Dielectric Spectroscopy: a Rapid Method for the Determination of Solvent Biocompatibility During Biotransformations

Dielectric spectroscopy provides a convenient means of determining the degree of intactness of biological cells. 4-terminal dielectric measurements of suspensions of Saccharomyces cerevisiae at 0.4 MHz show that, as with all other biological cells, these organisms possess a substantial β-dispersion. The additional of octanol to such suspensions causes a rapid decrease in the electrical capacitance of the suspension, which parallels the cellular viability as determined by methylene blue staining. The kinetics of cell death are determined in part by the rate of dissolution of the organic solvent in the aqueous phase. The toxicity of several organic solvents to S. cerevisiae is studied using this technique, and is found to be dependent upon the polarity of the solvent. The present method provides a simple and rapid means for assessing the biocompatibility of solvents used in biotransformations.     

Chlorophyll fluorescence images demonstrate variable pathways in the effects of plasma membrane excitation on electron flow in chloroplasts of <Emphasis Type=Italic>Chara</Emphasis> cells

Chlorophyll fluorescence Imaging and Microscopy PAM fluorometry were applied to study spatial dynamics of photosystem II quantum yield ( DF/F_m￠ ) left( {Delta F/F_m^prime } right) and non-photochemical quenching (NPQ) in resting and electrically stimulated Chara corallina cells in the absence and presence of the hydrophilic electron acceptor methyl viologen (MV) in the external medium. Electrical excitation of the plasma membrane temporarily enhanced the heterogeneity of photosynthetic patterns under physiological conditions (in the absence of MV), but irreversibly eliminated these patterns in the presence of MV. These findings suggest that the action potential (AP) of the excitable plant cell affects the spatial patterns of photosynthesis and chlorophyll fluorescence through different pathways operated in the absence and presence of MV. Based on the extent of NPQ as an indicator of MV-dependent electron flow, it is supposed that MV cannot permeate into the chloroplasts of photosynthetically active “acid cell regions” but gains an immediate access to the stroma of these chloroplasts after triggering of an AP. The AP-triggered MV-dependent non-photochemical quenching in the chloroplasts of acidic cell regions was routinely observed at 0.1 mM Ca²⁺ in the medium but not at elevated (2 mM) external Ca²⁺ concentration. The results are interpreted in terms of competition between two permeant divalent ion species, Ca²⁺ and MV²⁺, for their passage through the voltage-gated calcium channels of the plasma membrane. It is proposed that the herbicidal activity of MV in characean cells, here serving as model object, can be manipulated by triggering AP and varying Ca²⁺ concentration in the environmental medium.     

ELECTROKINETIC PHENOMENA : IV. A COMPARISON OF ELECTROPHORETIC AND STREAMING POTENTIALS

1. The conditions are described which are necessary for the comparison of certain types of electrokinetic potentials. An experimental comparison is made of (a) electrophoresis of quartz particles covered with egg albumin; and (b) similar experiments by Briggs on streaming potentials. A slight, consistent, difference is found between the electrophoretic potential and the streaming potential. This difference is probably due to the difference in the protein preparations used rather than to real difference in the electrophoretic and streaming potentials. 2. Data are given which facilitate the measurements and enhance the precision of the estimation of electrical mobilities of microscopic particles.     

Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.     

Hormonal Profiling Reveals a Hormonal Cross-Talk During Fruit Decay in Sweet Cherries

Journal of Plant Growth Regulation - Although fruit decay in sweet cherries has been studied in some detail, information is still scarce about the possible role of phytohormones during postharvest....     

Novel nuclear-encoded subunits of the chloroplast NAD(P)H dehydrogenase complex

The NAD(P)H dehydrogenase (NDH) complex functions in photosystem I cyclic electron transfer in higher plant chloroplasts and is crucial for plant responses to environmental stress. Chloroplast NDH complex is a close relative to cyanobacterial NDH-1L complex, and all fifteen subunits so far identified in NDH-1L have homologs in the chloroplast NDH complex. Here we report on the identification of two nuclear-encoded proteins NDH48 and NDH45 in higher plant chloroplasts and show their intimate association with the NDH complex. These two membrane proteins are shown to interact with each other and with the NDH complex enriched in stroma thylakoids. Moreover, the deficiency of either the NDH45 protein or the NDH48 protein in respective mutant plants leads to severe defects in both the accumulation and the function of the NDH complex. The NDH48 and NDH45 proteins are not components of the hydrophilic connecting domain of the NDH complex but are strongly attached to the hydrophobic membrane domain. We conclude that NDH48 and NDH45 are novel nuclear-encoded subunits of the chloroplast NDH complex and crucial both for the stable structure and function of the NDH complex.     

The synthesis and characterization of a series of cobalt(II) β-ketoaminato complexes and their cytotoxic activity towards human tumor cell lines

A series of square planar cobalt(II) compounds bearing tetradentate β-ketoaminato ligands with variation in the number of ―CF₃ ligand substituents has been prepared and structurally and spectroscopically characterized. The fluorinated β-ketoamine ligands were prepared utilizing a multistep reaction sequence employing a silylenol protecting group. An additional tetrahedral cobalt compound bearing two bidentate β-ketoaminato ligands was also prepared and characterized.Cytotoxic activity of the cobalt-containing complexes was evaluated using six human cell lines; including two different prostate cancer cell lines (PC-3 and VCaP), acute monocytic leukemia (THP-1), astrocytoma (U-373 MG), hepatocellular carcinoma (HepG2), and neuroblastoma (SH-SY5Y) cells. The cobalt compounds are more active than their corresponding ligands. The activity is cell type specific; the cobalt compounds exhibit strong activity against human prostate cancer and monocytic leukemia cells but weak or no activity against neuroblastoma, astrocytoma, and liver carcinoma cells. Activity generally increases with a greater number of ―CF₃ substituents, and square planar complexes exhibit greater activity than the tetrahedral derivative. The mechanisms of activity against human PC-3 prostate cancer cells involve caspase-3 and two different mitogen-activated protein kinases. The addition of a thiol antioxidant reduced cytotoxicity, suggesting the possible involvement of reactive oxygen species. These cobalt complexes may represent a novel class of cytotoxic drugs selective towards certain types of tumors.