Lipase-catalyzed transamidation of non-activated amides in organic solvent

A novel biocatalytic reaction of transamidation of non-activated amides with amines is reported. Among 45 different lipolytic and proteolytic enzymes tested, only the lipase from Candida antarcticawas able to catalyze this reaction. The reaction proceeded with up to ca. 80% conversion in anhydrous methyl tert-butyl ether and worked with both N-substituted and unsubstituted amides. The biocatalytic transamidation is an equilibrium process and, therefore, higher conversions to the desired amide were achieved by using increased concentrations of the amine nucleophile.     

Interconnection of physico-chemical characteristics of organic solvents and their denaturing ability in relation to proteins]

Reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was studied. The regularities of this process were analyzed, employing both experimental and literary data based on the results of kinetic and spectroscopic measurements. In all the systems under study denaturation proceeded in a threshold manner, i. e., an abrupt change in the catalytic and/or spectroscopic properties of the dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of reversible protein denaturation by organic solvents was proposed. This model is based on the widely accepted viewpoint that the undisturbed water shell around the protein globule is necessary for maintaining the dissolved protein in the native state. Quantitative analysis of the model led to an equation establishing a relationship between the threshold concentration of an organic solvent and its physico-chemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation fits well in the experimental data for all the proteins tested. Based on the above thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents (termed as the denaturation capacity or DC) was proposed. Different organic solvents arranged according to their DC values form the DC scale of organic solvents which permits to predict theoretically the threshold concentration of any organic solvent for a given protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphogenesis of potato plants in vitro. II. Endogenous levels,distribution, and metabolism of IAA and cytokinins

The levels of endogenous IAA and cytokinins (zeatin, zeatin riboside, isopentenyladenine, and isopentenyladenosine) were determined in potato plants cultured in vitro under red light (R) and blue light (B) on medium with or without hormones. On medium without hormones in B, plants contained much higher cytokinin levels, particularly in leaves and roots, and also slightly elevated IAA levels. Kinetin in the medium in B changed the distribution of cytokinins and significantly increased IAA level in roots. In R, the presence of kinetin led to an increased cytokinin level in the whole plant, while the IAA level was slightly lower. IAA in the medium in B decreased cytokinin level in all plant parts, while the IAA level did not change significantly. In R, the presence of IAA in the medium led to a moderate increase of CK level and to a significant increase in IAA level, especially in roots. Uptake of 1-¹⁴C-IAA and of ³H-zeatin was generally higher in B than in R. Higher percentage of IAA taken up in B was converted to conjugates in the roots. Metabolism of ³H-zeatin was similar in R and B with only slight differences in metabolite amounts.Thus, in all experimental situations in which tuber formation was stimulated, IAA level in roots and stolons rose significantly, stressing the importance of an IAA gradient for tuber formation.     

Cladistic analysis of the Cirripedia Thoracica

We present a cladistic analysis of the Cirripedia Thoracica using morphological characters and the Acrothoracica and Ascothoracida as outgroups. The list of characters comprised 32 shell and soft body features. The operational taxonomic units (OTUs) comprised 26 well-studied fossil and extant taxa, principally genera, since uncertainty about monophyly exists for most higher ranking taxonomic units. Parsimony analyses using PAUP 3.1.1 and Hennig86 produced 189 trees of assured minimal length. We also examined character evolution in the consensus trees using MacClade and Clados. The monophyly of the Balanomorpha and the Verrucomorpha sensu stricto is confirmed, and all trees featured a sister group relationship between the ‘living fossil Neoverruca and me Brachylepadomorpha. In the consensus trees the sequential progression of ‘pedunculate‘sister groups up to a node containing Neolepas also conforms to current views, but certain well-established taxa based solely on plesiomorphies stand out as paraphyletic, such as Pedunculata (= Lepadomorpha); Eolepadinae, Scalpellomorpha and Chthamaloidea. The 189 trees differed principally in the position of shell-less pedunculates, Neoverruca, the scalpelloid Capitulum, and the interrelationships within the Balanomorpha, although the 50% majority rule consensus tree almost fully resolved the latter. A monophyletic Sessilia comprising both Verrucomorpha and Balanomorpha appeared among the shortest trees, but not in the consensus. A tree with a monophyletic Verrucomorpha including Neoverruca had a tree length two steps longer than the consensus trees. Deletion of all extinct OTUs produced a radically different tree, which highlights the importance of fossils in estimating cirripede phylogeny. Mapping of our character set onto a manually constructed cladogram reflecting die most recent scenario of cirripede evolution resulted in a tree length five steps longer than any of our shortest trees. Our analysis reveals that several key questions in cirripede phylogeny remain unsolved, notably the position of shell-less forms and the transition from ‘pedunculate‘to ‘sessile‘barnacles. The inclusion of more fossil species at this point in our understanding of cirripede phylogeny will only result in even greater levels of uncertainty. When constructing the character list we also identified numerous uncertainties in the homology of traits commonly used in discussing cirripede evolution. Our study highlights larval ultrastructure, detailed studies of early ontogeny, and molecular data as the most promising areas for future research.     

Experimental characteristics of the antileishmaniasis action of amphotericin B]

The efficacy of amphotericin B powder of the Soviet production was studied as a therapeutic agent in experimental zoonotic leishmaniosis of the skin form of mice and hamsters. Amphotericin B was administered per os and its action was compared with that of monomycin injected subcutaneously (50 mg/kg). Amphotericin B in a dose of 150 mg/kg administered for 30 days protected 96+/-0.07 and 76.4+/-0.9 per cent of the mice and hamsters respectively from development of the leishmaniosis clinical signs, 68.8+/-0.04 per cent of the animals being cured (in the treatment experiments). The effect of the antibiotic was analogous to that of monomycin administered parenterally. The study of the kinetics of amphotericin B showed that the antibiotic was well absorbed from the digestive tract of the animals into the blood which provided the chemotherapeutic effect.     

Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis.

The process of reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N-methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so-called 'bad' solvents; reasons for the occurrence of 'bad' solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the well-known ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/water-immiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.     

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Characteristics of DNA in vegetative specimens of Bursaria truncatella]

DNA from Bursaria truncatella was isolated and purified by conventional methods. The DNA base content was calculated from both centrifugation in CsCl and melting. The GC-content is 24%. In CsCl density gradient 3H-DNA is banded as a single peak at a range 1.682--1.688 g/cm3 with the maximum at 1.684 g/cm3. The Tm in 0.12 M FB (pH 6.8) was 79 degrees C. About 50% of DNA seems to be represented by highly repetitive sequences, another 50% being made of single-copy sequences (a preliminary data on DNA-DNA reassotiation kinetics). For the estimation of the molecular weight of DNA, the cells were lysed immediately before the centrifugation, at the surface of the alkaline isokinetic sucrose gradient solution. Two components of DNA were detected with molecular weights 10-10(6) and 100-10(6) daltons. It is likely that these two components belong to the macronuclear DNA, because according to cytophotometrical evidence the DNA content in the macronucleus of B. truncatella is 2500 times as much as that in its micronucleus.