Actin filament-membrane attachment: are membrane particles involved?

The association of actin filaments with membranes is an important feature in the motility of nonmuscle cells. We investigated the role of membrane particles in the attachment of actin filaments to membranes in those systems in which the attachment site can be identified. Freeze fractures through the end-on attachment site of the acrosomal filament bundles in Mytilus (mussel) and Limulus (horseshoe crab) sperm and the attachment site of the microvillar filament bundles in the brush border of intestinal epithelial cells were examined. There are no particles on the P face of the membrane at these sites in the sperm systems and generally none at these sites in microvilli. In microvilli, the actin filaments are also attached along their lengths to the membrane by bridges. When the isolated brush border is incubated in high concentrations of Mg++ (15 mM), the actin filaments form paracrystals and, as a result, the bridges are in register (330 A period). Under these conditions, alignment of the particles on the P face of the membrane into circumferential bands also occurs. However, these bands are generally separated by 800-900 A, indicating that all the bridges cannot be directly attached to membrane particles. Thus membrane particles are not directly involved in the attachment of actin filaments to membranes.     

Hydrolysis of starch particles using immobilized barley α-amylase

Barley α-amylase has been immobilized on silica particles with diameters between 0.5 and 10 μm using a covalent binding method. Immobilization procedures were adjusted to optimize enzyme activity. The effects of product inhibition, thermal stability and operational stability have been determined. The feasibility of using the immobilized enzyme to hydrolyze wheat starch particles at temperatures below the gelatinization temperature (<55 °C) was proven. The optimal conditions for the hydrolysis were found to be: pH 4.5, 40 °C, calcium ion concentration 0.002 M and immobilized enzyme loading of 30 mg/ml. At these conditions, the immobilized enzyme was able to hydrolyze wheat starch particles at concentrations as high as 100 mg/ml with a final conversion of 90% after 24 h of operation. Maltose and glucose were found to inhibit the immobilized enzyme in a similar manner as reported previously using soluble enzyme. Although the thermostability of the immobilized enzyme was superior to the soluble enzyme, the immobilized enzyme degraded at the same rate as the soluble enzyme during cold wheat starch hydrolysis (operational stability unchanged). Model equations are presented for product inhibition, hydrolysis kinetics and enzyme degradation. Using best-fit parameters, the equations are shown to fit the experimental data well.     

Decay of γ particles in germinating zoospores of Blastocladiella emersonii

Summary The breakdown and disappearance of particles was followed by electron microscopy on synchronously germinating populations of B. emersonii zoospores. The sequence of morphological changes, unaffected by the method used to induce germination, was divided into four stages. Initially, the particle's surrounding unit membrane becomes amorphous and buds off vesicles that fuse with the plasma membrane. Simultaneously, ca. 80 m vesicles are visible either partially embedded in, or lying free along, the inner surface of the electron dense matrix. These small vesicles seem to originate from a newly detected membrane system in the electron dense matrix. In the next stage, particles characteristically adhere to various organelles, especially the nuclear cap. In subsequent stages the particles continue to decay by releasing 80 m vesicles from their matrices and by budding off vesicles from their surrounding membranes. Often, multivesicular bodies are formed when the 80 m vesicles become enclosed by the particle's budding outer membrane. In the final stage, the surrounding membranes of the different particles fuse and produce large vacuoles containing many of the electron dense matrices which, though by now partially fragmented, continue to release 80 m vesicles. The relationship of the above events to the formation of the cell wall is discussed.     

Polarographic studies of membrane particles containing Na−K ATPase

Summary The properties of a suspension of membrane particles containing Na–K ATPase have been investigated with the aid of d–c and a–c polarography. In particular, we have studied the interaction of three cations, two very effective enzyme inhibitors and one activator, with the enzyme preparation. Ag⁺ and Cu⁺⁺, which inhibit the enzyme at very low concentrations, bind very strongly. No binding could be found with the activating ion, Tl⁺, however. Adsorption of a substance with an isoelectric point between pH 4 and pH 5.5 occurred at the electrode surface between –0.1 and –1.2 V at pH 7, and was associated with the random currents that appear during the measurements. The random currents arise when the membrane particles collide with the electrode and cause changes in the structure of the electrical double layer. (Added substances that adsorb more strongly at the mercury/water interface eliminate the random currents.) The adsorbed film impedes the flow of the free Ag⁺ and Cu⁺⁺ ions, and to a smaller extent, the flow of Tl⁺ ions. The differences between the binding of inhibiting and activating ions are correlated with their effects on the ATPase enzyme activity.     

