Lipases from different sources vary widely in dependence of catalytic activity on water activity.

We have measured the rates of esterification in hexane catalysed by suspended immobilised lipases (triacylglycerol acylhydrolase, EC 3.1.1.3), with pre-equilibration to known thermodynamic water activity (a(w)). There were important differences between the enzymes from five different microbes in their retention of activity at low a(w). That from Rhizomucor miehei showed over 40% maximal activity at an a(w) of 0.12, and that from Rhizopus niveus was also fairly active at low a(w). Lipases from other sources required higher a(w) values to show good activity, increasing in the sequence Humicola sp., Candida rugosa and Pseudomonas cepacia. The behaviour was generally similar to two very different support materials, anion-exchange resin and macroporous polypropylene. Comparison of the sequences of the homologous enzymes from Rh. miehei, Rh. niveus and Humicola sp. suggests that changes in charged residues in the 'hinge and lid' region of the structure may be significant in low a(w) tolerance.     

Lipases in lipophilization reactions

Lipases are used in various sectors, as pharmaceutical, food or detergency industry. Their advantage versus classical chemical catalysts is that they exhibit a better selectivity and operate in milder reaction conditions. Theses enzymes can also be used in lipophilization reactions corresponding to the grafting of a lipophilic moiety to a hydrophilic one such as sugar, amino acids and proteins, or phenolic compounds. The major difficulty to overcome in such enzyme-catalyzed reaction resides in the fact that the two involved substrates greatly differ in term of polarity and solvent affinity. Therefore, several key parameters are to be considered in order to achieve the reaction in satisfactory kinetics and yields. The present review discusses the nature of such parameters (eg solvent nature, water activity, chemical modification of substrates) and illustrates their effect with examples of lipase-catalyzed lipophilization reactions of various sugar, amino acids or phenolic derivatives.     

Picornaviruses of two different genera have similar structures

Crystals of Mengo virus were used to collect three-dimensional X-ray diffraction data to 7 Å resolution. A self-rotation function showed the precise orientation of the Mengo particles in the crystal unit cell. A cross-rotation function against similar data of cubic rhinovirus crystals showed a peak when the orientations of these two icosahedral viruses were superimposed. This demonstrates similarity of capsid construction between two picornaviruses of different taxonomic genera.     

Lipases in catalytic reactions of organic chemistry

Aspects of enzymatic catalysis in lipase-catalyzed reactions of organic synthesis are discussed in the review. The data on modern methods of protein engineering and enzyme modification allowing a broader range of used substrates are briefly summarized. The application of lipase in the preparation of pharmaceuticals and agrochemicals containing no inactive enantiomers and in the synthesis of secondary alcohol enantiomers and optically active amides is demonstrated. The subject of lipase involvement in the C-C bond formation in the Michael reaction is discussed. Data on the enzymatic synthesis of construction materials—polyesters, siloxanes, etc.—are presented. Examples demonstrating the application of lipase enzymatic catalysis in industry are given.     

Structurally similar diazenes exhibit significantly different biological activity

We have previously synthesized various diazenecarboxamides (subsequently referred to as diazenes) that were cytotoxic to several tumor cell lines. To increase their biological activity, the structure has been modified appropriately. In the present study we examined the effects of N¹-phenyl-N²-(2-pyridinylmethyl)diazenedicarboxamide (RL-337) obtained from the previously examined cytotoxic compound N¹-phenyl-N²-(2-pyridinyl)diazenecarboxamide (JK-279), and compared them with those of diazene JK-279. Using a modified colorimetric MTT assay, the cytotoxicity of RL-337 was determined on human cervical carcinoma HeLa cells, glioblastoma A1235 cells, and prostate adenocarcinoma PC-3 cells. The possible synergistic effect of diazene RL-337 with cisplatin, doxorubicin, and vincristine, and its influence on intracellular GSH content was examined on HeLa cells. Diazene RL-337 was cytotoxic against all three human tumor cell lines, being more cytotoxic to HeLa cells than diazene JK-279. The higher efficacy of RL-337 than of JK-279 can be connected with higher basicity of the 2-picoline moiety present in the former diazene comparing with the pyridine fragment that is a part of the latter. The diazene RL-337 acted synergistically with cisplatin, doxorubicin, and vincristine (diazene JK-279 exhibited synergistic effect only with cisplatin). Glutathione (determined by Tietze's method) was not a target molecule of diazene RL-337 (but was for JK-279, as shown earlier). After just 1 h treatment with diazene RL-337, the cells started to lose membrane integrity. There was no cleavage of caspase-3 in RL-337-treated samples, and the majority of cells died 6 h after the treatment through necrosis (previously, apoptosis-like cell death was detected for diazene JK-279). Thus, although diazenes JK-279 and RL-337 are very similar in their structure, they exhibit widely different biological activity.     

