Solubility of proteins

A theory is presented on the solubility of proteins, in the hydrated as well as in the dry state, and in water as well as in organic solvents. To this effect, colloidal stability is assimilated with the solubility of the proteins, considered as hydrated entities. By means of a surface thermodynamic approach it can be shown that an increase in size of a hydrated protein must lead to insolubility, even in the absence of any change in a protein's surface properties. This can be substantiated experimentally by comparing the surface properties of immune complexes with those of their constituent immunoglobulins, as well as by comparing some of the properties of intact tobacco mosaic virus with those of its monomeric capsid subunits. Insolubilization of proteins by means of charge interactions as well as by dehydration is studied; an explanation is given of why precipitation caused by charge interactions is more likely to lead to partial irreversible denaturation than precipitation caused by protein-protein interactions brought about by partial dehydration (e.g., by “salting-out”). A link is established between the smallness (or even the negative value) of the interfacial tension between given proteins and various solvents and their solubility in these solvents. The energy of hydration of proteins can also be measured, and the differences between the free energies of interaction of dried and hydrated proteins with water point toward the additional processes underlying the solubilization, i.e., toward the conformational change of a protein in the process of becoming hydrated. The parameter of conformational change of a protein, while becoming hydrated, appears to be more closely linked to its degree of hydration than to its hydration energy.     

Yellow Fever Vaccine. II. Antigenicity and Neurovirulence of a Vaccine Seed Free from Avian Leukosis Virus

Avian leukosis virus (ALV)-free candidate primary and secondary seed lots were indistinguishable from corresponding ALV-contaminated lots with respect to (i) potency as measured by titration in newborn and weanling mice and in the MA-104 plaque system, (ii) degree of viscerotropism as measured by viremia in monkeys, (iii) neurotropism as determined by the monkey neurovirulence test, and (iv) potency as determined by antibody response in monkeys inoculated by the intracerebral route.     

On the levels of phenylalanine, tyrosine and tetrahydrobiopterin in the blood of tumor-bearing organisms

The levels of free phenylalanine and free tyrosine (and m-tyrosine) as well as of protein-bound tyrosine were determined by means of automated aminoacid analysis and by reaction with 1-nitrosonaphthol(2). Their absolute values as well as their percentages relative to total aminoacids of the blood were markedly increased in all tumor patients tested, as well as in experimental tumor rats. Although tetrahydrobiopterin could scarcely be detected by fluorometric methods in the blood of control persons, it is accumulated blood of all tumor patients tested. The major part was found in the erythrocyte fraction. The results are discussed with respect to decreased phenylalanine hydroxylation rate and to depressed tyrosine catabolism recently found by means of in vivo experiments in tumor-bearing rats.     

Genetic networks regulated by ASYMMETRIC LEAVES1 (AS1) and AS2 in leaf development in Arabidopsis thaliana: KNOX genes control five morphological events

The asymmetric leaves 1 ( as1 ) and as2 mutants of Arabidopsis thaliana exhibit pleiotropic phenotypes. Expression of a number of genes, including three class-1 KNOTTED -like homeobox ( KNOX ) genes ( BP , KNAT2 and KNAT6 ) and ETTIN / ARF3 , is enhanced in these mutants. In the present study, we attempted to identify the phenotypic features of as1 and as2 mutants that were generated by ectopic expression of KNOX genes, using multiple loss-of-function mutations of KNOX genes as well as as1 and as2 . Our results revealed that the ectopic expression of class-1 KNOX genes resulted in reductions in the sizes of leaves, reductions in the size of sepals and petals, the formation of a less prominent midvein, the repression of adventitious root formation and late flowering. Our results also revealed that the reduction in leaf size and late flowering were caused by the repression, by KNOX genes, of a gibberellin (GA) pathway in as1 and as2 plants. The formation of a less prominent midvein and the repression of adventitious root formation were not, however, related to the GA pathway. The asymmetric formation of leaf lobes, the lower complexity of higher-ordered veins, and the elevated frequency of adventitious shoot formation on leaves of as1 and as2 plants were not rescued by multiple mutations in KNOX genes. These features must, therefore, be controlled by other genes in which expression is enhanced in the as1 and as2 mutants.     

