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.     

Ontologies in radiation oncology

Ontologies are a formal, computer-compatible method for representing scientific knowledge about a given domain. They provide a standardized vocabulary, taxonomy and set of relations between concepts. When formatted in a standard way, they can be read and reasoned upon by computers as well as by humans. At the 2019 International Conference on the Use of Computers in Radiation Therapy, there was a session devoted to ontologies in radiation therapy. This paper is a compilation of the material presented, and is meant as an introduction to the subject. This is done by means of a didactic introduction to the topic followed by a series of applications in radiation therapy. The goal of this article is to provide the medical physicist and related professionals with sufficient background that they can understand their construction as well as their practical uses.     

Role of taurine in the central nervous system

Taurine demonstrates multiple cellular functions including a central role as a neurotransmitter, as a trophic factor in CNS development, in maintaining the structural integrity of the membrane, in regulating calcium transport and homeostasis, as an osmolyte, as a neuromodulator and as a neuroprotectant. The neurotransmitter properties of taurine are illustrated by its ability to elicit neuronal hyperpolarization, the presence of specific taurine synthesizing enzyme and receptors in the CNS and the presence of a taurine transporter system. Taurine exerts its neuroprotective functions against the glutamate induced excitotoxicity by reducing the glutamate-induced increase of intracellular calcium level, by shifting the ratio of Bcl-2 and Bad ratio in favor of cell survival and by reducing the ER stress. The presence of metabotropic taurine receptors which are negatively coupled to phospholipase C (PLC) signaling pathway through inhibitory G proteins is proposed, and the evidence supporting this notion is also presented.     

Glutamine transport in brain mitochondria

Gln is transported into rat brain synaptic and non-synaptic mitochondria by a protein catalyzed process. The uptake is significantly higher in synaptic than in non-synaptic mitochondria. The transport is inhibited by the amino acids Glu, Asn and Asp, and by the TCA cycle intermediates succinate, malate and 2-OG. The inhibition by 2-OG is counteracted by AOA and is therefore assumed to be due to transamination of 2-OG, whereby Glu is formed. This presumes that Glu also binds to an inhibitory site on the matrix face of the inner membrane. The transport is complex and cannot be explained by the simple uniport mechanism which has been proposed for renal (Schoolwerth and LaNoue, 1985), and liver mitochondria (Soboll et al., 1991). Thus, Gln transport is stimulated by respiration and by the proton electrochemical gradient. Since it is indicated that both the neutral Gln zwitterion and the Gln anion are transported, there are probably different uptake mechanisms, but not necessarily different carriers. Gln may be transported by an electroneutral mechanism as a proton compensated anion, as well as electrophoretically as a zwitterion with a proton, and probably also by diffusion as a zwitterion. The properties of the brain mitochondrial Gln uptake mechanisms are also not identical with those of a purified renal Gln transporter. It is possible that the Gln transport is controlled by more than one protein, which may be situated on distinct species in a heterogeneous mitochondrial population. Since Gln is assumed to participate in energy production as well as in the synthesis of nucleic acid components and proteins in brain mitochondria, the control of Gln uptake in these organelles may be important.     

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.     

<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.     

Microbial metabolism of methanesulfonic acid

Methanesulfonic acid is a very stable strong acid and a key intermediate in the biogeochemical cycling of sulfur. It is formed in megatonne quantities in the atmosphere from the chemical oxidation of atmospheric dimethyl sulfide (most of which is of biogenic origin) and deposited on the Earth in rain and snow, and by dry deposition. Methanesulfonate is used by diverse aerobic bacteria as a source of sulfur for growth, but is not known to be used by anaerobes either as a sulfur source, a fermentation substrate, an electron acceptor, or as a methanogenic substrate. Some specialized methylotrophs (including Methylosulfonomonas, Marinosulfonomonas, and strains of paragraph signHyphomicrobium and Methylobacterium) can use it as a carbon and energy substrate to support growth. Methanesulfonate oxidation is initiated by cleavage catalysed by methanesulfonate monooxygenase, the properties and molecular biology of which are discussed.     

Cadmium-induced changes in the growth and oxidative metabolism of pea plants

The effect of growing pea (Pisum sativum L.) plants with CdCl(2) (0-50 microM) on different plant physiological parameters and antioxidative enzymes of leaves was studied in order to know the possible involvement of this metal in the generation of oxidative stress. In roots and leaves of pea plants Cd produced a significant inhibition of growth as well as a reduction in the transpiration and photosynthesis rate, chlorophyll content of leaves, and an alteration in the nutrient status in both roots and leaves. The ultrastructural analysis of leaves from plants grown with 50 microM CdCl(2), showed cell disturbances characterized by an increase of mesophyll cell size, and a reduction of intercellular spaces, as well as severe disturbances in chloroplast structure. Alterations in the activated oxygen metabolism of pea plants were also detected, as evidenced by an increase in lipid peroxidation and carbonyl-groups content, as well as a decrease in catalase, SOD and, to a lesser extent, guaiacol peroxidase activities. Glutathione reductase activity did not show significant changes as a result of Cd treatment. A strong reduction of chloroplastic and cytosolic Cu,Zn-SODs by Cd was found, and to a lesser extent of Fe-SOD, while Mn-SOD was only affected by the highest Cd concentrations. Catalase isoenzymes responded differentially, the most acidic isoforms being the most sensitive to Cd treatment. Results obtained suggest that growth of pea plants with CdCl(2) can induce a concentration-dependent oxidative stress situation in leaves, characterized by an accumulation of lipid peroxides and oxidized proteins as a result of the inhibition of the antioxidant systems. These results, together with the ultrastructural data, point to a possible induction of leaf senescence by cadmium.     

