Non-covalent enzyme-polyelectrolyte complexes as self-buffered catalysts for use in low-water organic media

Summary When free chymotrypsin is used to catalyse hydrolysis of N-acetyl tyrosine ethyl ester in 90% acetonitrile, the reaction rate soon falls because of the accumulation of the acidic product. If the enzyme is used in the form of a suspended complex with polyacrylic acid, the polyelectrolyte acts as an acid-base buffer to permit extended reaction.     

Selection for β2-microglobulin mutation in mismatch repair-defective colorectal carcinomas

Novel peptide antigens complexed with human leukocyte antigen (HLA) and β₂-microglobulin (β2M) molecules are presented at the cell surface to cytotoxic T lymphocytes (CTLs), provoking lysis of the antigen-presenting cell [1]. In tumour cells, genetically altered or abnormally expressed proteins provide a source of peptides that can be presented to CTLs; the resulting anti-tumour CTL responses may provide part of the body's defence against cancer. Disabling mutations in the HLA and β2M proteins required for peptide presentation allow a tumour cell to escape destruction by CTLs. Cells with deficient DNA mismatch repair have high spontaneous mutation rates [2] and produce many altered proteins that are a potential source of numerous unique peptides. Mutator tumour cells might therefore be particularly vulnerable to immune surveillance and CTL attack. Mutator phenotypes [3] and [4] and loss of β2M (or HLA) expression [5] and [6] are both relatively common among sporadic colorectal tumours. We have compared the frequency of β2M mutations in sporadic colorectal and other tumours with and without a mutator phenotype. Mutations were more frequent among colorectal tumours with the microsatellite instability indicative of a defect in DNA mismatch repair. The inactivating β2M mutations were predominantly frameshifts, which is consistent with the underlying mismatch repair defects. Evasion of immune surveillance by acquiring β2M mutations therefore occurs at high frequency in tumour cells with a mutator phenotype due to defective DNA mismatch repair.     

Chick begging as a signal: are nestlings honest?

Begging by dependent avian offspring is known to correlate withhunger level, and parents use this as a signal of brood demandto adjust their chick feeding behavior. While there is informationon how each chick adjusts its begging to its own condition,little is known of how chicks adjust to the state of their nestmates. In two experiments we manipulated the competitive environmentof individual European starling (Sturnus vulgaris) chicks byaltering the state of nest mates while holding the state oftarget chicks constant In the first experiment we placed thetarget chick's nest mates in neighboring nests with brood sizesof two, five, or eight chicks. Following the manipulation wereturned them to their own nests and recorded begging behavioron videotape. In the second experiment we separated a targetchick from its siblings and manipulated feeding level in thelaboratory. The siblings were fed at one of three levels; meanwhile,all the target chicks were fed at the intermediate level. Afterthe manipulation we placed the target chicks with their siblingsand recorded their begging in response to an artificial stimulus.In neither experiment was the begging effort of the unmanipulatedtarget chicks affected by the changes in begging behavior oftheir siblings. This result supports the view that begging isa reliable signal of individual chick state and does not involveresponses to the effort of nest mates.     

No evidence of inbreeding avoidance or inbreeding depression in a social carnivore

Dispersal by young mammals away from their natal site is generallythought to reduce inbreeding, with its attendant negative fitnessconsequences. Genetic data from the dwarf mongoose, a pack-livingcarnivore common in African savannas, indicate that there areexceptions to this generalization. In dwarf mongoose populationsin the Serengeti National Park, Tanzania, breeding pairs arecommonly related, and close inbreeding has no measurable effecton offspring production or adult survival. Inbreeding occursbecause average relatedness among potential mates within a packis high, because mating patterns within the pack are randomwith respect to the relatedness of mates, and because dispersaldoes little to decrease the relatedness among mates. Young femalesare more likely to leave a pack when the dominant male is aclose relative but are relatively infrequent dispersers. Youngmales emigrate at random with respect to the relatedness ofthe dominant female and tend to disperse to packs that containgenetically similar individuals.[Behav Ecol 7: 480–489(1996)]     

