Role of Tim4 in the regulation of ABCA1+ adipose tissue macrophages and post-prandial cholesterol levels.

Dyslipidemia is a main driver of cardiovascular diseases. The ability of macrophages to scavenge excess lipids implicate them as mediators in this process and understanding the mechanisms underlying macrophage lipid metabolism is key to the development of new treatments. Here, we investigated how adipose tissue macrophages regulate post-prandial cholesterol transport. Single-cell RNA sequencing and protected bone marrow chimeras demonstrated that ingestion of lipids led to specific transcriptional activation of a population of resident macrophages expressing Lyve1, Tim4, and ABCA1. Blocking the phosphatidylserine receptor Tim4 inhibited lysosomal activation and the release of post-prandial high density lipoprotein cholesterol following a high fat meal. Both effects were recapitulated by chloroquine, an inhibitor of lysosomal function. Moreover, clodronate-mediated cell-depletion implicated Tim4+ resident adipose tissue macrophages in this process. Thus, these data indicate that Tim4 is a key regulator of post-prandial cholesterol transport and adipose tissue macrophage function and may represent a novel pathway to treat dyslipidemia.

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants.

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.

Tabashir: an opal of plant origin.

A specimen of tabashir, a variety of opal found in bamboo, contained more water but smaller amounts of alkalis and alkaline earths than most opals. It consisted of particles of about 100 angstroms in diameter, linked together in clumps, which appeared in fractured surfaces as irregularities. The tabashir was amorphous, but its microstructure differed from that of silicagel and amorphous opal of inorganic origin.

Antibody catalysis of the oxidation of water.

Recently we reported that antibodies can generate hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*). We now show that this process is catalytic, and we identify the electron source for a quasi-unlimited generation of H2O2. Antibodies produce up to 500 mole equivalents of H2O2 from 1O2*, without a reduction in rate, and we have excluded metals or Cl- as the electron source. On the basis of isotope incorporation experiments and kinetic data, we propose that antibodies use H2O as an electron source, facilitating its addition to 1O2* to form H2O3 as the first intermediate in a reaction cascade that eventually leads to H2O2. X-ray crystallographic studies with xenon point to putative conserved oxygen binding sites within the antibody fold where this chemistry could be initiated. Our findings suggest a protective function of immunoglobulins against 1O2* and raise the question of whether the need to detoxify 1O2* has played a decisive role in the evolution of the immunoglobulin fold.

Reaction mechanism for the inactivation of the quinoprotein methylamine dehydrogenase by phenylhydrazine.

Phenylhydrazine has previously been shown to be an irreversible inactivator of the tryptophan tryptophylquinone (TTQ) enzyme methylamine dehydrogenase [Davidson, V.L. and Jones, L.H. (1992) Biochim. Biophys. Acta 1121, 104-110]. Structure-reactivity correlations have been performed to elucidate the mechanism by which this inactivation occurs. The reactions of a series of p-substituted phenylhydrazines with methylamine dehydrogenase were examined. Correlation with electronic substituent effects was observed. A Hammett plot of the second order inactivation rate constants versus sigma p exhibited a positive slope. Plots of these rate constants against substituent constants which reflected either resonance or field/inductive parameters for each p-substituent indicated that the rate was primarily influenced by resonance electronic effects. A Brønsted plot of the inactivation rate constant against pKa of each substituted phenylhydrazine yielded a beta-value (slope) of 0.7. Based upon these results, a reaction mechanism is proposed for the inactivation of methylamine dehydrogenase by phenylhydrazines, and a structure is proposed for the putative transition state for the rate-limiting step in the overall processes of binding and adduct formation by phenylhydrazine. The relevance of these results to the process of imine formation between substrate amines and TTQ during the normal catalytic process is also discussed.

Cofactor-directed inactivation by nucleophilic amines of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans.

Phenylhydrazine, semicarbazide, aminoguanidine, hydrazine, and hydroxylamine each irreversibly inactivated methylamine dehydrogenase from Paracoccus denitrificans and caused changes in the absorbance spectrum of the protein-bound tryptophan tryptophylquinone [TTQ] prosthetic group. Different spectral perturbations were observed on reaction with each of these inactivators. In each case a stoichiometry of 2 mol per mol of enzyme (1:1 per cofactor) was required to observe complete modification of the absorbance spectrum. Identical changes were observed in the presence and absence of oxygen. The reactions of hydrazine and hydroxylamine were very rapid, with stoichiometric inactivation occurring in less than 30 s. Inactivation by phenylhydrazine and semicarbazide exhibited apparent bimolecular kinetics and second order rate constants for inactivation, respectively, of 25 min-1 mM-1 and 39 min-1 mM-1. In contrast, inactivation by aminoguanidine exhibited saturation behavior and kinetic parameters of KI = 2.5 mM and kinact = 0.5 min-1 were obtained. Ammonium salts did not inactivate the enzyme, but were reversible competitive inhibitors with respect to methylamine. A Ki of 20 mM was obtained for ammonium chloride. A mechanism for the reactions of these compounds with the TTQ cofactor of methylamine dehydrogenase is proposed, and the relationship of these data to the mechanisms of interaction of these compounds with o-quinones and other quinoproteins which possess TTQ and other quinone cofactors is discussed.

