Second virial coefficient of alpha-crystallin

Light scattering studies were performed on bovine alpha-crystallin measuring the scattering intensities as a function of scattering angle, concentration, and temperature. The data yielded the molecular weight, radius of gyration, and second virial coefficient of alpha-crystallin at different temperatures. The second virial coefficient increased with increasing temperature. Both the enthalpy and entropy of solution of alpha-crystallin are positive. The Flory theta temperature was found to be 271 K.     

Expression of the Ganglioside GD3 in Human Meningiomas Is Associated with Monosomy of Chromosome 22

The ganglioside composition of 59 meningiomas has been compared with a molecular genetic analysis of chromosome 22 in the same specimens. Major gangliosides were GM3 (II3NeuAc-LacCer) and/or GD3 [II3(NeuAc)2-LacCer]. In specimens with no or partial deletions of chromosome 22, the GM3 ganglioside predominated, and the mean value for GM3, 61% of total sialic acid, was around four times higher than that for GD3. A loss of chromosome 22, found in 56% of the specimens, was shown to be associated with an increase in the proportion of ganglioside GD3, with the ratio between mean values of GM3 and GD3 being approximately 1:1. Logistic regression revealed that the probability of predicting monosomy of chromosome 22 by the GD3 proportion was 66%.     

Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus

Summary Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H₂ at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H₂ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H₂ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H₂ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H₂ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H₂ases.     

Influence of cytosolic pH changes on the organisation of the endoplasmic reticulum in epidermal cells of onion bulb scales: Acidification by loading with weak organic acids

Summary The anastomosing ER system of epidermal cells of onion bulb scales is composed of three modifications: lamellar and tubular elements, located in the cell periphery, and long tubular stands located deeper in the cytoplasm. Cytoplasmic acidification of epidermal cells by loading with weak organic acids like acetic or propionic acid causes the decay of the lamellar elements and the disappearance of long tubular strands. Organelle movement is also inhibited. The effects depend on the pH of the incubation medium and on the administered acid concentration, and are characterized by a distinct lag phase of about 7 min. The induced ER changes are transient with adaptation starting after about 50min. Buffer components alone have little influence on the cellular ER organization within a pH-range of 4.0–8.0. However, the pH of the medium strongly affects the time course of the effects as well as recovery after omitting the administered acid. Both modulation and recovery occur more rapidly at neutral or slightly alkaline pH. Actin filaments, which play a major role in ER organization and organelle movement, are not affected by cytosolic acidification.Dedicated to the memory of Professor Oswald Kiermayer     

Continuous stereospecific Δ-3-keto-steroid reduction by PAAH bead-entrapped Clostridium paraputrificum cells

The development of a continuous anaerobic process for stereospecific Δ⁴-3-keto-steroid reduction by immobilized Clostridium paraputrificum cells cells is described. Following a study on conditions for cell growth and sporulation, spores of C. paraputrificum were aseptically immobilized in PAAH beads. Conditions for cell growth and induction in the immobilized state were determined, as well as the medium composition required to maintain a stabilized immobilized cell population. The effect of the concentration of ethylene glycol added as selected cosolvent on reaction kinetics, substrate solubility, specific activity, and cell growth, was investigated. A 10% (v/v) cosolvent input provided maximal activity along with enhanced solubility of the steroidal substrate. It was shown that cell growth was enhanced in the presence of the added cosolvent in addition to its effect on substrate solubility and enzymic activity. The immobilized cells readily performed Δ⁴, as well as 3-keto steroid reduction of several steroids, including ADD, AD, 16-dehydroprogesterone, progesterone, and hydrocortisone. It was shown that repeated batch-wise reduction cycle—in the presence of the cosolvent—resulted in rapid loss of activity, while the continuous uninterrupted process permitted the attaining of full bioconversion level, maintained stable for at least the period of 5 days of continuous operation tested.     

The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

Analysis of time-resolved fluorescence anisotropy in lipid-protein systems

Fluorescent probes located in heterogeneous environments give rise to anomalous time-resolved fluorescence anisotropy. A simple analytical expression of anisotropy has been derived for the case of a small difference in local fluorescence lifetimes. The expression has the diagnostic advantage that the time dependence of the fluorescence anisotropy can be predicted from the differences in fluorescence lifetimes and residual anisotropies of the probes located in different sites. Using this model, the local fluorescence anisotropy parameters and the relative contributions of the lipid probe octadecyl rhodamine B in a lipid environment and in the vicinity of bacteriophage M13 coat protein reconstituted in phospholipid bilayers, composed of 80% 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 20% 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol have been determined experimentally. At 40°C, the correlation times for bound and free probes are 2.3 and 3.0 ns, respectively, while the corresponding order parameters are 0.85 and 0.62, respectively.Abbreviations ESR electron spin resonance - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol - L/P ratio phospholipid to coat protein molar ratio - <> average fluorescence lifetime - r(0) initial anisotropy - r() residual anisotropy On leave of Shanghai Medical Equipment Research Institute, 77 Jiang Ning Rd. Shanghai, People's Republic of China Offprint requests to: M. A. Hemminga     

Application of Deuterated A-Tocopherols to the Biokinetics and Bioavailability of Vitamin E

-Tocopherol, a superior chain-breaking, peroxyl radical-trapping antioxidant and the most active component of vitamin E, is elevated in liver tumor cells, contributing to their greater resistance towards lipid peroxidation compared to cells from normal tissues. Also, in regenerating rat liver the level of vitamin E has been found to fluctuate in phase with the rate of cell division. In order to study the biokinetcis and mechanisms of the distribution of vitamin E in organs and within tissues of animals, deuterated forms of -tocopherol have been synthesized and their uptake into blood and tissues has been measured by gas chromatography-mass spectrometry. Measurement of the competitive uptake from a mixture of the RRR-and SRR--tocopherol stereoisomers labelled with different amounts of deuterium shows that the liver exerts a strong preference for secretion of the natural (RRR) stereoisomer into the plasma. It is suggested that a tocopherol-binding protein plays a key role in this process.     

Impact of sar and agr on methicillin resistance in Staphylococcus aureus

Abstract The global regulators agr and sar control expression of cell wall and extracellular proteins. Inactivation of either sar and/or agr in a typical heterogeneously methicillin-resistant Staphylococcus aureus resulted in a small but reproducible decrease in the number of cells in the subpopulation expressing high methicillin resistance. The amount of low affinity penicillin-binding protein PBP2', the prerequisite for methicillin resistance, was apparently not affected, however, a reduction in PBP1 and PBP3 production was observed, suggesting that these resident PBPs of the cells might be involved somehow together with PBP2' in high level methicillin resistance.