EPR Spin Trapping Study of the Decomposition of Azo Compounds in Aqueous Solutions by Ultrasound: Potential for Use as Sonodynamic Sensitizers for Cell Killing

Sonodynamic therapy, a promising new approach to cancer treatment, is based on synergistic cell killing by combination of certain drugs (sonosensitizers) and ultrasound. Although the mechanism of sonodynamic action is not understood, the role of free radicals produced from sonosensitizers by ultrasound is implicated. In this work, we studied formation of free radicals during the decomposition of several water-soluble azo compounds by 50 kHz ultrasound in aqueous solutions. Using the spin trap 3, 5-dibromo-4-nitrosobenzene sulfonate (DBNBS) tertiary carbon-centered radicals from 2, 2'-azobis (N,N'-dimethyl-eneisobutyramidine) dihydrochloride (VA-044), 2-(carbamoylazo)-isobutyronitrile (V-30), and 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and CH3 radicals from 1, 1'-azobis (N,N'-dimethylformamide) (ADMF) were detected in argonsaturated solutions and the corresponding oxygen-centered radicals (alkoxyl and peroxyl) from VA-044, V-30, and AAPH were identified using the spin trap 5, 5'-dimethyl-l-pyrroline-N-oxide (DMPO) in aerated sonicated solutions. No free radicals from 4, 4'-dihydroxyazobenzene-3, 3'-dicarboxylic acid, disodium salt (DHAB) could be found in either system. While VA-044 and AAPH could also be readily decomposed by heat (42.5°C and 80°C), V-30 decomposition only occurred in the ultrasound-exposed solutions. The most likely mechanism of decomposition of azo compounds by ultrasound is their thermolysis in the heated shell of the liquid surrounding ca vita ting bubbles driven by ultrasound and/or by pyrolysis inside these bubbles. Experiments using scavengers of ·OH and ·H, which are produced by sonolysis in aqueous solutions, demonstrated that these radicals are not involved in the ultrasound-mediated radical production from the azo compounds. Due to the known cytotoxic potential of free radicals produced from azo compounds, the use of these compounds as ultrasound sensitizers appears to be a promising approach for sonodynamic cell killing.     

Isolation and propagation of yolk-sac-derived endothelial cells from a hypervascular transgenic mouse expressing a gain-of-functionFPS/FES proto-oncogene

Summary We report on the isolation and propagation of endothelial cells from the mouse embryonic yolk sac, the earliest site of blood vessel development, and on the advantages of a hypervascular transgenic mouse source of these cells. These transgenic mice express multiple copies of an activated allele of the humanfps/fes proto-oncogene and display hypervascularity progressing to multifocal hemangiomas. This phenotype suggested a role of thefps/fes proto-oncogene in vasculogenesis and angiogenesis and led us to investigate the growth characteristics of yolk-sac-derived endothelial cells from transgenicfps/fes embryos. We have established eight independent cell clones from a mixture of transgenic and control yolk sacs from Day 12 embryos. Southern blot hybridization analysis showed all eight clones to be derived from transgenic cells suggesting a growth advantage of cells carrying the activatedfps/fes gene. A cell line, Clone 166 (C166), established from one of these clones, was more fully characterized. C166 exhibits normal endothelial characteristics, such as rearrangement into tubelike structures when placed on Matrigel, expression of angiotensin converting enzyme, retention of cobblestone morphology at confluence, and the presence of cell surface receptors for acetylated low density lipoprotein. The cells constitutively express murine endothelial cell adhesion molecule VCAM-1 and the vascular addressin identified by antibody MECA-99. As expected, the cell line expresses high levels of the cytoplasmic protein-tyrosine kinase encoded by thefps/fes proto-oncogene. The clone we have described as well as other endothelial cell lines that we have established from the mouse embryonic yolk sac should prove useful for the study of endothelial cell differentiation and for the determination of the mechanisms underlying the establishment of organ-specific endothelial cell heterogeneity.     