Binding constants for a physiologic electron-transfer protein complex between methylamine dehydrogenase and amicyanin. Effects of ionic strength and bound copper on binding.

Two soluble proteins, methylamine dehydrogenase and amicyanin, form a physiologically relevant complex in which intermolecular electron transfer occurs. To characterize and quantitate the binding of these two weakly-associating proteins, an ultrafiltration binding assay has been developed which involves brief centrifugation of mixtures of proteins in centrifuge concentrators followed by quantitation of proteins on each side of the filtration membrane by HPLC. Under low ionic strength conditions which are optimal for the redox reaction between these proteins, a Kd of 4.5 microM was measured for the methylamine dehydrogenase-amicyanin complex. The Kd increased by 8-fold in the presence of added salt. Apoamicyanin, which is known from crystallographic analysis to be structurally very similar to amicyanin, exhibited a much higher Kd and much less specific binding than did the holoprotein. Apoamicyanin also exhibited apparent self-association at low ionic strength which was not observed with amicyanin. These observations are correlated with the known crystal structures of these proteins, free and in complex, and with the available biochemical information on the interactions of these two proteins.

Tyr(30) of amicyanin is not critical for electron transfer to cytochrome c-551i: implications for predicting electron transfer pathways.

A Pathways analysis of the methylamine dehydrogenase-amicyanin-cytochrome c-551i protein electron transfer (ET) complex predicts two sets of ET pathways of comparable efficiency from the type I copper of amicyanin to the heme of cytochrome c-551i. In one pathway, the electron exits copper via the Cys(92) copper ligand, and in the other, it exits via the Met(98) copper ligand. If the Pathways algorithm is modified to include contributions from the anisotropy of metal-ligand coupling, independent of differences in copper-ligand bond length, then the pathways via Cys(92) are predicted to be at least 100-fold more strongly coupled than the pathways via any of the other copper ligands. All of the favored pathways via Cys(92) include a through-space jump from Cys(92) to the side chain of Tyr(30). To determine whether or not the pathways via Cys(92) are preferentially used for ET, Tyr(30) was changed to other amino acid residues by site-directed mutagenesis. Some mutant proteins were very unstable suggesting a role for Tyr(30) in stabilizing the protein structure. Y30F and Y30I mutant amicyanins could be isolated and analyzed. For the Y30I mutant, the modified Pathways analysis which favors ET via Cys(92) predicts a decrease in ET rate of at least two orders of magnitude, whereas the standard Pathways analysis predicts no change in ET rate since ET via Met(98) is not affected. Experimentally, the ET rates of the Y30I and Y30F mutants were indistinguishable from that of wild-type amicyanin. Likely explanations for these observations are discussed as are their implications for predicting pathways for ET reactions of metalloproteins.

Plant cell cloning and culture products.

Biotechnological developments in the use of plant cells as sources of biochemicals have now brought several applications within commercial range. Plant propagation has developed faster commercially, but now requires similar biotechnology to enable the automated production of large numbers of plants at low cost. Although some of the enabling technology has been developed for pregerminated seeds this has not been coupled to production of plantlets in bioreactors. Our present lack of knowledge of how to control gene expression in plant cells, which stems from our ignorance of the organization and regulation of the plant genome, is a critical factor limiting all applications of plant cell cultures.

Mineral components of plant cell walls.

Plant cell walls that are secondarily thickened contain silicon and metal cations. The silicon occurs predominantly as silica (SiO2.nH2O) deposited in intimate association with the organic components of the walls and, according to recent evidence, as an integral constituent of polyuronides. Relatively large amounts of deposited (i.e., solid) silica are found in rice and other cereals and in grasses. When ingested by ruminant animals, practically all the solid silica may be recovered in the feces. However, microscopic particles of silica from plants are, to a small extent, absorbed as such through the gastrointestinal wall in both man and ruminant animals. It has now been shown that silicon is essential for animals, and that it is a constituent of certain mucopolysaccharides, thereby contribution to the architecture of connective tissues. The acidic silanol group of solid silica in plant cell walls may be involved in binding metal cations, but carboxyl and phenolic hydroxyl groups of the organic components of the walls are probably mainly responsible. Binding of metal cations by these components of plant cell walls, and possibly by silica, is likely to reduce availability of the cations for intestinal absorption.

The therapeutic potential for catalytic antibodies: from a concept to a promise.

More than ten years have now elapsed since the first reports confirmed that antibodies not only label antigenic targets but can also perform catalytic functions. Much of the initial research in this area focussed on exploring the scope and utility of these biocatalysts both as enzyme mimics and as programmable protein catalysts. However, their potential in the biomedical field has also been probed. This review details the present perspective of catalytic antibodies as new tools for immunotherapy and specifically focuses on their application to prodrug activation and drug inactivation.

Factors affecting the stability of methanol dehydrogenase from Paracoccus denitrificans.

