Cytoskeletal mechanics in adherent human airway smooth muscle cells: probe specificity and scaling of protein-protein dynamics

We probed elastic and loss moduli in the adherent human airway smooth muscle cell through a variety of receptor systems, each serving as a different molecular window on cytoskeletal dynamics. Coated magnetic microbeads were attached to the cell surface via coating-receptor binding. A panel of bead coatings was investigated: a peptide containing the sequence RGD, vitronectin, urokinase, activating antibody against ₁-integrin, nonactivating antibody against ₁-integrin, blocking antibody against ₁-integrin, antibody against ₁-integrin, and acetylated low-density lipoprotein. An oscillatory mechanical torque was applied to the bead, and resulting lateral displacements were measured at baseline, after actin disruption by cytochalasin D, or after contractile activation by histamine. As expected, mechanical moduli depended strongly on bead type and bead coating, differing at the extremes by as much as two orders of magnitude. In every case, however, elastic and loss moduli increased with frequency f as a weak power law, f ^x–1. Moreover, with few exceptions, data could be scaled such that elastic and frictional responses depended solely on the power law exponent x. Taken together, these data suggest that power law behavior represents a generic feature of underlying protein-protein dynamics. actin; cytoskeleton; magnetic twisting cytometry; scale free; viscoelasticity     

Evidence of programmed cell death in post-phloem transport cells of the maternal pedicel tissue in developing caryopsis of maize

We present cellular- and ultracellular-level studies here to show developmental programmed cell death (PCD) of placento-chalazal (P-C) cell layers in maternal pedicel tissue in developing caryopses of normal seed (Mn1) and in the invertase-deficient miniature (mn1) seed mutant in maize (Zea mays). PCD was evidenced by loss of nuclei and all subcellular membranous organizations in many P-C layers. The terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) stain that is diagnostic of apoptotic-like PCD identified spatially and temporally two distinctive subdomains, which coincided with nucellar and integumental P-C layers based on their developmental origins. The early phase of PCD in the nucellar P-C was TUNEL negative and was specific to only the fertilized caryopses, indicating that the signaling for PCD in these maternal cells originated in the zygotic tissues. In fact, the initiation of PCD coincided with endosperm cellularization and was rapidly and coordinately completed prior to the beginning of the major storage phase in endosperm. Cell shape in these cell layers was also influenced by the genotype of filial endosperm. The later phase of PCD was restricted to the integumental P-C layers underneath the nucellar cells and was TUNEL positive in both genotypes. The two subdomains of the P-C layers were also distinguishable by unique cell wall-associated phenolic compounds. Based on collective evidence, we infer that the nucellar PCD may have osmolytic etiology and may lead to activation of the post-phloem transport function of the P-C layer, whereas the integumental PCD was senescent related, in particular, protecting the maturing seed against microbes that may be transported from the maternal tissue.     

The influence of detergent, the active component of detergent and sodiumtripolyphosphate on the biochemical process of some fungi.

Detergent "Merix" (Merima-Krusevac) and its components, sodiumtripolyphosphate and ethoxyled oleyl-cetyl alcohol in defined concentrations have influence on the enzymatic activity, bioproduction of amino acids and proteins and total biomass of species Aspergillus niger (A. niger) and Fusarium oxysporum (F. oxysporum). Detergent in concentration of 1% vol., in some cases, significantly stimulated production of the majority of amino acids. Detergent and its components (ethoxyled oleyl-cetyl alcohol and sodiumtripolyphosphate) in concentration of 1% vol. showed different influences on the production of proteins by the species of fungi A. niger and F. oxysporum. The enzymatic activity of fungi A. niger and F. oxysporum changed from total inhibition up to partial or total stimulation under influence of investigated pollutants. Detergent, sodiumtripolyphosphate and ethoxyled oleyl-cetyl alcohol, all in concentration of 1% vol. have reduced the production of the biomass of fungi A. niger and F. oxysporum. Investigated fungi have shown different response to the used pollutants.     

Formation of Palmitic Acid/Ca2+ Complexes in the Mitochondrial Membrane: A Possible Role in the Cyclosporin-Insensitive Permeability Transition

A possible role of palmitic acid/Ca2+ (PA/Ca2+) complexes in the cyclosporin-insensitive permeability transition in mitochondria has been studied. It has been shown that in the presence of Ca2+, PA induces a swelling of mitochondria, which is not inhibited by cyclosporin A. The swelling is accompanied by a drop in membrane potential, which cannot be explained only by a work of the Ca2+ uniporter. With time, the potential is restored. Evidence has been obtained indicating that the specific content of mitochondrial lipids would favor the PA/Ca2+ -induced permeabilization of the membrane. In experiments with liposomes, the PA/Ca2+ -induced membrane permeabilization was larger for liposomes formed from the mitochondrial lipids, as compared to the azolectin liposomes. Additionally, it has been found that in mitochondria of the TNF (tumor necrosis factor)-sensitive cells (WEHI-164 line), the content of PA is larger than in mitochondria of the TNF-insensitive cells (C6 line), with this difference being mainly provided by PA incorporated in phosphatidylethanolamine and especially, cardiolipin. The PA/Ca2+ -dependent mechanism of permeability transition in mitochondria might be related to some pathologies, e.g. myocardial ischemia. The heaviness of myocardial infarction of ischemic patients has been demonstrated to correlate directly with the content of PA in the human blood serum.     

