of tumourigenic line SK-UT-1B combine features characteristic both of normal (diploid karyotype, a low level of polyploid cells, absence of chromosomal marker) and tumour cells (high level of chromosomal instability, high malignancy). We suggest that maintenance of diploid karyotype in this line is controlled via the p53/p21 pathway. We demonstrate that the amount of p53 increases following γ-irradiation and accumulated p53 protein seems to be functional as p53-luc and p21/Waf-luc reporter plasmids were found to be activated. However, γ-irradiation-induced increase of p53 was not accompanied by increase of p21/Waf on the protein level. Apparently this is one of the reasons for G1/S and G2/M checkpoint control disruption. The absence of these checkpoints could not prevent the proliferation of cells with intrachromosomal rearrangements. The only effective checkpoint in SK-UT-1B is the p53-dependent M checkpoint, which directed the cells with changed chromosome numbers to apoptosis and therefore strictly guarded the diploidy of the cell population. This indicates that p53 can control the preservation of genetic stability at different levels via different pathways.

Thrombin, the ultimate protease in the blood coagulation cascade, mediates its known cellular effects by unique proteolytic activation of G-protein-coupled protease-activated receptors (PARs), such as PAR1, PAR3, and PAR4, and a “tethered ligand” mechanism. PAR1 is variably expressed in subpopulations of neurons and largely determines thrombin's effects on morphology, calcium mobilization, and caspase-mediated apoptosis. In spinal cord motoneurons, PAR1 expression correlates with transient thrombin-mediated [Ca²⁺]i flux, receptor cleavage, and elevation of rest [Ca²⁺]i activating intracellular proteases. At nanomolar concentrations, thrombin retracts neurites via PAR1 activation of the monomeric, 21 kDa Ras G-protein RhoA, which is also involved in neuroprotection at lower thrombin concentrations. Such results suggest potential downstream targets for thrombin's injurious effects. Consequently, we employed several G-protein-specific modulators prior to thrombin exposure in an attempt to uncouple both heterotrimeric and monomeric G-proteins from motoneuronal PAR1. Cholera toxin, stimulating Gs, and lovastatin, which blocks isoprenylation of Rho, reduced thrombin-induced calcium mobilization. In contrast, pertussis toxin and mastoparan, inhibiting or stimulating Go/Gi, were found to exacerbate thrombin action. Effects on neuronal rounding and apoptosis were also detected, suggesting therapeutic utility may result from interference with downstream components of thrombin signaling pathways in human motor neuron disorders, and possibly other neurodegenerative diseases. Published 2001 John Wiley & Sons, Inc. J Neurobiol 48: 87–100, 2001

Metal response element-binding transcription factor-1 (MTF-1) is a unique, zinc-inducible transcription factor that binds to metal response elements in the metallothionein promoter and activates transcription in response to metals and oxidative stress. MTF-1 contains six zinc fingers of the Cys2-His2type. It was previously shown that MTF-1 is reversibly activated to bind DNA in response to changes in zinc status, unlike other zinc finger transcription factors, which do not appear to be reversibly activated by zinc in the cellular environment. Here we show that zinc fingers 2–4 constitute the core DNA-binding domain, whereas fingers 5 and 6 appear to be unnecessary for DNA binding in vitro. Deletion of finger 1 resulted in a protein that bound DNA constitutively in vitro. Furthermore, transfer of MTF-1 finger 1 to a position immediately preceding the three zinc fingers of Sp1 resulted in a chimeric protein that required exogenous zinc to activate DNA binding in vitro, unlike native Sp1, which binds DNA constitutively. Transient transfection experiments demonstrated that intact MTF-1 activated a reporter 2.5–4-fold above basal levels after metal treatment in mouse MTF-1 knockout cells, DrosophilaSL2 cells, and yeast. However, the metal response was lost in all three systems when finger 1 was deleted, but was unaffected by deletion of fingers 5 and 6. These data suggest that finger 1 of MTF-1 constitutes a unique metal-sensing domain that, in cooperation with the transactivation domains, produces a zinc-sensing metalloregulatory transcription factor.