A set of modular binary vectors for transformation of cereals

Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well as the exploitation of transgenes for targeted trait improvement. However, in most cereals, this option has long been compromised by tedious and low-efficiency transformation protocols, as well as by the lack of versatile vector systems. After having adopted and further improved the protocols for Agrobacterium-mediated stable transformation of barley (Hordeum vulgare) and wheat (Triticum aestivum), we now present a versatile set of binary vectors for transgene overexpression, as well as for gene silencing by double-stranded RNA interference. The vector set is offered with a series of functionally validated promoters and allows for rapid integration of the desired genes or gene fragments by GATEWAY-based recombination. Additional in-built flexibility lies in the choice of plant selectable markers, cassette orientation, and simple integration of further promoters to drive specific expression of genes of interest. Functionality of the cereal vector set has been demonstrated by transient as well as stable transformation experiments for transgene overexpression, as well as for targeted gene silencing in barley.     

DNA damage by carbonyl stress in human skin cells

Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the alpha-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N(epsilon)-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.     

Membrane Raft Disruption Promotes Axonogenesis in N2a Neuroblastoma Cells

Membrane rafts are discrete microdomains found in cell membranes that contain cholesterol and glycosphingolipids such as gangliosides. As cholesterol is a major component of membrane rafts, its sequestration by the polyene filipin can be used to disrupt them. In previous work we observed that membrane raft disruption by filipin treatment of murine neuroblastoma N2a cells led to changes in expression of cell processes. In this study, we determined the type of process formation induced by filipin treatment as well as whether their expression was accompanied by changes in ganglioside content or subcellular distribution. The results indicate that the processes formed were axonal in nature and their expression was accompanied by changes in both ganglioside content as well as the subcellular localization of GM1. Special issue article in honor of Dr. George DeVries.     

Protective effects of green tea catechins on alveolar macrophages against bacterial infections

Bacterial pneumonia in immunocompromised patients as well as elderly persons often becomes a life threatening disease, even when effective antibiotics are used extensively. In addition, the appearance of antibiotic-resistant bacteria in medical facilities as well as in patients requires another approach to treat such patients besides treatment with antibiotics. In this regard, green tea catechins, such as epigallocatechin gallate (EGCg), may be one of the potential agents for such purpose due to its possible potential immunomodulatory as well as antimicrobial activity. The studies by us showed that EGCg enhanced the in vitro resistance of alveolar macrophages to Legionella pneumophila infection by selective immunomodulatory effects on cytokine formation. Furthermore, the tobacco smoking-induced impairment of alveolar macrophages regarding antibacterial as well as immune activity was also recovered by EGCg treatment. These results indicate that EGCg may be a possible potential immunotherapeutic agent against respiratory infections in immunocompromised patients, such as heavy smokers.     

Sorption of hydrophobic organic contaminants and trace metals on phytoplankton and implications for toxicity assessment

The partitioning of trace metals and hydrophobic organic contaminants to phytoplankton determines their toxicity as well as their fate and transport in aquatic ecosystems. Accurate impact assessments, therefore, depend on a good understanding of the factors regulating the sorption of these compounds to biotic particles. The accumulation of chlorinated organic compounds in phytoplankton is generally considered as being due solely to physical sorption, described by reversible equilibrium models based on Langmuir or Freundlich isotherms. On the other hand, the uptake of trace metals is a two phase process: a fast sorption component viewed as an ionexchange or a covalent bonding process with cell surface ligands, followed by an intracellular transport phase that is dependent on cellular metabolic activity. The uptake of inorganic and hydrophobic organic pollutants and their bioaccumulation are influenced in a complex manner by duration of exposure and cell density, by environmental factors such as pH, the concentration of cations and of dissolved and colloidal organic matter, as well as by phytoplankton physiological condition. High concentrations of H⁺, Ca²⁺, and Mg²⁺ ions will reduce trace metal sorption by directly competing for uptake sites on the cell's surface, whereas the presence of dissolved organic carbon such as natural and synthetic chelators and phytoplankton exudates will reduce the bioavailability of both trace metals and hydrophobic organic contaminants. Thus, the impact of toxic contaminants on phytoplankton may be determined as much by the factors influencing uptake and partitioning as by the potency of the toxicants and interspecies differences in sensitivity. Recommendations for improving toxicity assessments are presented.     

Degradation of aqueous 4-chloroaniline by ozonolysis and combined gamma-rays-ozone processing

The decomposition of 4-chloroaniline (4-ClA), used as a model for water pollutants, was studied by ozonolysis as well as by gamma-rays in the presence of ozone under comparable conditions. The degradation process was followed by absorption spectroscopy and by HPLC-method as well. Depending on the ozone concentration (mg O3/min) introduced into the aqueous solution the substrate is decomposed to a mixture of carboxylic acids, which can be entirely degradated by prolonged treatment. The combined processing of 4-ClA by gamma-irradiation in the presence of ozone proved to lead even to more efficient degradation of the substrate. Some primary reaction steps are briefly discussed.     