Effects of Cr(VI) long-term and low-dose action on mammalian antioxidant enzymes (an in vitro study)

In order to investigate the low-dose long-term Cr(VI) action on antioxidant enzymes in cultured mammalian cells we estimated the activity of glutathione dependent antioxidant enzymes, catalase and superoxide dismutase (SOD) under various chromium concentrations in human epithelial-like L-41 cells. The long-term action of 20 microM causes the toxicity that results in losing of the cell viability by activating the apoptotic process, as identified by morphological analysis, the activation of caspase-3, and DNA fragmentation. The toxic chromium concentration totally destroys glutathione antioxidant system, and diminishes the activity of catalase and cytosolic Cu, ZnSOD. The non-toxic concentration (2 microM) causes the activation of the antioxidant defense systems, and they neutralize the oxidative impact.     

Morphometric variability between and within species of Ligophorus Euzet & Suriano, 1977 (Monogenea: Ancyrocephalidae) in the Mediterranean Sea

Monogeneans belonging to the genus Ligophorus Euzet & Suriano, 1977 are parasitic on the gills of several fish species of the family Mugilidae (mullets). At present, 12 species belonging to this genus have been described in the Mediterranean Sea and each host species is infected by a number of Ligophorus species that are not found on other mullets. A multivariate study using principal component and canonical variate analyses was carried out, with the aim of investigating the extent of morphometric variability within and between the 12 Mediterranean species of Ligophorus. Significant intraspecific morphometric variation was detected between two allopatric populations of L. angustus Euzet & Suriano, 1977 parasitic on Chelon labrosus from Sardinia; however, the existence of a new species of Ligophorus needs further evidence. Analysis of interspecific variation confirms the validity of the morphological keys used at present for the identification of Ligophorus at the species level and provides a picture of the relationships between species, indicating that the two species parasitising Mugil cephalus, L. chabaudi Euzet & Suriano, 1977 and L. mugilinus (Hargis, 1955) are the most differentiated. The hypothesis that strict host-parasite ecological relationships may be the result of co-speciation processes is briefly discussed.     

Heterologous expression of bovine and porcine beta-lactoglobulins in Pichia pastoris: towards a comparative functional characterisation

Bovine and porcine beta-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity. The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures. Both proteins were obtained in the culture medium at the concentration of 200 microg/ml. Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium. A preliminary NMR characterisation showed that both proteins were correctly folded.     

Quantification of D-dimer and soluble fibrin in blood plasma of people with ischemic heart disease and hypertension

The method of D-dimer quantification in the human blood plasma has been developed using monoclonal antibodies 111-3b and II-4d. The method has been verified on the blood plasma of the patients with ischemic heart disease with and without stenocardia and with hypertension. The results showed that at ischemic heart disease with and without stenocardia and at hypertension the quantities of D-dimer in the blood plasma were generally less than the highest normal level 500 ng/ml (64.3%, 76.2% and 95%, correspondingly). The semiquantitative measurements of soluble fibrin levels in blood plasmas of the patients with ischemic heart disease and hypertension have been performed. It has been shown that the quantity of soluble fibrin at these diseases range greatly from < 0.03 mg/ml to 0.15 mg/ml. There was no correlation between the quantities of D-dimer and soluble fibrin in blood plasmas of the patients. Electrophoresis in PAAG with SDS showed that the soluble fibrin at these diseases had the mo- lecular mass of the fibrin (ogen). Thus the soluble fibrin in blood plasmas analysed consisted mainly of fibrin desAA oligomers (may be with fibrinogen incorporation) which are not stabilized by the factor XIIIa.     

Effects of high static magnetic field exposure on different DNAs

The effects of magnetic fields produced by permanent magnets on different DNA sources were investigated in vivo and in vitro. Escherichia coli DNA, plasmid, and amplification products of different lengths were used as the magnetic field target. The in vivo assays did not reveal any DNA alterations following exposure, demonstrating the presence of cell dependent mechanisms, such as the repair system and the buffering action of the heat shock proteins DNA K/J (Hsp 70/40). The in vitro assays displayed interactions between the magnetic field and DNA, revealing principally that magnetic field exposure induces DNA alterations in terms of point mutations. We speculate that the magnetic field can perturb DNA stability interacting with DNA directly or potentiating the activity of oxidant radicals. This genotoxic effect of the magnetic field, however, is minimized in living organisms due to the presence of protective cellular responses.     

Defective dendritic cell migration and activation of adaptive immunity in PI3Kgamma-deficient mice

Gene-targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) in dendritic cell (DC) migration and induction of specific T-cell-mediated immune responses. DC obtained from PI3Kgamma-/- mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kgamma-/- mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8alpha- DC. Furthermore, PI3Kgamma-/- mice showed a defective capacity to mount contact hypersensitivity and delayed-type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen-loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kgamma plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kgamma may be considered a new target to control exaggerated immune reactions.