Dynamic features of cells expressing macrophage properties in tissue cultures of dissociated cerebral cortex from the rat

Summary Two previously identified forms of macrophage were investigated in primary cultures of cerebral cortical cells. Dynamic features were revealed through time-lapse video recording and aspects of macrophage function were assessed. The two cell forms were shown to be different pre-mitotic stages of a single cell type. The cell cycle for these cells involved an initial large, flat, quiescent cell which retracted to yield a slightly rounded form with numerous processes. This latter form lost processes and developed profuse filopodia as it became very rounded just prior to division; both resulting daughter cells then regained the initial large flat appearance. These cells possessed several properties of macrophages, including phagocytosis, nucleoside diphosphatase enzyme, and CR3 receptors. These properties were transient, expressed just before and after mitosis, but subsequently down-regulated in the flat daughter cells. Because of this feature, it was difficult to determine the exact size of this cell population; however, the observed rate of proliferation suggests it may be substantial. It is suggested that these cells correspond to non-microglial macrophages of brain tissue and, because of their significant down-regulation, they may be difficult to detect. This may be important in studies of brain accessory immune cells in tissue culture.     

Molecular and immunological characterization of the major outer membrane proteins of Brucella

Abstract Saccharomyces cerevisiae exponentially growing in basic or 0.7 M NaCl medium were isotopically labelled with ³⁵S-methionine, followed by protein separation and quantification by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with computerised image analysis. The electrophoretic separation resolved about 650 proteins of which 13 displayed significant and at least 2-fold changes in rate of synthesis during saline growth. By sequencing of 2D-PAGE resolved proteins, one of the 8 induced spot, p42.9/5.5, was shown to correspond to the full length (containing the N-terminal extension) product of the GPD 1 gene encoding the cytoplasmic glycerol 3-phosphate dehydrogenase. The expression of the TDH 3 gene, glyceraldehyde 3-phosphate dehydrogenase, and the ENO 2 gene, enolase, decreased during growth in NaCl medium, declines hypothesised to have an impact on the flux to glycerol.     

Reaction profile of the last step in cytochrome P-450 catalysis revealed by studies of model complexes

 A series of oxoiron(IV) porphyrin cation radical complexes was investigated as compound I analogs of cytochrome P-450. Both the spectroscopic features and the reactivities of the complexes in oxygen atom transfer to olefins were examined as a function of only one variable, the axial ligand trans to the oxoiron(IV) bond. The results disclosed two important kinetic steps – electron transfer from olefin to oxoiron(IV) and intramolecular electron transfer from metal to porphyrin radical – which are affected differently by the axial ligands. The large kinetic barrier of the latter step in the reaction of olefins with the perchlorato-bound oxoiron(IV) porphyrin cation radical complex enabled the trapping of a reaction intermediate in which the metal, but not the porphyrin radical, is reduced. The first electron transfer step is probably followed by σ-bond formation, which readily accounts for formation of isomerized organic products at low temperatures. It is finally postulated that part of the enhanced oxygenation activities of cytochrome P-450 monooxygenases and chloroperoxidases is due to a lowering of the energy barrier for the second electron transfer step via participation of their redox-active cysteinate ligand. Received: 16 January 1997 / Accepted: 24 May 1997     

Insulin-releasing and cytotoxic properties of the frog skin peptide,tigerinin-1R: a structure–activity study

The frog skin host-defense peptide tigerinin-1R (RVCSAIPLPICH.NH₂) is insulinotropic both in vitro and in vivo. This study investigates the effects on insulin release and cytotoxicity of changes in cationicity and hydrophobicity produced by selected substitutions of amino acids by l-arginine, l-lysine and l-tryptophan. The [A5W], [L8W] and [I10W] analogs produced a significant (P < 0.01) increase in the rate of insulin release from BRIN-BD11 rat clonal β cells at concentration of 0.01 nM compared with 0.1 nM for tigerinin-1R. The increase in the rate of insulin release produced by a 3 μM concentration of the [S4R], [H12K], and [I10W] analogs from both BRIN-BD11 cells and mouse islets was significantly greater (P < 0.05) than that produced by tigerinin-1R. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 μM indicating that plasma membrane integrity had been preserved. [A5W] tigerinin-1R was the only analog tested that showed cytotoxic activity against human erythrocytes (LC₅₀ = 265 ± 16 μM) and inhibited growth of Escherichia coli (MIC = 500 μM) and Staphylococcus aureus (MIC = 250 μM). The circular dichroism spectra of tigerinin-1R and [A5W] tigerinin-1R indicate that the peptides adopt a mixture of β-sheet, random coil and reverse β-turn conformations in 50% trifluoroethanol/water and methanol/water. Administration of [S4R] tigerinin-1R (75 nmol/kg body weight) to high-fat fed mice with insulin resistance significantly (P < 0.05) enhanced insulin release and improved glucose tolerance over a 60 min period following an intraperitoneal glucose load. The study supports the claim that tigerinin-1R shows potential for development into novel therapeutic agents for treatment of type 2 diabetes mellitus.     