Background: Mechanisms underlying neurodegeneration are actively sought for new therapeutic strategies. Transgenic, knockout and genetic mouse models greatly aid our understanding of the mechanisms for neuronal cell death. A naturally occurring, autosomal recessive mutant, known as wobbler, and mice transgenic for familial amyotrophic lateral sclerosis (FALS1) superoxide dismutase (SOD)1 mutations are available, but the molecular mechanisms remain equally unknown. Both phenotypes are detectable after birth. Wobbleris detectable in the third week of life, when homozygotes (wr/wr) exhibit prominent gliosis and significant motor neuron loss in the cervical, but not in lumbar, spinal cord segments. To address molecular mechanisms, we evaluated “death signals” associated with the multifunctional serine protease, thrombin, which leads to apoptotic motor neuronal cell death in culture by cleavage of a G-protein coupled, protease-activated receptor 1 (PAR-1). Materials and Methods: Thrombin activities were determined with chromogenic substrate assays, Western immunoblots and immunohistochemistry were performed with anti-PAR-1 to observe localizations of the receptor and anti-GFAP staining was used to monitor astrocytosis. PAR-1 mRNA levels and locations were determined by reverse transcription polymerase chain reaction (qRT-PCR) and insitu hybridizations. Cell death was monitored with in situ DNA fragmentation assays. Results: In preliminary studies we found a 5-fold increase in PAR-1 mRNA in cervical spinal cords from wr/wr, compared with wild-type (wt) litter-mates. Our current studies suggested that reactive astrocytosis and motor neuron cell death were causally linked with alterations in thrombin signaling. PAR-1 protein expression was increased, as demonstrated by immunocytochemistry and confirmed with in situ hybridization, in phenotypic wr/wrmotor neurons, compared with wt, but not in astrocytes. This increase was much greater in cervical, compared with lumbar, segments, paralleling motor neuron degeneration. We also found, using reverse transcription polymerase chain reaction (qRT-PCR) with RNA from genotyped embryos, that PAR-1 was already increased in wr/wrcords at E12, the earliest time examined. Conclusions: Thus, motor neuron degeneration and death follows PAR-1 expression both temporally and topographically in wobblermice. Since our culture studies show that thrombin mobilized [Ca2+]iby activating PAR-1, eventually leading to motor neuron apoptosis, up-regulation of PAR-1 during development may contribute both to “appropriate” as well as “inappropriate” neuronal death in wobbler.

Metal response element-binding transcription factor-1 (MTF-1) is a six-zinc finger protein that plays an essential role in activating metallothionein expression in response to the heavy metals zinc and cadmium. Low affinity interactions between zinc and specific zinc fingers in MTF-1 reversibly regulate its binding to the metal response elements in the mouse metallothionein-I promoter. This study examined the subcellular distribution and DNA binding activity of MTF-1 in cells treated with zinc or cadmium. Immunoblot analysis of cytosolic and nuclear extracts demonstrated that in untreated cells, about 83% of MTF-1 is found in the cytosolic extracts and is not activated to bind to DNA. In sharp contrast, within 30 min of zinc treatment (100 μm), MTF-1 is detected only in nuclear extracts and is activated to bind to DNA. The activation to bind to DNA and nuclear translocation of MTF-1 occurs in the absence of increased MTF-1 content in the cell. Furthermore, immunocytochemical localization and immunoblotting assays demonstrated that zinc induces the nuclear translocation of MTF-1-FLAG, expressed from the cytomegalovirus promoter in transiently transfected dko7 (MTF-1 double knockout) cells. Immunoblot analysis of cytosolic and nuclear extracts from cadmium-treated cells demonstrated that concentrations of cadmium (10 μm) that actively induce metallothionein gene expression cause only a small increase in the amount of nuclear MTF-1. In contrast, an overtly toxic concentration of cadmium (50 μm) rapidly induced the complete nuclear translocation and activation of DNA binding activity of MTF-1. These studies are consistent with the hypothesis that MTF-1 serves as a zinc sensor that responds to changes in cytosolic free zinc concentrations. In addition, these data suggest that cadmium activation of metallothionein gene expression may be accompanied by only small changes in nuclear MTF-1.

