Gelatin degradation at elevated temperature

Four gelatin types (A, B, C and AB), two different samples of each, were subjected to temperature treatments with the incubation temperature, incubation time, gelatin concentration and solvent (type and concentration of salt ions and pH) as variables. Degradation was studied by means of fast protein liquid chromatography and sodium dodecylsulphate polyacrylamide gel electrophoresis. All the variables tested seemed to be critical.

Addition of a protease inhibitor cocktail confirmed that the observed degradation was not due to the action of proteases.

Fluorescence measurements indicated that during the temperature treatment pentosidine and pyridinoline cross-links can be broken, while the cleavage of peptide bonds was verified by ninhydrin tests and N-terminal amino-acid analyses with phenyl isothiocyanate.     

A rapid solid-phase fluorimetric assay for measuring bacterial adherence,using DNA-binding stains

In this report, we describe the validation of a rapid, single-step, microtiter plate method for quantifying bacterial adherence, based on fluorescent labeling of microorganisms with cell-permeable fluorescent DNA-binding probes. We have tested the binding to saliva-coated microtiter plates of bacteria, including Helicobacter pylori and viridans streptococci (S. mitis, S. gordonii, S. sanguis), known to interact with salivary components. Furthermore, we tested the short-term and longer-term temporal stability of a saliva-mediated adherence of these bacteria in a healthy population (N=30). The assay exhibited excellent reliability statistics, yielding within-assay variability coefficients ranging from 4.9% to 11%. A range of approximately 5 x 10(4)-1 x 10(7) cells could be detected. This method may be generally applicable to study surface binding of virtually any microbial species, while obviating the need of radioactive materials or specific antibodies for quantification, thus providing a procedure that is useful to both basic and clinical research.     

Neutrophil adhesion is impaired in the mesentery but not in the liver sinusoids of portal hypertensive rats

Altered leukocyte/cytokine response to inflammation has been observed in human and experimental portal hypertension. The aim of this study was to characterize leukocyte adhesion in portal hypertensive (PPVL) rats stimulated with endotoxin. Leukocyte rolling, adhesion, and migration assessed by intravital microscopy were impaired in mesenteric venules after lipopolysaccharide administration (150 microg/kg) in PPVL vs. sham-operated rats. Analysis of leukocyte L-selectin expression and soluble L-selectin showed that this defective adhesion was related to increased L-selectin shedding. In vitro experiments using isolated leukocytes treated with phorbol 12-myristate 13-acetate showed that monocytes and neutrophils but not lymphocytes were hyperreactive to cell activation, as measured by CD11b overexpression and increased L-selectin shedding in PPVL rats. However, neutrophil emigration in liver sinusoids and in the lung 3 h after endotoxin injection were similar in both groups of animals. Thus the alterations in leukocyte activation and adhesion molecule expression observed in this study may contribute to a better understanding of the higher susceptibility and severity of bacterial infections in cirrhotic patients with portal hypertension.     

Adsorption properties of proteins at and near the air/water interface from IRRAS spectra of protein solutions

A detailed study is performed using infrared reflection absorption spectroscopy (IRRAS) to characterize the molecular behaviour of proteins at and near the air/water interface of protein solutions. IRRAS spectra of beta-casein solutions in H2O and D2O show spectral shifts and derivative-like features not commonly observed in monomolecular layer systems. They can be fully understood using optical theory. Fair agreement between experimental and simulated IRRAS spectra over a broad spectral range (4000-1000 cm(-1)) is obtained using a stratified layer model. An attenuated total reflection and transmission spectrum is used to represent the protein extinction coefficient in H2O and D2O, respectively. It is shown that the derivative-like features observed result from the reflective properties of the proteins themselves. Furthermore, both concentration and film thickness could be fitted. At high protein concentrations (100 mg/mL) the spectrum is that of a single homogeneous protein solution. At 0.1 mg/mL, beta-casein is accumulated at the surface in a thin layer of approximately 10 nm thickness, with a concentration about 2500 times higher than in the sub-phase. At an initial concentration of 10 mg/mL, the concentration in the surface layer is about 15 times higher than in the subphase, while the thickness is about 30 nm.     

Class III pistil-specific extensin-like proteins from tobacco have characteristics of arabinogalactan proteins

Class III pistil-specific extensin-like proteins (PELPIII) are specifically localized in the intercellular matrix of tobacco (Nicotiana tabacum) styles. After pollination the majority of PELPIII are translocated into the callosic layer and the callose plugs of the pollen tubes, which could suggest a function of PELPIII in pollen tube growth. PELPIII may represent one of the chemical and/or physical factors from the female sporophytic tissue that contributes to the difference between in vivo and in vitro pollen tube growth. PELPIII glycoproteins were purified and biochemically characterized. Because of their high proline (Pro) and hydroxy-Pro (Hyp) content, PELPIII proteins belong to the class of Pro/Hyp-rich glycoproteins. The carbohydrate moiety of PELPIII is attached through O-glycosidic linkages and comprises more than one-half the total glycoprotein. Deglycosylation of PELPIII revealed two backbones, both reacting with PELPIII-specific antibodies. N-terminal amino acid sequencing of these backbones showed that PELPIII is encoded by the MG14 and MG15 genes. Two heterogeneous N-terminal sequences of MG14 and MG15, both starting downstream of the predicted signal peptide cleavage site, seem to be present, which indicates a novel N-terminal processing. Monosaccharide analysis showed that the carbohydrate moiety of PELPIII almost completely consists of arabinose and galactose in an equal molar ratio. Carbohydrate linkage analysis showed terminal and 2-linked arabinofuranosyl residues, as well as terminal and 6-, 3-, and 3,6-linked galactopyranosyl residues to be present, indicating the presence of both extensin-like and Type II arabinogalactan oligosaccharide units. The ability of beta-glucosyl Yariv reagent to bind with PELPIII confirmed the arabinogalactan protein-like characteristics of these proteins.     

Mathematical conservation ecology: A one-predator-two-prey system as case study

A method is presented to analyse the long-term stochastic dynamics of a biological population that is at risk of extinction. From the full ecosystem the method extracts the minimal information to describe the long-term dynamics of that population by a stochastic logistic system. The method is applied to a one-predator-two-prey model. The choice of this example is motivated by a study on the near-extinction of a porcupine population by mountain lions whose presence is facilitated by mule deer taking advantage of a change in land use. The risk of extinction is quantified by the expected time of extinction of the population.