Use of a copper-phthalocyanine membrane electrode for rapid preliminary detection of polycyclic mutagens

Preliminary screening of polycyclic mutagens is achieved within 20 min by using a biomimetic electrode composed of an oxygen electrode and a copper-phthalocyanine membrane. When benzo[alpha]pyrene (0.05 mM) was added to the buffer solution in the presence of 0.98 M hydrogen peroxide, the current of the phthalocyanine electrode decreased. A linear relationship was obtained between the current decrease and the benzo[alpha]pyrene concentration over the range 0.19-0.60 mM. The minimum measurable concentration for benzo[alpha]pyrene was 0.01 mM. Such responses were not obtained for other organic compounds such as alcohol, ether, n-hexane and cyclohexane. The copper-phthalocyanine membrane electrode has selectively detected polycyclic mutagens such as amino acid pyrolysis products. The current decrease was 1.18-1.46 microA when 0.05 mM amino acid pyrolysis products were employed.     

Induction of the acrosome reaction in goat spermatozoa in simple physiological salt solution

The present study was carried out to determine whether K+, Mg2+, PO4(3-), and HCO3- in a medium are necessary for inducing the acrosome reaction in ejaculated goat spermatozoa. Washed goat spermatozoa were resuspended in K-1 medium, containing NaCl, KCl, CaCl2, MgCl2, NaH2PO4, and NaHCO3; in K-2 medium, containing NaCl, CaCl2, NaH2PO4, and NaHCO3; in K-3 medium, containing NaCl, CaCl2, and NaHCO3; and in K-4 medium, containing only NaCl and CaCl2, followed by preincubation in a sealed glass tube at 39.5 degrees C for 1, 2, or 3 h. The sperm acrosome reaction was evaluated by the trypan blue-Giemsa method and hamster test. The results were essentially the same in all cases. Following preincubation for 1 h, however, the percentage of acrosome-reacted spermatozoa, the proportion of zona-free hamster eggs penetrated by spermatozoa, and the average number of spermatozoa in the vitellus of these penetrated eggs were low; all values indicated a significant increase with preincubation for 2 and 3 h. The presence of K+, Mg2+, PO4(3-), and HCO3- in the medium thus does not appear necessary for inducing the acrosome reaction in goat spermatozoa, since they can undergo the reaction during preincubation in a simple physiological salt solution containing only NaCl and CaCl2 when preincubated in sealed glass tubes at 39.5 degrees C.     

Characterization of hemocytes from the marine amphipod Parhyale hawaiensis (Dana 1853): Setting the basis for immunotoxicological studies

Hemocytes are circulating blood cells that play a crucial function in amphipods and other crustacean immune systems. The hemocytes of the marine tropical amphipod Parhyale hawaiensis have been used for the evaluation of DNA damage and micronuclei, but they have not been characterized in the scientific literature. The aim of this study was to describe the hemolymph cells of P. hawaiensis and study their phagocytotic activity. Basic dyes were used to differentiate the cell types and the presence of lipids. The total hemocyte counts (THCs) and the proportion and sizes of the hemocyte types were determined. Hemolymph was exposed to Escherichia coli for verification of the presence of phagocytosis. Three cell types, all containing lipids, were identified in P. hawaiensis: granulocytes (oval shape, 13.4 × 7.6 μm), semi-granulocytes (oval shape, 14.1 × 7.2 μm), and hyalinocytes (round shape, 9.6 × 7.2 μm). Those three cell types were found in different percentages in males (64.8%, 31.1%, and 4.2%) and females (70.1%, 28.2%, and 1.7%). THCs for males were 9007 ± 3800 cells per individual and 4695 ± 1892 cells per individual for females. The cells of E. coli were phagocytized by the hemocytes. Our findings increased the knowledge of hemocytes in P. hawaiensis and is a step forward in using hemocyte-based immune responses as an endpoint in ecotoxicology.     

Ligand binding to heparan sulfate proteoglycans induces their aggregation and distribution along actin cytoskeleton.