Proceeding from our observation that LPS-unresponsive mice of the strain C57BL/10ScCr mice fail to express the Tlr4gene [Poltorak A, He X. Smirnova I et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4gene. Science1998; 282: 2085], we have defined the exact limits of a deletion encompassing Tlr4in the C57BL/10ScCr genome. The deletion removes 74723 bp of DNA, with reference to the control strain 129/J (from which the complete sequence of the Tlr4locus was obtained). There is no inserted element, and no re-arrangement of the chromosome (e.g. inversion or translocation) in the immediate region of Tlr4; the deletion removes only one recognizable gene. Hence, other immunological anomalies that have been identified in C57BL/10ScCr mice (a non-healing phenotype in Leishmaniainoculation and failure to produce interferon-γ in response to numerous microbial infections) must be ascribed to one of two causes. Mutation(s) at other loci may be responsible for these defects. Alternatively, Tlr4locus deletion may have phenotypic consequences that exceed the well known blockade of LPS signal transduction.

Cytochrome c oxidase is a membrane‐bound redox‐driven proton pump. The coupling of the exergonic electron‐transfer reactions from cytochrome cto oxygen to proton translocation across the membrane requires control of internal electron‐ and proton‐transfer reactions. In this work, we focus on the kinetics of electron and proton transfer during those reaction steps that are coupled to proton pumping in cytochrome coxidase. The results show that during the first pumping step (peroxy → oxoferryl transition), proton transfer regulates intramolecular electron transfer. The proton transfer takes place in two steps: (1) Internal proton transfer from a protonatable group, proposed to be Glu(I‐286), in the so‐called D‐pathway, to an oxygen intermediate at the binuclear center (τ ≅ 100 μs); (2) Rapid re‐protonation of Glu(I‐286) from the bulk solution (τ < 100 μs). Only after proton uptake from solution the last (fourth) electron is transferred “one step closer” towards the binuclear center (from CuAto heme a). During the second proton‐pumping step (oxoferryl → oxidized), this electron is transferred to the binuclear center, linked to the uptake of a proton through the D‐pathway. The electron‐transfer rate displays a kinetic‐isotope effect (kH/kD) of 6 ± 1 (in a pH range in which the pH dependence of the rate is small), which indicates that the electron transfer is rate‐limited by the proton transfer. The entry into the D‐pathway (around Asp(I‐132)) is composed of a cluster of negatively‐charged amino acid residues together with a number of histidines, forming a so‐called proton‐collecting antenna designed to allow rapid protonation of groups within the proton‐transfer pathway.

During vertebrate neuromuscular development, all muscle fibers are transiently innervated by more than one neuron. Among the numerous factors shown to potentially influence the passage from poly‐ to mononeuronal innervation, serine proteases and their inhibitors appear to play important roles. In this regard, protease nexin I (PNI), a potent inhibitor of the serine protease, thrombin, is highly localized to the neuromuscular junction (NMJ). In turn, thrombin is responsible for activity‐dependent synapse elimination both in an in vitro model, and in vivo. In the present study, we used a monospecific anti‐PNI polyclonal antibody to study the developmental kinetics of PNI expression in mouse leg skeletal muscle. By using immunoblotting, we detected PNI at embryonic day 16 (E16), as a 48‐kDa band. This 48‐kDa PNI band became prominent in leg muscle extracts at postnatal day 5 (P5) and remained so in extracts from adult muscle. In contrast, a higher molecular weight immunoreactive PNI band, which was sodium dodecyl sulfate– and β‐mercaptoethanol–resistant, was first detected at E16, increased at birth (P0), and then decreased at P15, i.e., after the wave of polyneuronal synapse elimination had occurred in these muscles. The results of an enzyme‐linked immunosorbent assay, measuring active, complexed, and truncated PNI, correlated with Western blot data. We used immunocytochemistry to probe the localization of PNI at the NMJ and found that PNI was present in the cytoplasm of myotubes at E16, but neither then nor at birth did it colocalize with acetylcholine receptors. PNI became localized at NMJs by P5 and increased by P15, after which it remained stably concentrated there in the adult. Finally, we studied the gene expression of PNI mRNA, by using Northern blotting, and showed that PNI mRNA was present in skeletal muscle and remained stable throughout the time‐course studies, suggesting that developmental regulation of muscle PNI occurs principally at the translational and/or post‐translational levels. These results suggest that the localization of PNI, through a binding site or “receptor” may play an important role in differentiation and maintenance of synapse. J. Comp. Neurol. 397:572–579, 1998. © 1998 Wiley‐Liss, Inc.