Reaction rate with suspended lipase catalyst shows similar dependence on water activity in different organic solvents.

We have studied the effect of thermodynamic water activity (a W) on the initial rate of esterification catalysed by an immobilised lipase (Lipozyme) suspended in an organic reaction mixture. The catalyst and the organic phase were separately pre-equilibrated to the same aw value. The rate shows similar dependence on aw in reaction mixtures based on five different organic solvents ranging in polarity from pentan-3-one to hexane, and in a liquid reactant mixture. There is a maximum at aw about 0.5, with a decline to 30-70% at aw of either 0.9 or less than 0.01. When the rates are presented in terms of water concentration in the organic phase (or total water content of the system), the maxima for the various solvents come at very different positions, reflecting the widely varying solubilities of water in the organic phase.     

The different receptor species of liver have similar complex insulin binding properties

Liver plasma membranes bind insulin in a complex fashion via three prominent disulfide-linked insulin receptor structures of 360K, 300K, and 260K molecular weight. To determine if the complex binding is explained by different binding affinities among the different structures, 125I-insulin was specifically cross-linked to the binding sites and the amount of radiolabeled insulin was determined after SDS-gel electrophoresis. The insulin binding characteristics of each structure were similar to the binding properties of the intact membrane. The Scatchard plot for each structure was curvilinear and the Kd values for the high and low affinity components were similar to the membrane values. Thus, the curvilinear Scatchard plot of insulin binding to liver membranes is also a feature of each receptor structure and is not a function of different receptors with different binding properties.     

Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism

Introduction

The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro. There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is to compare the metabolic profile of traditional and new generation NO donors to see which one points out the osteoarthritic process in the best way.

Methods

Human healthy and OA chondrocytes were isolated from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with NO donors (NOC-12 or SNP). NO production was evaluated by the Griess method, and apoptosis was quantified by flow cytometry. Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities by enzymatic assay, mitochondrial membrane potential (Δψm) by JC-1 using flow cytometry, and ATP levels were measured by luminescence assays. Glucose transport was measured as the uptake of 2-deoxy-[³H]glucose (2-[³H]DG). Statistical analysis was performed using the Mann-Whitney U test.

Results

NOC-12 liberates approximately ten times more NO₂^- than SNP, but the level of cell death induced was not as profound as that produced by SNP. Normal articular chondrocytes stimulated with NOC-12 had reduced activity from complexes I, III y IV, and the mitochondrial mass was increased in these cells. Deleterious effects on ΔΨm and ATP levels were more profound with SNP, and this NO donor was able to reduce 2-[³H]DG levels. Both NO donors had opposite effects on lactate release, SNP diminished the levels and NOC-12 lead to lactate accumulation. OA chondrocytes incorporate significantly more 2-[³H]DG than healthy cells.

Conclusions

These findings suggest that the new generation donors, specifically NOC-12, mimic the OA metabolic process much better than SNP. Previous results using SNP have to be considered prudently since most of the effects observed can be induced by the interactions of secondary products of NO.     

Maize and wheat mitochondrial tRNAPro coding regions have similar sequences but different organizations.

The nucleotide sequences of two maize mitochondrial DNA regions containing a tRNAPro gene and an incomplete tRNAPro gene have been compared with the corresponding regions of wheat mitochondrial DNA. These regions have similar sequences but their organization is modified due to different recombination events involving the tRNAPro immediate environment.     

Transforming genes among three different oncogenic subgroups of human adenoviruses have similar replicative functions.