Comparison between flow cytometry and image cytometry in ploidy distribution assessments in gynecologic cancer.

The DNA content in 37 tumors from 34 women with gynecological cancer was measured by flow cytometry (FCM) and interactive image cytometry (ICM). Agreement was obtained in 81% of cases as regards ploidy levels, but seven tumors (19%) showed different ploidies. Of these, five were classified as diploid by FCM but either aneuploid (three cases) or polyploid (two cases) by ICM. Two other tumors were aneuploid by ICM but polyploid (one case) and unclassifiable (one case) by FCM. All tumors classified as aneuploid by FCM were also aneuploid by ICM, and all tumors classified diploid by ICM were also diploid by FCM. Of six patients whose tumors were classified as euploid (five diploid and one polyploid) by FCM but classified as aneuploid by ICM, five relapsed, and three of these have died of disease. On the basis of these findings, it is concluded that ICM must be performed in cases classified as diploid by FCM to ensure that small subpopulations of aneuploid tumor cells are not overlooked.     

Differential effects of the diacylglycerol kinase inhibitor R59022 on thrombin versus collagen-induced human platelet secretion

R59022 is an inhibitor of the enzyme 1,2-diacylglycerol (DAG) kinase, which, by inhibiting the conversion of DAG to phosphatidic acid, causes an increase in endogenous DAG levels and the activity of the DAG-dependent enzyme protein kinase C. This property of the drug was utilized in the present study to assess the role of DAG, i.e., its relative importance as a potentiatory versus inhibitory mediator, in agonist-induced platelet activation. The phosphorylation of the 40-47-kDa protein by protein kinase C was monitored as an indicator of endogenous DAG levels and correlated with other agonist-induced platelet responses such as platelet aggregation, 5-hydroxytryptamine (5HT) secretion and arachidonate release, the agonists used being those that induce DAG formation, e.g., thrombin and collagen. Pretreatment of platelets with R59022 before agonist addition resulted in the potentiation of 5HT secretion as well as 45 kDa protein phosphorylation induced by thrombin and the DAG analogue, 1,2-dioctanoylglycerol (DiC8). However, collagen-induced 5HT secretion was significantly inhibited (70%) in the presence of R59022, which also had strong inhibitory effects on aggregation induced by collagen, as well as by thrombin and DiC8. The inhibition of collagen-induced secretion by R59022 was in contrast to the potentiatory effects of DiC8 on the same, suggesting that even although DAG acts as a potentiatory signal in this system, the inhibitory effects of R59022 on collagen-induced aggregation can mask any effects of endogenous DAG. This inhibitory effect of R59022 on agonist-induced platelet aggregation makes it unsuitable as a tool in studying the role of DAG in platelet activation induced by agonists such as collagen as well as the 'weak' agonists (ADP, adrenaline and platelet-activating factor), where aggregation mediates other responses such as arachidonate release and secretion. Furthermore, potentiatory effects of R59022 on 5HT secretion induced by phorbol 12-myristate 13-acetate and ionomycin, which are effects unlikely to be related to inhibition of DAG kinase was observed, and these effects further underline the non-specificity in the actions of R59022 and its limitations as a tool in studying platelet stimulus-response coupling.     

Effects of cycloheximide on abscisic Acid biosynthesis and stomatal aperture in bean leaves

Cycloheximide was shown to block abscisic acid (ABA) biosynthesis in nonstressed as well as in stressed Phaseolus vulgaris leaves. Leaf wilting caused by cycloheximide resulted from increased stomatal opening as judged by a decreased stomatal diffusion resistance. The inhibition of ABA biosynthesis by cycloheximide was at least partially responsible for the increase in stomatal opening as suggested by the cooccurrence of inhibition of ABA biosynthesis and increased stomatal opening, and the partial reversal of stomatal opening in cycloheximide-treated leaves by exogenous ABA. Dark treatment failed to close stomatal in cycloheximide-treated leaves, suggesting that stomatal closure in response to darkness may normally be mediated by ABA.     