Cell surface heparan sulfate proteoglycans (HSPGs) participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study demonstrates that soluble heparin-binding proteins or cross-linking antibodies induce the aggregation of cell surface HSPGs and their distribution along underlying actin filaments. Immunofluorescence and confocal microscopy and immunogold and electron microscopy indicate that, in the absence of ligands, HSPGs are irregularly distributed on the fibroblast cell surface, without any apparent codistribution with the actin cytoskeleton. In the presence of ligand (lipoprotein lipase) or antibodies against heparan sulfate, HSPGs aggregate and colocalize with the actin cytoskeleton. Triton X-100 extraction and immunoelectron microscopy have demonstrated that in this condition HSPGs were clustered and associated with the actin filaments. Crosslinking experiments that use biotinylated lipoprotein lipase have revealed three major proteoglycans as binding sites at the fibroblast cell surface. These cross-linked proteoglycans appeared in the Triton X-100 insoluble fraction. Platinum/carbon replicas of the fibroblast surface incubated either with lipoprotein lipase or antiheparan sulfate showed large aggregates of HSPGs regularly distributed along cytoplasmic fibers. Quantification of the spacing between HSPGs by confocal microscopy confirmed that the nonrandom distribution of HSPG aggregates along the actin cytoskeleton was induced by ligand binding. When cells were incubated either with lipoprotein lipase or antibodies against heparan sulfate, the distance between immunofluorescence spots was uniform. In contrast, the spacing between HSPGs on fixed cells not incubated with ligand was more variable. This highly organized spatial relationship between actin and proteoglycans suggests that cortical actin filaments could organize the molecular machinery involved in signal transduction and molecular movements on the cell surface that are triggered by heparin-binding proteins.     

Complete primary structure of a galactose-specific lectin from the venom of the rattlesnake Crotalus atrox. Homologies with Ca2(+)-dependent-type lectins

The complete primary structure of a galactose-specific lectin contained in the venom of the rattlesnake, Crotalus atrox, was determined. The lectin is composed of two covalently linked, identical subunits, each consisting of 135 amino acid residues. Under physiological conditions the lectin proved to be highly aggregated. The venom lectin contained 9 half-cystines, 8 of which formed four intrasubunit disulfide bridges (Cys3-Cys14, Cys31-Cys131, Cys38-Cys133, and Cys106-Cys123), while Cys86 was involved in an intersubunit disulfide bridge. Because of the high content of disulfide bridges, the intact lectin was extremely resistant to tryptic digestion. The determined amino acid sequence was found to be homologous with those of the so-called carbohydrate recognition domains of Ca2(+)-dependent-type lectins in animal. Among them, 8 amino acid residues (Cys31, Gly69, Trp92, Pro97, Cys106, Asp120, Cys123, and Cys131) were completely conserved. Leu40, Trp67, and Trp81 were also well conserved. The rattlesnake venom lectin showed high hemagglutinating activity. These results, together with the occurrence of similar lectins in crotalid venoms, suggest that these lectins have evolved in order to make the venom a more effective weapon to capture prey animals.     

Proposal of Mycobacterium peregrinum sp. nov., nom. rev., and elevation of Mycobacterium chelonae subsp. abscessus (Kubica et al.) to species status: Mycobacterium abscessus comb. nov.

We studied the taxonomic positions of the rapidly growing organism Mycobacterium fortuitum and phenotypically related organisms. We confirmed that "Mycobacterium peregrinum" ATCC 14467T (T = type strain) is genetically independent of M. fortuitum ATCC 6841T by using various DNA hybridization conditions. Strains that were genetically identified as "M. peregrinum" were phenotypically differentiated from M. fortuitum ATCC 6841T. Thus, we propose that "M. peregrinum" should be revived as an independent species, Mycobacterium peregrinum sp. nov., nom. rev. The type strain is strain ATCC 14467. M. fortuitum subsp. acetamidolyticum ATCC 35931T exhibited a high level of DNA relatedness to M. fortuitum ATCC 6841T. The hybridized DNAs maintained stable heteroduplexity at high stringency; thus, we confirmed that M. fortuitum subsp. acetamidolyticum is identical to M. fortuitum ATCC 6841T. We found that M. chelonae subsp. abscessus ATCC 19977T is genetically different from M. chelonae subsp. chelonae NCTC 946T on the basis of the results of quantitative hybridization even under optimal conditions. There was no reason to maintain this organism as a subspecies of M. chelonae. Thus, we propose that M. chelonae subsp. abscessus should be elevated to species status as Mycobacterium abscessus (Kubica et al.) comb. nov. The type strain is strain ATCC 19977.     