Cytochemical demonstration of catalasepositive particles (peroxisomes?) in fibroblasts

Summary The connective tissue of the mucosa of the respiratory tract, of the gastric mucosa and of the mucosa of the tongue was investigated in mice. The tissue was fixed in glutaraldehyde and incubated in an alkaline DAB-medium to demonstrate the peroxidatic activity of catalase. In fibroblasts and fibrocytes, as well as in lymphoid cells, membrane bounded particles from 0.10 to 0.25 m in diameter were found, whose matrices were intensely stained by the histochemical reaction. The reaction is inhibited by the addition of 2×10^–2 M 3-amino-1,2,4-triazole. In connective tissue cells of specimens, which were not reacted to demonstrate catalase activity, these organelles show a granular matrix of moderate electron density. They lack a crystalline core. The possibility that these catalase-positive particles (CPs) represent peroxisomes is discussed.This investigation was kindly supported by a grant of Hochschuljubiläumsstiftung der Stadt Wien.     

LATEX: A peculiar biological component in airborne particles?

In a study in the USA, latex allergens have beenidentified in airborne particles. Natural rubber(latex) is a product from the plant Heveabrasiliensis and is largely used in the tireindustry. In the vicinity of roads, latex can becomeairborne due to wear-off processes of tires.Sensitisation to latex has been increasing in the lastdecade; an important pathway for the sensitisation ismediated by the airways. It is not known, if thisconcerns mainly laboratory personnel or if this isalso an environmental problem.This study investigated the content of latex inairborne particles in the vicinity of a road withmoderate traffic, in comparison to a control site.Latex was determined in a competitive ELISA based onpolyclonal IgE antibodies. During spring and summer,collected airborne dust was analysed for the contentof protein and latex. Latex concentrations inPM₁₀ in the city were higher than at the controlsite. At both sites, latex levels were significantlylower in PM_2.5 than in PM₁₀.     

Phagocytosis of optically-trapped particles： delivery of the pure phagocytic signal

Phagocytosis is a fundamental cell biological process exhibited by a wide variety of cell types from single cell organisms, which rely on this for feeding, to phagocytes in higher animals, which rely on specialised immune cells for combating infecting micro-organisms. In the immune system, both macrophages and neutrophils play roles as phagocytes. Neutrophils are often called ＂professional phagocytes＂ because of their remarkable capacity for phagocytosis, being able to intemalise microscopic particles （diam 0.5-3 μm） of virtually any surface material. The efficiency and speed of phagocytosis is, however, increased by coating the surface of the particles with opsonins such as antibodies or the complement component C3bi （acting on J32 integrin receptors）, and C3bi-accelerated phagocytosis by neutrophils is the first line of defence by the innate immune system in vivo, operating in advance of the slower production of antibodies. Understanding the mechanism ofphagocytosis is, therefore, clearly an important goal.     

Colorful virus-like particles: fluorescent protein packaging by the Qβ capsid

Qβ virus-like particles encapsulating multiple copies of fluorescent proteins were generated in high yields using a modular system enhanced by specific engineered RNA--protein interactions. The resulting particles were structurally indistinguishable from recombinant Qβ alone. The encapsidated proteins were nearly identical in photochemical properties to monomeric analogues, were more stable toward thermal degradation, and were protected from proteolytic cleavage. Residues on the outer capsid surface were chemically derivatized by acylation and azide--alkyne cycloaddition without affecting the fluorescence properties of the packaged proteins. A high-affinity carbohydrate-based ligand of the CD22 receptor was thereby attached, and specific cell labeling by the particles was successfully detected by flow cytometry and confocal laser microscopy.     

17β-estradiol inhibits the production of infectious particles of hepatitis C virus

Persistent infection with hepatitis C virus causes serious liver diseases, such as chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma. The male gender is one of the critical factors in progression of hepatic fibrosis due to chronic HCV infection; thus female hormones may play a role in delaying the progression of hepatic fibrosis. It has also been reported that women are more likely than men to clear HCV in the acute phase of infection. These observations lead the present authors to the question: do female hormones inhibit HCV infection? In this study using HCV J6/JFH1 and Huh‐7.5 cells, the possible inhibitory effect(s) of female hormones such as 17β‐estradiol (the most potent physiological estrogen) and progesterone on HCV RNA replication, HCV protein synthesis and production of HCV infectious particles (virions) were analyzed. It was found that E₂, but not P₄, significantly inhibited production of the HCV virion without inhibiting HCV RNA replication or HCV protein synthesis. E₂–mediated inhibition of HCV virion production was abolished by a nuclear estrogen receptor (ER) antagonist ICI182780. Moreover, treatment with the ERα‐selective agonist 4, 4′, 4″‐ (4‐propyl‐[1H]‐pyrazole‐1, 3, 5‐triyl)trisphenol (PPT), but not with the ERβ‐selective agonist 2, 3‐bis (4‐hydroxyphenyl)‐propionitrile (DPN) or the G protein‐coupled receptor 30 (GPR30)‐selective agonist 1‐(4‐[6‐bromobenzo 1, 3 dioxol‐5‐yl]‐3a, 4, 5, 9b‐tetrahydro‐3H‐cyclopenta [c] quinolin‐8‐yl)‐ethanone (G‐1), significantly inhibited HCV virion production. Taken together, the present results suggest that the most potent physiological estrogen, E₂, inhibits the production of HCV infectious particles in an ERα–dependent manner.     