Two Phenol Sulfotransferase Species from One cDNA: Nature of the Differences

A phenol sulfotransferase from rat liver (EC 2.8.2.9), expressed inEscherichia colifrom a single cDNA, was purified as two separable but catalytically active proteins. The proteins appeared to be identical to each other and to the natural liver sulfotransferase by comparison of their amino acid constitution, amino-terminal end group, and interaction with a polyclonal antibody raised against the liver enzyme. Each of the recombinant forms, α and β, catalyzed the sulfuryl group transfer from 4-nitrophenylsulfate to an acceptor phenol, a reaction in which 3′-phospho-adenosine 5′-phosphate (PAP) is a necessary intermediate. Only form β, however, catalyzes the physiological transfer of a sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the free phenol. Evidence is presented that sulfotransferase α, but not β, has 1 mol of PAP tightly bound per enzyme dimer. The ability to utilize PAPS as a sulfate donor could be altered: form α could be treated and purified as form β to acquire the ability to use PAPS, whereas form β was treated by extended incubation with PAP, lost its ability to use PAPS, and was purified as form α.     

A Mild Purification Method for Polysaccharide Binding Membrane Proteins: Phase Separation of Digitonin Extracts to Isolate the Hyaluronate Synthase fromStreptococcussp. in Active Form

A new method was developed to purify the streptococcal hyaluronate synthase in active form to electrophoretic homogeneity. The method is based on the extraction of protoplast membranes with digitonin and a phase separation into an aqueous and a detergent phase induced by addition of polyethylene glycol 6000 at 0°C. Proteins bound to hyaluronate were enriched in the aqueous phase, whereas other membrane proteins resided in the detergent phase. Final purification of the hyaluronate synthase was achieved by ion exchange chromatography.     

Immune Evasion by Helicobacter pylori: Gastric Spiral Bacteria Lack Surface Immunoglobulin Deposition and Reactivity with Homologous Antibodies

Background. Helicobacter pylori infection persists in the presence of potent serum and gastric mucosal anti-body responses against bacterial antigens. The aim of this article is to report on a study determine whether there is antibody deposition on H. pylori in vivo in the stomach of infected patients and whether gastric and cultured forms of H. pylori differ in their antibody reactivity.
Materials and Methods. Serum, gastric biopsies, and antral brushings were obtained from 10 patients having endoscopy. H. pylori was cultured from gastric biopsies. Bacterial samples were stained directly for immunoglobulin deposition and indirectly using rabbit antiurease serum or patient serum. Samples were examined by immunofluorescence microscopy and flow cytometry.
Results. Although spiral bacteria could be identified easily by acridine orange staining and antiurease staining of gastric brushings from H. pylori infected patients, gastric bacteria did not have detectable IgG or IgA present, and only one of five samples could be stained for IgG and IgA indirectly using patient serum. In contrast, cultured bacteria could be stained readily with homologous serum for IgG and IgA in the majority of cases. Low pH inhibited immunoglobulin reactivity with cultured H. pylori.
Conclusions. Gastric H. pylori may evade humoral defense owing to poor deposition of immunoglobulin in the gastric environment or failure to express surface antigens that are present on cultured forms of H. pylori.     

Spatial patterns in a two-tiered semi-arid shrubland in southeastern Spain

Abstract. Single species and bivariate distribution patterns in a semi-arid shrubland in southeastern Spain, dominated by the tall leguminous shrub Retama sphaerocarpa, were investigated by second-order spatial analysis based on Ripley's K-function. Shrubs were significantly clumped because of a strong association of dwarf shrubs, mostly Artemisia barrelieri, under the canopy of Retama. Retama shrubs were randomly distributed, but when different size-classes were analysed separately, the pattern changed from significantly clumped to random and then to regular with increasing canopy diameter, suggesting increasing intraspecific competition with shrub size. Artemisia was significantly clumped at all scales because of aggregation under the canopy of large Retama shrubs. The association between the species became stronger with increasing canopy diameter of Retama shrubs, suggesting that facilitation prevailed over interspecific competition because of niche separation in different tiers, both above and below ground. Retama shrub size thus determined both the type of pattern for its own size class and tier, and the scale and intensity of the association with its understorey shrubs.