<Emphasis Type="Italic">γ</Emphasis>-Fluorinated analogues of glutamic acid and glutamine

Gamma-fluorinated analogues of glutamic acid and glutamine are compounds of biological interest. Syntheses of such compounds are extensively reviewed in this article. 4-fluoroglutamic acid was prepared as a mixture of racemic diastereomers by Michael reaction, inverse-Michael reaction or by electrophilic / nucleophilic fluorination. Optically enriched 4-fluoroglutamic acids were obtained by several resolution techniques as well as by asymmetric methodologies using the chiral pool. 4-fluoroglutamine was prepared as a mixture of stereoisomers as well as in racemic erythro and threo forms from the corresponding 4-fluoroglutamic acids using aminolysis and conventional protection and deprotection strategies. Racemic 4,4-difluoroglutamic acid was synthesized by a nitroaldol reaction and its L-enantiomer obtained via three different asymmetric routes. Racemic 4,4-difluoroglutamic acid was converted into the corresponding 4,4-difluoroglutamine using a protection / aminolysis / deprotection sequence while N-Boc-L-4,4-difluoroglutamine was prepared directly from (R)-Garner's aldehyde using a Reformatsky reaction as the key step.     

Inhibition of leukocyte adhesion by the in vivo and in vitro administration of cannabinoids

Cannabinoids have been shown to affect various aspects of arachidonic acid metabolism both in vivo and in vitro. Eicosanoid metabolites of arachidonate and related octadecanoate are believed to be involved in cell adhesion processes as agonists in some instances and as antagonists in other cases. This report shows data in which cannabinoids exhibit marked inhibitory effects on the adhesion of mouse peritoneal cells to polystyrene culture dishes. The effects could be seen by in vivo administration of the drugs as well as by direct exposure of the cells in vitro. The data suggest that this inhibition of adhesion is mediated by one or more products generated by stimulation of a lipoxygenase pathway.     

Fungal leaching of nickeliferous laterites

Extraction of nickel by microbial leaching of Greek laterites is feasible by using ofAspergillus andPenicillium. The effectiveness was found to depend on the ability of the microorganism to produce hydroxycarboxylic acids, especially citric acid, as well as other metabolites. The nickel recoveries achieved were as high as 60%, in 48 d, when the ore was leached in the presence of the living fungi, in a sucrose medium, and as high as 70%, in a much shorter time, when the ore was leached by their metabolic products after pH adjustment by means of sulfuric acid. The use of much cheaper, factory grade, Greek beet molasses as a growth medium proved promising, giving the possibility of making the process more attractive in economic terms.     

Evidence for generation of a large amount of nitric oxide-like vascular smooth muscle relaxant by cholesterol-rich neutrophils

To determine the effect of cholesterol incorporation on the ability of neutrophils to generate superoxide radicals and nitric oxide-like vasorelaxant material, isolated human neutrophils were incubated with cholesterol-rich liposomes, which increased total cholesterol content by 141% and esterified cholesterol content by 523%. Cholesterol loading resulted in 5 to 7 fold increase in cytosolic calcium in resting as well as in PMA or f-MLP-stimulated cells, but a marked (P less than 0.01) reduction in both PMA- and f-MLP-stimulated superoxide radical generation by these cells. Nitric oxide-like activity measured as relaxation of rat aortic rings was more pronounced (P less than 0.02) in cholesterol-rich than in cholesterol-poor cells. The greater relaxation of aortic rings in response to cholesterol-rich neutrophils was observed in rings with or without intact endothelium, and was potentiated by superoxide dismutase and inhibited by oxyhemoglobin as well as L-NMMA, thus suggesting that the vasorelaxant material was nitric oxide. The greater generation of nitric oxide by cholesterol-rich neutrophils occurs perhaps in response to increased cytosolic calcium.     

Carbopol-Based Gels for Nasal Delivery of Progesterone

The purpose of this study was to investigate the nasal absorption of progesterone from carbopol-based nasal gels in rabbits. Progesterone nasal gels were prepared by dispersing carbopol 974 (1%, 1.5%, and 2%) in distilled water followed by addition of progesterone/progesterone–β cyclodextrin complex dissolved in propylene glycol then neutralization. The potential use of β cyclodextrin (CD) as nasal absorption enhancer by simple addition, as a physical mixture and as a complex with progesterone was investigated. The absolute bioavailability of progesterone from nasal gels in rabbits was studied by estimating the serum progesterone level by competitive solid-phase enzyme immunoassay in comparison to intravenous injection. The carbopol gel formulations produced a significant increase in bioavailability. CD complex promotes the nasal absorption of progesterone from carbopol gels as compared with gels where the CD is added by simple addition and gels which do not contain CD. This method of addition of CD as an inclusion complex in the gels could be considered as a preferred platform in nasal drug administration.