Iron components in PS II particles of spinach by Mössbauer spectroscopy

Mössbauer spectrum measured for the iron components of photosystem II (PS II) particles of spinach is a superposition of 4 doublets. Quadrupole splitting and chemical shifting of doublets I–IV are characteristics of proteins with oxidized cytochrome b-559, reduced cytochrome b-559, Fe³⁺-Q complex and Fe²⁺ -Q complex respectively. After the PS II particles are treated with La³⁺, two doublets of Fe²⁺ disappear and Fe²⁺ is converted into Fe³⁺, indicating that the reduced cytochrome b-559 has been converted into the oxidized cytochrome b-559, and Fe²⁺ -Q complex into Fe³⁺ -Q complex. The Mössbauer spectrum of PS II particles treated with La³⁺ and Ca²⁺ shows that Ca²⁺ can weaken the inhibitory effect of La³⁺ in part, and a portion of the reduced cytochrome b-559 and Fe-Q complex still exist.     

Influencing the direct formation of ‘Janus’ particles in molecular dynamics simulations

The binary nucleation of phase-separated Lennard-Jones clusters was analysed under various system conditions using molecular dynamics simulations. The modified potential model provides a simple gateway to observe non-wetting behaviour and imitates the more complex interactions of non-miscible substances. Thus, not only the transition from ideally mixed clusters to so-called ‘Janus’ particles, but also the structural aspects and dynamic formation processes of nanoscopic droplets are directly observable from the gas phase. Various shapes and sizes of these inhomogeneous clusters have been found via simple tuning of system parameters. From this analysis, we gained further insight into the direct formation of ‘Janus’ particles from the gas phase.     

Human parvovirus B19 virus-like particles: In vitro assembly and stability

Virus-like particles (VLPs) are biological nanoparticles identical to the natural virions, but without genetic material. VLPs are suitable for the analysis of viral infection mechanisms, vaccine production, tissue-specific drug delivery, and as biological nanomaterials. Human parvovirus B19 (B19) infects humans; therefore VLPs derived from this virus have enormous potential in medicine and diagnostics. Current production of self-assembled VLPs derived from B19 is typically carried out in eukaryotic expression systems. However many applications of VLPs require access to its internal core. Consequently, the processes of disassembly and further reassembly of VLPs are critical both for purification of viral proteins, and for encapsulation purposes. Herein we report the in vitro self-assembly of B19 VLPs derived from the recombinant VP2 protein expressed in Escherichia coli and the effects of pH and ionic strength on the assembly process. Our results demonstrate that VP2 is able to form VLPs completely in vitro. At neutral pH, homogeneous VLPs assemble, while at acidic and basic pHs, with low ionic strength, the major assemblies are small intermediates. The in vitro self-assembled VLPs are highly stable at 37 °C, and a significant fraction of particles remain assembled after 30 min at 80 °C.     

Normal-phase HPLC quantitation of chlorophyll a′ and phylloquinone in Photosystem I particles

Fractionated Photosystem (PS) I particles consisting of six, five or two core proteins were analyzed by HPLC for chlorophyll (Chl) a and phylloquinone (PhQ). Each particle had a Chl a/P700 molar ratio of 50–55 and contained ca. 2 molecules of Chl a per P700. Deliberate control of eluent composition led to isolated elution of PhQ and -carotene in the normal-phase chromatogram. Based on these a simple HPLC procedure has been established to determine the PhQ/P700 molar ratio, which was ca. 2 for the larger two PS I particles and ca. 1 for the smallest particle, in line with previous reports.     

α-Chymotrypsin immobilized on ferromagnetic particles coated with titanium oxide: Production and catalytic characterization

Immobilization of α-chymotrypsin on magnetic particles with stable coat with titanium oxides as a main constituent allowed the biocatalytic system to be quickly and qualitatively separated into the components after completion of the enzymatic reaction. X-ray phase analysis demonstrated that the coat of magnetic particles is composed mainly of titanium dioxide in brookite modification. The maximal capacity of the particles amounted to 0.3 mg protein/mg particles. It was demonstrated that the reaction catalyzed by immobilized α-chymotrypsin proceeds in a kinetic mechanism due to a high dispersion of the ferromagnetic particles. The catalytic constant (25 s⁻¹) andK _M (0.17 mM) for the immobilized enzyme for the hydrolysis of N-acetyl-L-tyrosine ethyl ester are comparable to the corresponding characteristics for the free enzyme.     