Methanol dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methanol-grown cells. The enzyme was composed of subunits of M(r) 67,000 and 12,000, and non-covalently bound pyrroloquinoline quinone. It exhibited a pH optimum at pH values of 9.0 and above. It was not stable at pH greater than 9.0, but exhibited little loss of activity after prolonged incubation at pH values as low as 4.5. Methyl dehydrogenase was relatively stable to thermal denaturation. The thermal stability was enhanced by the presence of Ca2+ and diminished by the presence of EDTA. These data suggest a structural role for Ca2+ in this enzyme, similar to what has been observed with quinoprotein glucose and ethanol dehydrogenases.

Hemodynamics of the femoral head.

UNLABELLED: We have determined the pressure-flow relationships of the canine femoral head during venous tamponade of the hip capsule. Intra-osseous pressures were determined before and after infusion of the joint with saline solution to a pressure of sixty-five centimeters of water. Femoral head blood flow was simultaneously determined by the indicator-dilution technique utilizing isotopically labeled microspheres. In puppies, pressure in the femoral head rose 248 per cent while flow dropped by 60 per cent after inflation of the capsule. In adults, no statistically significant change in either pressure or flow was seen. Thus, in the immature animal, venous tamponade results in increased pressure and decreased blood flow. In the mature animal, venous tamponade does not alter intra-osseous pressures or blood-flow rates due to the intact intramedullary venous drainage of the adult femoral head. CLINICAL RELEVANCE: In the immature individual, venous tamponade may well be involved in the development of Legg-Perthes disease. A bout of nonspecific synovitis may elevate intracapsular pressure sufficiently to obstruct venous outflow. This creates an increase in intra-osseous pressure and a decrease in femoral head blood flow. Hemodynamic changes of this magnitude have not been shown to induce Legg-Perthes disease; however, a strong suspicion exists that such alterations may be linked to the disease. In the adult, venous tamponade probably is not involved in the pathogenesis of avascular necrosis. The maintenance of intramedullary venous drainage of the epiphysis into the metaphysis may account for the fact that vascular necrosis of the femoral head rarely develops in adult patients with osteoarthritis or rheumatoid arthritis plus an inflammatory synovitis of the hip.

Continuity of care. Development and implementation of a shared patient data base.

Although the inpatient Oncology Unit, the Medical Oncology Clinic, and Radiation Oncology provided care for many of the same patients, there was no mechanism for sharing nursing information, and little colleague input from one area to another. In order to meet this need, a nurse from each of the clinic areas was added to the inpatient unit's Patient Care Evaluation Committee. Working through this committee, these nurses developed an Inpatient/Outpatient Data Flow Sheet, which could be initiated in any oncology area to implement information flow when a patient was to be seen in a different setting. It proved to be an effective tool. The flow sheet, along with our rationale, was then presented for consideration as a computerized program to be used between the three areas. After careful investigation, it was approved. This provided the oncology areas with the first data storage capability for nursing in the hospital. It offered oncology nurses in distinct and separate areas access to obtain and update information on shared patients. This manuscript will focus on the computer program and the data base designed for the oncology department and its impact on nurses and patients.

HIV infection: educational programs and policies for school personnel.

Acquired Immunodeficiency Syndrome (AIDS) challenges schools as the frontline defense against this epidemic. School personnel should become knowledgeable about human immunodeficiency virus (HIV) infection and get involved in presenting quality HIV/AIDS education programs. This paper provides an epidemiological update on AIDS as it relates to the school-age population. Evidence establishing modes of transmission is emphasized, and recommendations are presented for handling students infected with HIV. Information is provided to assist school staff implement HIV/AIDS education programs, including core concepts to be emphasized.

Specific inhibition of ectodomain shedding of glycoprotein Ibα by targeting its juxtamembrane shedding cleavage site.

BACKGROUND: Ectodomain shedding of glycoprotein Ibα (GPIbα), a proteolytic event in which metalloprotease ADAM17 cleaves the Gly464-Val465 bond and releases glycocalicin to the plasma, is considered a critical step in mediating clearance of stored platelets. Supporting evidence has largely come from studies using ADAM17 inhibitors. However, the definitive proof is lacking due to the broad substrate specificity of ADAM17. AIM: To achieve substrate-specific inhibition of GPIbα shedding. METHODS: Development of monoclonal antibodies that directly bind the sequence around the GPIbα shedding cleavage site and inhibit GPIbα shedding by blocking ADAM17 access to the cleavage site. RESULTS: Six anti-GPIbα monoclonal antibodies with varying binding affinities were obtained. The prototypic clone, designated 5G6, and its monomeric Fab fragment bind specifically purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα. The clone 5G6 showed similar inhibitory potency as a widely used shedding inhibitor GM6001 in both constitutive and induced GPIbα shedding in human platelets. It does not recognize mouse GPIbα or inhibit shedding of other platelet receptors. Finally, 5G6 binding displays no detectable effect on platelet activation and aggregation. CONCLUSIONS: The clone 5G6 specifically inhibits GPIbα shedding with no detectable effect on platelet functions. The method of substrate-specific shedding inhibition by macromolecular binding of the shedding cleavage site can be applicable to many other transmembrane receptors undergoing ectodomain shedding.