Expression and intracellular localization of the human N-acetylmuramyl-L-alanine amidase, a bacterial cell wall-degrading enzyme

N-acetylmuramyl-L-alanine amidase (NAMLAA) specifically degrades peptidoglycan, which is a major component of bacterial cell walls with strong inflammatory properties. For instance, peptidoglycan is capable of stimulating peripheral blood cells to release pro-inflammatory cytokines and is capable of inducing chronic arthritis in an animal model. In a previous study we found that degradation of peptidoglycan by purified NAMLAA reduced its inflammatory effects. To determine where NAMLAA is located in tissues, monoclonal antibodies against purified NAMLAA were produced for use in immunohistochemistry, immunoelectron microscopy, flow cytometric analysis, and Western blotting. The immunohistochemical studies showed NAMLAA-positive cells in human spleen, liver, arthritic synovial tissues, and lymph nodes. In flow cytometric studies of blood and bone marrow, neutrophilic and eosinophilic granulocytes proved to be positive. Monocytes were negative, although they do contain lysozyme, the other important peptidoglycan-degrading enzyme. However, mature macrophages obtained by bronchoalveolar lavage and subsequent selection based on autofluorescence did possess NAMLAA. In immunocytochemical staining of blood smears, thrombocytes were also positive for NAMLAA. Western blot analysis and immunoelectron microscopy of neutrophils and eosinophils showed that NAMLAA is located in azurophilic granules of neutrophils and in secretory vesicles and crystalloid-containing granules of eosinophils. Flow cytometric analysis of blood and bone marrow from different French-American-British-classified acute myeloid leukemia (AML) patients showed that AML-M2 myeloblasts were the first in the granulocyte maturation lineage that were positive for NAMLAA. The more immature AML, such as AML-M0 and AML-M1, did not express NAMLAA. CD15- and CD13-negative megakaryoblasts, corresponding to AML-M7, were also positive for NAMLAA. The expression pattern of NAMLAA in the myeloid lineage suggests that the monoclonal antibody AAA4, recognizing NAMLAA, is useful for discrimination between AML in the monocyte lineage and in the granulocyte lineage.     

A second order monotone upwind scheme

We analyze a special finite difference scheme of upwind type for an ordinary singularly perturbed nonlinear boundary value problem. In particular we prove the uniqueness and monotone dependence upon the right hand sides of the discrete solutions and the second order accuracy in the global domain.     

A strategy for sampling on a sphere applied to 3D selective RF pulse design

Conventional constant angular velocity sampling of the surface of a sphere results in a higher sampling density near the two poles relative to the equatorial region. More samples, and hence longer sampling time, are required to achieve a given sampling density in the equatorial region when compared with uniform sampling. This paper presents a simple expression for a continuous sample path through a nearly uniform distribution of points on the surface of a sphere. Sampling of concentric spherical shells in k-space with the new strategy is used to design 3D selective inversion and spin-echo pulses. These new 3D selective pulses have been implemented and verified experimentally.     

Extent of radial sarcomere coupling revealed in passively stretched cardiac myocytes

We report a simple method, the PinPoint assay, for detecting and identifying single-base variations (polymorphisms) at specific locations within DNA sequences. An oligonucleotide primer is annealed to the target DNA immediately upstream of the polymorphic site and is extended by a single base in the presence of all four dideoxynucleotide triphosphates and a thermostable DNA polymerase. The extension products are desalted, concentrated, and subjected to delayed-extraction MALDI-TOF mass spectrometry. The base at the polymorphic site is identified by the mass added onto the primer. Heterozygous targets produce two mass-resolved species that represent the addition of both bases complementary to those at the polymorphic site. The assay is suitable for double-stranded PCR products without purification or strand separation. More than one primer can be simultaneously extended and then mass-analyzed. The mass spectrometric method thus shows promise for high-volume diagnostic or genotyping applications.     

Evaluation of different fracture-mechanical J-integral initiation values with regard to their usability in the safety assessment of components

Determining fracture-mechanical material characteristic values on the basis of the J-integral is described and stipulated in a variety of standards and guidelines. The individual specifications differ in terms of procedure when determining the characteristic values and, therefore, also in terms of the meaningfulness of the results. This paper presents the different procedures, suggested in the course of the development of test methods in the field of elastic—plastic fracture mechanics, used to characterize crack initiation behaviour with regard to their features as material characteristic values and their usability in the safety assessment of components.     

Water sorption isotherms of freeze-dried milk products: applicability of linear and non-linear regression analysis in modelling

Summary  Water sorption of various freeze-dried milk products was modelled using several water sorption isotherm models, most of which proved to be applicable. Sorption models were fitted to experimental data using linear and non-linear regression analysis. Both methods gave almost the same prediction of water sorption when the model had a good fit. The GAB model was considered to be the most applicable in predicting water sorption in practical applications, as the use of one universal model is desirable. Time-dependent changes, e.g. lactose crystallization above glass transition, were taken into account in the water sorption modelling. Water contents and relative humidities which allowed changes in physico-chemical properties were not included in the modelling, because of unsteady amounts of sorbed water.     

A comparison of subcellular element concentrations in frozen-dried, plastic-embedded, dry-cut sections and frozen-dried cryosections

Biological X-ray microanalysis of diffusible elements within cellular and subcellular compartments requires preparation methods to retain electrolytes in the compartments they occupied in vivo. X-ray microanalysis of frozen-dried, plastic-embedded samples has been used to quantitate electrolytes at the cellular level. We have compared the subcellular elemental distribution in dry cut sections from such samples with that in ultrathin frozen-dried cryosections. Rat pancreases were quench-frozen onto a helium-vapor-cooled copper block. Cryosections were cut at 130-150 K, transferred using a Gatan cold stage, frozen-dried in the column and analysed at 190 K. Tissue fragments were frozen-dried at 190 K, and cut on a dry knife at 293 K. Both samples provided images permitting unambiguous identification of all major compartments except the Golgi complex. Intracellular potassium-to-sodium ratios obtained on frozen-dried plastic-embedded sections were lower than for cryosections (e.g. 1.77 in basal cytoplasm in plastic sections as compared to 4.34 for cryosections) and varied with the pre-embedding procedure (e.g. 1.77 in formaldehyde-fixed as compared to 2.87 in osmium-fixed plastic sections). Potassium gradients between adjacent organelles were large in cryosections and insignificant in plastic-embedded material. Higher cytoplasmic phosphorus, potassium and sulfur concentrations were observed in cryosections. Therefore, a redistribution of electrolytes and covalently bound elements occurred subcellularly in the plastic sections. Calcium was quantifiable in most organelles in cryosections but the plastic lowered sensitivity too much to permit routine calcium quantification. We conclude that in our hands frozen-dried, plastic-embedded samples were compromised and provided lower sensitivity than cryosections.(ABSTRACT TRUNCATED AT 250 WORDS)