Suppression of the NF-κB pathway by diesel exhaust particles impairs human antimycobacterial immunity

Epidemiological studies suggest that chronic exposure to air pollution increases susceptibility to respiratory infections, including tuberculosis in humans. A possible link between particulate air pollutant exposure and antimycobacterial immunity has not been explored in human primary immune cells. We hypothesized that exposure to diesel exhaust particles (DEP), a major component of urban fine particulate matter, suppresses antimycobacterial human immune effector cell functions by modulating TLR-signaling pathways and NF-κB activation. We show that DEP and H37Ra, an avirulent laboratory strain of Mycobacterium tuberculosis, were both taken up by the same peripheral human blood monocytes. To examine the effects of DEP on M. tuberculosis-induced production of cytokines, PBMC were stimulated with DEP and M. tuberculosis or purified protein derivative. The production of M. tuberculosis and purified protein derivative-induced IFN-γ, TNF-α, IL-1β, and IL-6 was reduced in a DEP dose-dependent manner. In contrast, the production of anti-inflammatory IL-10 remained unchanged. Furthermore, DEP stimulation prior to M. tuberculosis infection altered the expression of TLR3, -4, -7, and -10 mRNAs and of a subset of M. tuberculosis-induced host genes including inhibition of expression of many NF-κB (e.g., CSF3, IFNG, IFNA, IFNB, IL1A, IL6, and NFKBIA) and IFN regulatory factor (e.g., IFNG, IFNA1, IFNB1, and CXCL10) pathway target genes. We propose that DEP downregulate M. tuberculosis-induced host gene expression via MyD88-dependent (IL6, IL1A, and PTGS2) as well as MyD88-independent (IFNA, IFNB) pathways. Prestimulation of PBMC with DEP suppressed the expression of proinflammatory mediators upon M. tuberculosis infection, inducing a hyporesponsive cellular state. Therefore, DEP alters crucial components of antimycobacterial host immune responses, providing a possible mechanism by which air pollutants alter antimicrobial immunity.     

Characterization of the α-cyanocinnamate binding site in rat heart mitochondria and in submitochondrial particles

The effect of pH and substrates on the binding of radiolabelled α-cyanocinnamate to mitochondria and submitochondrial particles has been investigated. It has been found that the binding is strongly influenced by the pH of the medium (it decreases on increasing the pH of the medium). The inhibition of pyruvate oxidation by this inhibitor follows the same pH dependence. The pH affects only the affinity of the α-cyanocinnamate binding site without changing their total number. A similar pH dependence has been found in inside-out submitochondrial particles where the binding sites are directly accessible. The quantitative parameters of the binding of α-cyanocinnamate in submitochondrial particles have been determined. The binding can be prevented or displaced by pyruvate and other substrates of the carrier. The turnover number for pyruvate transport in rat-heart mitochondria has been determined.     

Genotoxicity of fractionated organic material in airborne particles from São Paulo, Brazil

Particulate matter less than 10 microns aerodynamic diameter (PM10) is associated with adverse health effects including increased respiratory problems and mortality. PM10 is also associated with increases in cancer in some urban areas. Identification of toxic compounds in PM10 is a step toward estimating exposure to these compounds and evaluating their public health risk. However, the toxic compounds on PM10 are part of a highly complex mixture of compounds that makes chemical characterization difficult. Before this study, there has been little investigation of genotoxic compounds in particulate matter from Latin American cities. Here, both bioassay (mutagenicity) and chemical analyses were conducted with organic solvent extracts of PM10 collected from S?o Paulo, a major Brazilian city. Sequential extraction in dichloromethane (DCM) followed by acetone (ACE) yielded 20.3% and 10.2% of the total mass, respectively. Non-polar and moderately polar organic material solubilized in DCM. ACE extracted more polar organic species and some inorganic ions. Both extracts were fractionated separately using cyanopropyl-bonded silica chromatography with organic solvents of increasing polarity. The mass distribution among the fractions was measured. The mutagenic activity of the fractions was assayed using the microsuspension procedure with the Salmonella typhimurium tester strain TA98, with and without addition of metabolic enzymes (S9). The DCM extract had about four times higher mutagenic activity than the ACE extract. In general, addition of S9 resulted in an increase in mutagenicity of DCM fractions, but a decrease for the ACE extract. Most of the activity was concentrated in fractions in the mid-range of polarity within both the DCM and ACE extracts. The fractions were analyzed by gas chromatography with mass selective detection (GC/MS) without derivatization. The most mutagenic fractions in the DCM extract contained ketones, aldehydes, and quinolines. The most mutagenic ACE fraction had ketones, carboxylic acids, and aldehydes.