We have examined the functional similarity of the transforming genes for replicative functions among three different subgroups of human adenoviruses (A, B, and C), using mutant complementation as an assay. A host range deletion mutant (dl201.2) of Ad2 (nononcogenic subgroup C) lacking about 5% of the viral DNA covering two early gene blocks (E1a and E1b) involved in cellular transformation was isolated and tested for its ability to replicate in nonpermissive KB cells in the presence of Ad7 (weakly oncogenic group B) or ad12 (highly oncogenic group A). The complementation of the mutant defect was demonstrated by cleaving the viral DNA extracted from mixed infected cells or the DNA extracted from purified virions from mixed infected cells with restriction endonuclease BamHI, which produces a different cleavage pattern with the DNA of each serotype. It was found that the defects in E1a plus E1b of dl201.2 could be complemented by Ad7 and Ad12, indicating that these genes in Ad2, Ad7, and Ad12 have similar functions during productive infection.     

Microbial communities in bulk fluids and biofilms of an oil facility have similar composition but different structure

The oil-water-gas environments of oil production facilities harbour abundant and diverse microbial communities that can participate in deleterious processes such as biocorrosion. Several molecular methods, including pyrosequencing of 16S rRNA libraries, were used to characterize the microbial communities from an oil production facility on the Alaskan North Slope. The communities in produced water and a sample from a 'pig envelope' were compared in order to identify specific populations or communities associated with biocorrosion. The 'pigs' are used for physical mitigation of pipeline corrosion and fouling and the samples are enriched in surface-associated solids (i.e. paraffins, minerals and biofilm) and coincidentally, microorganisms (over 10(5) -fold). Throughout the oil production facility, bacteria were more abundant (10- to 150-fold) than archaea, with thermophilic members of the phyla Firmicutes (Thermoanaerobacter and Thermacetogenium) and Synergistes (Thermovirga) dominating the community. However, the structure (relative abundances of taxa) of the microbial community in the pig envelope was distinct due to the increased relative abundances of the genera Thermacetogenium and Thermovirga. The data presented here suggest that bulk fluid is representative of the biofilm communities associated with biocorrosion but that certain populations are more abundant in biofilms, which should be the focus of monitoring and mitigation strategies.     

BCG preparations,cultured homogeneously dispersed or as a surface pellicle,elicit different immunopotentiating effects but have similar antitumor activity in a murine fibrosarcoma

Summary Four preparations of Bacillus Calmette-Guérin (BCG), cultured either homogeneously dispersed or as a surface pellicle, were compared with reference to their immunomodulating capacities and antitumor effects. The BCG preparations included two vaccines that originated from the same seed strain, but one had subsequently been produced in each way. The immunological assays included in vivo stimulation of lymph nodes in a mouse model, in vitro stimulation of murine spleen lymphocytes, and in vivo stimulation of macrophages in a Listeria monocytogenes clearance model in the mouse. The antitumor effect was determined in a non-immunogenic, non-metastasizing murine fibrosarcoma. The results indicated that vaccines produced as a homogeneous culture in general induced a higher lymphocyte stimulation both in vivo and in vitro. In the Listeria clearance model a markedly enhanced clearance was established with three of the four preparations, the phenomenon being related to the number of culturable particles administered. This difference was not attributed to the production method, but to other factors, including the actual composition of the vaccine.The results found in the immunological assays were not connected to the observed antitumor activities, as for each preparation a combination of route, dose, and time interval for tumor regression was found. Prophylactic administration of BCG had no effect at all; enhancement was observed after intratumoral administration of the two preparations prepared as surface pellicles.It is concluded that a protocol for the quality control of bacterial vaccines used for cancer immunotherapy should include both immunological assays and a range of different animal tumor models.Fellow of the Koningin Wilhelmina Fonds of the National Cancer League of the Netherlands The Abbreviations used are: BCG, Bacillus Calmette-Guérine; c.p., culturable particles; Con A, Concanavalin A; MEM, Minimum Essential Medium; FCS, fetal calf serum; cpm, counts per minute; IT, intratumoral     

Characterization of Alternaria and Penicillium species from similar substrata based on growth at different temperature,pH and water activity

Fifty-eight Alternaria isolates representing 10 species or species-groups and 66 Penicillium isolates representing 18 species were examined for their growth response to the combined effects of water activity, temperature and pH in an extended Central Composite Design. Growth responses were recorded as colony diameter after one and two weeks of growth and analysed using different multivariate statistical analyses. The isolates, when analysed by Principal Component Analysis, clustered according to their genus and to some degree to species or species groups and not according to substratum as excepted. Soft Independent Modelling of Class Analogy and Response Surface Analysis showed that growth responses or growth profiles may be used as classification tool. Partial Least Squares Regression showed that a combination of two different media based on Dichloran Rose bengal Yeast Extract Sucrose agar incubated at two different temperatures were enough to get genus segregation and to some extent species segregation. The results also showed that water activity, temperature and pH interact strongly in their effect on growth rates and that the squared products (optima) of water activity, temperature and pH for each isolate were important for modelling the data sufficiently.     