Structural and functional modifications of the manganese cluster in Ca(2+)-depleted S1 and S2 states: electron paramagnetic resonance and X-ray absorption spectroscopy studies.

The effect of extraction of weakly bound Ca2+ by low-pH treatment on the O2-evolving apparatus was studied by use of low-temperature electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy. In low-pH-treated PSII membranes, an S2 EPR multiline signal with modified line shape was induced by illumination at 0 degrees C, but its signal amplitude decreased upon lowering the excitation temperature with concomitant oxidation of cytochrome (cyt) b-559 in place of Mn. The half-inhibition temperature for formation of the modified multiline signal was found at -33 degrees C, which was much higher than that for formation of the normal S2 state in untreated control membranes. Signal IIf was normally induced down to -30 degrees C, but its dependence on excitation temperature was different from that for modified S2. This was interpreted as indicating that the low-temperature blockage of modified S2 formation is due to the incapability of electron abstraction from the Mn cluster. The Mn K-edge of X-ray absorption near-edge structure (XANES) spectrum shifted to lower energy by 0.8 eV after low-pH treatment, but the shift was reversed by addition of Ca2+. Upon illumination at 0 degrees C of treated membranes, the K-edge energy was up-shifted by 0.8 eV, but was not upon illumination at 210 K. These results were interpreted as indicating that extraction of weakly bound Ca2+ by low-pH treatment gives rise to structural and functional modulations of the Mn cluster.     

Structure of genomic DNA for rat platelet phospholipase A2

A genomic DNA for rat platelet phospholipase A2 was isolated by screening a rat genomic library with oligonucleotide probes based on its published amino acid sequence. The rat platelet phospholipase A2 gene had a total length of about 2.5 kb and contained five exons and four introns. The intron-exon structure of the rate gene was similar to that of human non-pancreatic phospholipase A2.     

Expression of lipoprotein lipase in ovaries of the guinea pig

Guinea pig ovaries were found to have significant lipoprotein lipase (LPL) activity, corresponding to almost one-tenth the activity in paraovarian adipose tissue and in heart per gram of tissue. Northern blot analysis demonstrated the same three species of LPL mRNA in ovaries (1.8, 3.1, and 3.5 kb) as in adipose tissue. In situ hybridization showed LPL mRNA in cells of the follicular wall, and in granulosa and theca lutein cells of the mature corpus luteum. By immunolocalization, LPL was visualized in the vascular endothelium throughout the ovary, but with highest concentration in the endothelium of capillaries and large vessels of the cortical region and capillaries in the stroma of the corpus luteum. These results suggest that in the guinea pig LPL may have a function for the delivery of lipids from lipoproteins to ovarian cells.     

A Design Pattern for Decentralised Decision Making

The engineering of large-scale decentralised systems requires sound methodologies to guarantee the attainment of the desired macroscopic system-level behaviour given the microscopic individual-level implementation. While a general-purpose methodology is currently out of reach, specific solutions can be given to broad classes of problems by means of well-conceived design patterns. We propose a design pattern for collective decision making grounded on experimental/theoretical studies of the nest-site selection behaviour observed in honeybee swarms (Apis mellifera). The way in which honeybee swarms arrive at consensus is fairly well-understood at the macroscopic level. We provide formal guidelines for the microscopic implementation of collective decisions to quantitatively match the macroscopic predictions. We discuss implementation strategies based on both homogeneous and heterogeneous multiagent systems, and we provide means to deal with spatial and topological factors that have a bearing on the micro-macro link. Finally, we exploit the design pattern in two case studies that showcase the viability of the approach. Besides engineering, such a design pattern can prove useful for a deeper understanding of decision making in natural systems thanks to the inclusion of individual heterogeneities and spatial factors, which are often disregarded in theoretical modelling.