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.     

Suppression of the primary cell-mediated immune response by human alpha1-fetoprotein in vitro.

A possible immunoregulatory role of human α₁-fetoprotein (HAFP) was investigated. HAFP-enriched fractions as well as pure HAFP were obtained by means of two different procedures, as follows. After passage of HAFP-containing ascites of patients with primary liver carcinoma (PLC) on an anti-HAFP immunosorbent column, the retained proteins were eluted first by a glycine-NaOH buffer, pH 10.0 (resulting in HAFP I), and second by NaSCN (HAFP II). HAFP was further purified by passage of the HAFP-containing fractions an on anti-human whole serum (anti-HWS) immunosorbent column. This resulted in semipurified HAFP I and II. HAFP, pure by means of SDS-disc gel electrophoresis, Ouchterlony gel diffusion, and immunoelectrophoresis, was obtained by recycling on the anti-HWS immunosorbent column, as well as by a final Sephadex G-100 gel filtration. A possible immunoregulatory activity was assessed by testing the influence of semipurified as well as pure HAFP I and II on the uptake of tritiated thymidine by human lymphocytes stimulated by allogeneic lymphocytes in vitro. Only HAFP I, semipurified as well as pure, consistently exerted a profound suppressive effect on this primary cell-mediated immune response at concentrations of 150–200 μg/ml. In contrast, HAFP II did not show a comparable immunoregulatory effect either because there are two biologically different HAFPs or because of a loss of biological activity from HAFP II due to the use of the sodium thiocyanate elution technique.     

Noise, sensitivity, and resolution of flow cytometers.

The sensitivity and resolution of flow cytometers are functions of the signal produced by a given particle as well as by the noise in the presence of which the signal is detected. The noise is primarily due to the fact that emission of light as well as its detection by photoelectric devises are stochastic processes. This fact leads to equations describing how resolution and sensitivity are limited by the magnitude of the signal, the background, and the photoelectron quantum yield of the detector. The equations are pointing to a method by which the signal and noise of a flow cytometer can be measured in absolute terms, as well as a way to determine fluorescence sensitivity without having to extrapolate to the noise level. The equations appear to be validated when applied to measuring data obtained with two different flow cytometers.     

Recombinant human IFN-gamma, but not IFN-alpha or IFN-beta, enhances MHC- and non-MHC-encoded glycoproteins by a protein synthesis-dependent mechanism

Despite quantitative as well as qualitative differences, all three types of IFN (IFN-alpha, IFN-beta, and IFN-gamma) modulate the synthesis as well as the expression of class I and class II histocompatibility Ag and a melanoma-associated Ag located in the plasma membrane as well as the cytoplasm of human melanoma cells. By employing inhibitors of RNA and protein synthesis it was demonstrated that IFN-alpha and -beta increase the expression of histocompatibility products and this tumor-associated Ag by a process not requiring new protein synthesis. In contrast, IFN-gamma does require de novo protein synthesis for its modulatory activity. Thus, it appears that IFN might trigger various adaptive functions in different cell lineages by inducing at least two separate sets of responses specific for either IFN-alpha and -beta or IFN-gamma. Because the induction requirements for (2'-5')-oligoadenylate synthetase as well as for the development of a cellular antiviral state by different IFN also display a similar protein synthesis dependence pattern, the present results suggest that a similar set of cellular mediators may be involved in the modulation of antigenic expression by IFN-gamma in human melanoma cells.     

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.     

Interaction of complement and leukocytes in severe acute pancreatitis: potential for therapeutic intervention

In acute pancreatitis, local as well as systemic organ complications are mediated by the activation of various inflammatory cascades. The role of complement in this setting is unclear. The aim of the present study was to determine the level of complement activation in experimental pancreatitis, to evaluate the interaction of complement and leukocyte-endothelium activation, and to assess the effects of complement inhibition by soluble complement receptor 1 (sCR1) in this setting. Necrotizing pancreatitis was induced in Wistar rats by the combination of intravenous cerulein and retrograde infusion of glycodeoxycholic acid into the biliopancreatic duct; edematous pancreatitis was induced by intravenous cerulein only. In control animals, a sham operation (midline laparotomy) was performed. Complement activation, leukocyte sequestration, and pancreatic as well as pulmonary injury were assessed in the presence/absence of sCR1. Increased levels of C3a were found in necrotizing but not in edematous pancreatitis. When complement activation in necrotizing pancreatitis was blocked by sCR1, levels of C3a and total hemolytic activity (CH50) were decreased. Leukocyte-endothelial interaction, as assessed by intravital microscopy, and pancreatic as well as pulmonary organ injury (wet-to-dry weight ratio, MPO activity, and histology) were ameliorated by sCR1. As a result of the present study, necrotizing but not edematous pancreatitis is characterized by significant and early complement activation. Based on the interaction of complement and leukocytes, complement inhibition by sCR1 may be a valuable option in the treatment of leukocyte-associated organ injury in severe pancreatitis.     