Two human colon tumor cell lines with similar nuclease sensitivities have different ethidium bromide binding characteristics

Chromatin from two human colon adenocarcinoma cell lines (HT-29 and LoVo) showed similar digestion kinetics when sensitivities to DNase I and micrococcal nuclease were examined. Chromatin conformations were probed by examining the binding of ethidium bromide. A Scatchard plot revealed that both chromatins bound the same amount of ethidium bromide per mole of DNA, but the DNA from LoVo cells was more accessible to the intercalator. The results indicate that differences in chromatin conformation are not necessarily accompanied by different nuclease sensitivities.     

Sympatric natural Saccharomyces cerevisiae and S. paradoxus populations have different thermal growth profiles

Saccharomyces cerevisiae and its close congener S. paradoxus are typically indistinguishable by the phenotypic criteria of classical yeast taxonomy, but they are evolutionarily distinct as indicated by hybrid spore inviability and genomic sequence divergence. Previous work has shown that these two species coexist in oak-associated microhabitats at natural woodland sites in North America. Here, we show that sympatric populations of S. cerevisiae and S. paradoxus from a single natural site are phenotypically differentiated in their growth rate responses to temperature. Our main finding is that the S. cerevisiae population exhibits a markedly higher growth rate at 37 degrees C than the S. paradoxus population; we also find possible differences in growth rate between these populations at two lower temperatures. We discuss the implications of our results for the coexistence of these yeasts in natural environments, and we suggest that thermal growth response may be an evolutionarily labile feature of these organisms that could be analyzed using genomic approaches.     

Two major medicinal honeys have different mechanisms of bactericidal activity

Honey is increasingly valued for its antibacterial activity, but knowledge regarding the mechanism of action is still incomplete. We assessed the bactericidal activity and mechanism of action of Revamil® source (RS) honey and manuka honey, the sources of two major medical-grade honeys. RS honey killed Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa within 2 hours, whereas manuka honey had such rapid activity only against B. subtilis. After 24 hours of incubation, both honeys killed all tested bacteria, including methicillin-resistant Staphylococcus aureus, but manuka honey retained activity up to higher dilutions than RS honey. Bee defensin-1 and H₂O₂ were the major factors involved in rapid bactericidal activity of RS honey. These factors were absent in manuka honey, but this honey contained 44-fold higher concentrations of methylglyoxal than RS honey. Methylglyoxal was a major bactericidal factor in manuka honey, but after neutralization of this compound manuka honey retained bactericidal activity due to several unknown factors. RS and manuka honey have highly distinct compositions of bactericidal factors, resulting in large differences in bactericidal activity.     

Anatomical profiles validate gall morphospecies under similar morphotypes

Plant galls are generated by the stimuli of gall-inducing organisms on their hosts, creating gall morphotypes that vary in color, shape, size, and tissue organization. Herein, we propose to compare the structural features of gall morphotypes on the superhost Croton floribundus (Euphorbiaceae) in order to recognize gall morphospecies, i.e., galls with similar shapes but different internal structures. Non-galled leaves and galls were analyzed macroscopically, histologically, and histochemically for the detection of primary metabolites, and the results obtained were used for statistical analyses of similarity. Among the eight gall morphospecies, four are globoid, two are lenticular, one is fusiform and one is marginal leaf rolling. Stomatal differentiation and the occurrence of different types of trichomes were impaired in some gall morphospecies. Three patterns of organization of the ground system are recognized, ranging from the maintenance of mesophyll cells that differentiate into palisade and spongy cells dorsiventrally to the formation of a complex cortex with three morphofunctional layers. The marginal leaf rolling galls have the simplest anatomical structures, quite similar to those of the non-galled host leaf, while lenticular, globoid (types I to IV), and fusiform galls are anatomically more complex. Herein, we report on eight gall morphospecies occurring on C. floribundus, which are distinguished by morpho-anatomical attributes and show the disruption of the morphogenetic patterns of the host leaf toward the morphogenesis of unique gall features.