Determination of G-protein levels,ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db)

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of G_i α-2, G_i α-3 and G-protein β-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of G_s α-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of G_s α-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 μM), GTP (100 μM), p[NH]ppG (100 μM), NaF (10 mM) and glucagon (10 μM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 μM) was lower by about 22%. Lower concentrations (EC₅₀-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC₅₀-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional G_i activity. The lower levels of expression of G-protein β-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

The role of lipid second messengers in aldosterone synthesis and secretion

Second messengers are small rapidly diffusing molecules or ions that relay signals between receptors and effector proteins to produce a physiological effect. Lipid messengers constitute one of the four major classes of second messengers. The hydrolysis of two main classes of lipids, glycerophospholipids and sphingolipids, generate parallel profiles of lipid second messengers: phosphatidic acid (PA), diacylglycerol (DAG), and lysophosphatidic acid versus ceramide, ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate, respectively. In this review, we examine the mechanisms by which these lipid second messengers modulate aldosterone production at multiple levels. Aldosterone is a mineralocorticoid hormone responsible for maintaining fluid volume, electrolyte balance, and blood pressure homeostasis. Primary aldosteronism is a frequent endocrine cause of secondary hypertension. A thorough understanding of the signaling events regulating aldosterone biosynthesis may lead to the identification of novel therapeutic targets. The cumulative evidence in this literature emphasizes the critical roles of PA, DAG, and sphingolipid metabolites in aldosterone synthesis and secretion. However, it also highlights the gaps in our knowledge, such as the preference for phospholipase D-generated PA or DAG, as well as the need for further investigation to elucidate the precise mechanisms by which these lipid second messengers regulate optimal aldosterone production.     

Tropical terrestrial invertebrates—Where to from here?

There are over one million described invertebrate species on Earth, the majority of which are likely to inhabit the highly biodiverse rain forests around the equator. These are some of the most vulnerable ecosystems on Earth due to the pressures of deforestation and climate change with many of their inhabitants at risk of extinction. Invertebrates play a major role in ecosystem functioning from decomposition and nutrient cycling to herbivory and pollination; however, while our understanding of these roles is improving, we are far from being able to predict the consequences of further deforestation, climate change, and biodiversity loss due to the lack of comparative data and the high proportion of species which remain to be discovered. As we move into an era of increased pressure on old-growth habitats and biodiversity, it is imperative that we understand how changes to invertebrate communities, and the extinction of species, affect ecosystems. Innovative and comprehensive methods that approach these issues are needed. Here, we highlight priorities for future tropical terrestrial invertebrate research such as the efficiency of sustainable land management, exploration of innovative methods for better understanding of invertebrate ecology and behavior, and quantifying the role of invertebrates in ecosystem functioning.     

Elasto-regenerative properties of polyphenols

Abdominal aortic aneurysms (AAA) are progressive dilatations of infra-renal aorta causing structural weakening rendering the aorta prone to rupture. AAA can be potentially stabilized by inhibiting inflammatory enzymes such as matrix metalloproteinases (MMP); however, active regression of AAA is not possible without new elastic fiber regeneration. Here we report the elastogenic benefit of direct delivery of polyphenols such as pentagalloyl glucose (PGG), epigallocatechin gallate (EGCG), and catechin, to smooth muscle cells obtained either from healthy or from aneurysmal rat aorta. Addition of 10 μg/ml PGG and ECGC induce elastin synthesis, organization, and crosslinking while catechin does not. Our results indicate that polyphenols bind to monomeric tropoelastin and enhance coacervation, aid in crosslinking of elastin by increasing lysyl oxidase (LOX) synthesis, and by blocking MMP-2 activity. Thus, polyphenol treatments leads to increased mature elastin fibers synthesis without increasing the production of intracellular tropoelastin.     

The antitumor agent 3-bromopyruvate has a short half-life at physiological conditions

Clinical research is currently exploring the validity of the anti-tumor candidate 3-bromopyruvate (3-BP) as a novel treatment for several types of cancer. However, recent publications have overlooked rarely-cited earlier work about the instability of 3-BP and its decay to 3-hydroxypyruvate (3-HP) which have obvious implications for its mechanism of action against tumors, how it is administered, and for precautions when preparing solutions of 3-BP. This study found the first-order decay rate of 3-BP at physiological temperature and pH has a half-life of only 77 min. Lower buffer pH decreases the decay rate, while choice of buffer and concentration do not affect it. A method for preparing more stable solutions is also reported.