Purification of Gc-2 globulin from human serum by displacement chromatography: a model for the isolation of marker proteins identified by two-dimensional electrophoresis

A protein that was initially known only as a minor spot in two-dimensional electrophoresis patterns of serum obtained from certain psoriasis patients, particularly those with a pustular component to their disease, has been purified by two stages of ion-exchange displacement chromatography on DEAE-Sephacel at different pH levels, followed by elution chromatography on hydroxylapatite. The purification was followed by examining the column fractions directly by two-dimensional gel electrophoresis. The capacity of the displacement system, which utilized carboxymethyldextrans as displacers, was very high; 6 ml of dialyzed serum applied to a 7-ml column in the initial stage resulted in a very substantial enrichment of the target protein. The second displacement stage yielded a highly purified product, contaminated only by A-1 lipoprotein. The latter was removed by hydroxylapatite chromatography. The purified protein was subsequently identified as Gc-2 globulin, a vitamin D-binding protein, by immunological procedures. The results demonstrate the effectiveness of ion-exchange displacement chromatography in focusing resolving power on the relatively narrow range of affinities represented by the target protein and its immediate neighbors in a chromatogram, as well as the applicability of the system to the isolation of a protein known only by its position in a two-dimensional electrophoretic pattern.     

In vitro biological activities of magainin alone or in combination with nisin

Antimicrobial peptides have received increasing attention not only as potential candidates to their administration as antimicrobial agents, but also as potential drugs applied in cancer therapy. Here, we have examined the action of both nisin and magainin on human promyelocytic leukemia HL-60 cells. Cells were cultured in presence of either nisin or magainin 1 as well as in combination with both nisin and magainin 1. Results have revealed that magainin, but not nisin, produces a loss of cell viability in HL-60 cells, and a minor increase of hemolysis, whereas it is not responsible for cell membrane disruption and lactate dehydrogenase (LDH) leakage. In addition, magainin is involved in a significant generation of reactive oxygen species (ROS), as well as in an augment of caspase-3 activity. Magainin-induced apoptosis was verified by DNA fragmentation and annexin V-FITC/propidium iodide (PI) staining of the cells. Promotion of cell death by magainin occurs via cytochrome c release accompanied by a substantial increase of proteasome activity. These results underline the importance of magainin as a drug capable of exerting an in vitro antitumoral activity by triggering apoptosis.     

Preventive effects of a water-soluble derivative of chroman moiety of vitamin E on lipid hydroperoxide-induced cell injuries and DNA cleavages through repressions of oxidative stress in the cytoplasm of human keratinocytes

ChrCrx (6-hydroxy-2, 5, 7, 8-tetramethyl-chroman-2-carboxylic acid) is a water-soluble analog in which 4', 8', 12'-trimethyltridecyl chain is deleted from an alpha-tocopherol molecule known as a hydrophobic antioxidant. Cell viability of human skin epidermal keratinocytes HaCaT was lowered by treatment with tert-butylhydroperoxide (t-BuOOH) of 50 microM for 48 h, designated as a subacute cytotoxicity, which was prevented by previous administration with ChrCrx in a dose-dependent manner as estimated by mitochondrial function-based WST-1 assay and cell morphological microscopy. In contrast an acute cytotoxicity due to treatment with t-BuOOH as dense as 200 microM for a period as short as 2 h could be also prevented with ChrCrx that was administered before and after, but was eliminated during, treatment with t-BuOOH. In contrast alpha-tocopherol was not cytoprotective against t-BuOOH. DNA strand cleavages were induced with t-BuOOH in the keratinocytes, and could be prevented by ChrCrx more effectively than alpha-tocopherol as assayed by TUNEL stain. The intracellular reactive oxygen species (ROS) was accumulated in a manner dependent on periods of t-BuOOH treatment in the cytoplasm more abundantly rather than the nucleus of keratinocytes, and was markedly diminished by ChrCrx as shown by fluorography using the redox indicator dye. Thus t-BuOOH-induced cell injuries and DNA cleavages of the keratinocytes can be prevented at least in part through efficient diminishment of ROS generated in the cytoplasm, to which the preferred distribution of ChrCrx may be advantageous over to the nucleus or membrane owing to its molecular hydrophilicity relative to alpha-tocopherol.