Primary culture of chondrocytes after collagenase IA or II treatment of articular cartilage from elderly patients undergoing arthroplasty

Background Joint replacement surgery provides articular cartilage samples for chondrocyte isolation. To our knowledge, the effect of the collagenase type on releasing of chondrocytes from the extracellular matrix of cartilage is not reported. Objectives To determine whether cartilage digested with collagenase IA yielded more chondrocytes than that digested with collagenase II and determine whether chondrocytes isolated with collagenase IA could be cultured in vitro. Methods Cartilage slices collected from 18 elderly patients who received joint replacement surgery (16 hips, 2 knees) were digested sequentially with 0.4% pronase E and 0.02% collagenase IA, or with 0.15% collagenase II alone, or sequentially with 0.4% pronase E and 0.02% collagenase II. We compared cell yield from each method. Cell viability by the most effective method was calculated and plotted. The morphology of cultured monolayer chondrocytes was recorded with a light microscope. Results Sequential digestion with pronase E and collagenase IA yielded 2566 ± 873 chondrocytes per mg wet cartilage, which was more effective than the other isolation methods (P = 0.018). The average chondrocyte viability could reach 84% ± 8% (n = 11). Light microscopic images showed typical chondrocyte morphology in monolayer cultures. Conclusion Sequential digestion of human articular cartilage with pronase E and collagenase IA was more effective than collagenase II alone or collagenase II combined with pronase E for releasing chondrocytes from extracellular matrix of cartilage. Chondrocytes isolated with this method could be maintained in monolayer cultures for at least 2 passages with unaltered morphology.

Serum phenoloxidase activity in the hemolymph of the anomuran crab Albunea symmysta (Linnaeus, 1758) (Decapoda: Anomura: Albuneidae)

We characterized the optimal conditions for measuring serum phenoloxidase activity and its functional activity and susceptibility to an inhibitor and various activators in an anomuran crab, Albunea symmysta (Linnaeus, 1758). The substrate affinity of the phenoloxidase (PO) enzyme was determined using different phenolic substrates in which only diphenols were found to be oxidized. The enzyme was characterized as a catecholoxidase-type of PO and 3,4-dihydroxy-DL-phenylalanine (DL-Dopa), the enzyme showing the highest substrate affinity to the serum. The optimal enzyme activity was observed at 5 mM DL-Dopa in 10 mM Tris-HCl buffer at a pH of 7.5 at 25 °C for 10 min, and absorbance at 470 nm. Serum-PO activity was inhibited by 7 mM phenylthiourea (PTU), and activated by activators such as trypsin, chymotrypsin, pronase-E, and detergent-like sodium dodecyl sulfate (SDS). We also identified the chemicals causing in vitro inhibition or activation of the enzyme as a serum of the crab having a potent PO activity.

An Internally Quenched Fluorescent Peptide Substrate for Protealysin

Protealysin, a metalloprotease of Serratia proteamaculans, is the prototype of a subgroup of the M4 peptidase family. Protealysin-like proteases (PLPs) are widely spread in bacteria but also occur in fungi and certain archaea. The interest in PLPs is primarily due to their putative involvement in the bacterial pathogenesis in animals and plants. Studying PLPs requires an efficient quantitative assay for their activity; however, no such assay has been reported so far. Here, we used the autoprocessing site sequence of the protealysin precursor to construct an internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ε-2,4-dinitrophenyl)lysine. Protealysin and thermolysin, the prototype of the M4 family, proved to hydrolyze only the Ser-Val bond of the substrate. The substrate exhibited a KM = 35 ± 4 μM and kcat = 21 ± 1 s−1 for protealysin as well as a KM = 33 ± 8 μM and kcat = 7 ± 1 s−1 for thermolysin at 37 °C. Comparison of the effect of different enzymes (thermolysin, trypsin, chymotrypsin, savinase, and pronase E) on the substrate has demonstrated that it is not strictly specific for protealysin; however, this enzyme has higher molar activity even compared to the closely related thermolysin. Thus, the proposed substrate can be advantageous for quantitative studies of protealysin as well as for activity assays of other M4 peptidases.

Efecto del uso combinado de la Pronasa E y de un extracto fúngico (Mucor racemosus forma sphaerosporus) en la maduración de embutidos crudos curados / Effect of the combined use of Pronase E and a fungal extract (Mucor racemosus forma sphaerosporus) on the ripening of dry fermented sausages

The effect of the addition of 600 units of Pronase E/kg of sausage mixture and/or a fungal extract ( Mucor racemosus forma sphaerosporus) at two different concentrations (1.75 and 17.5 mg protein/kg sausage mixture) on dry fermented sausages was evaluated. The only addition of protease to sau sages led to an increase in the content of free amino acids and biogenic amines. When the protease and the fungal extract were added together at high concentration (17.5 mg protein/kg sausage mix ture), a decrease in the free amino acid content and a slight increase in the ammonia content were observed, together with a clear increase in the concentration of several volatile compounds, such as 2- and 3-methylbutanal, 2-methylpropanal and 2- and 3-methyl-1-butanol. The addition of the fungal extract only at high concentration (17.5 mg protein/kg sausage mixture) or the combined protease and fungal extract at low concentration (1.75 mg protein/kg sausage mixture), produced slight physi cochemical modifications compared with the corresponding controls. All sausages with Pronase E showed a clear modification of texture, with a marked decrease of hardness, adhesiveness, springi ness, cohesiveness, gumminess, chewiness, cutting force and cutting work. Sensory analysis of the different batches of sausages showed that the combined addition of Pronase E (600 units/kg of sau sage mixture) and the Mucor racemosus forma sphaerosporus extract at high concentration (17.5 mg protein/kg of sausage mixture) improved the odor, taste, texture and, in consequence, the global acceptability in relation to the control batch.

Role of Surface Proteins in Vibrio cholerae Attachment to Chitin

ABSTRACT The role of surface proteins in Vibrio choleraeattachment to chitin particles in vitro was studied. Treatment ofV. cholerae O1 ATCC 14034 and ATCC 14035 with pronase E reduced the attachment of bacteria to chitin particles by 57 to 77%. A statistically significant reduction was also observed when the attachment to chitin was evaluated in the presence of homologous Sarkosyl-insoluble membrane proteins (MPs) (67 to 84%),N-acetylglucosamine (GlcNAc) (62%), the sugar that makes up chitin, and wheat germ agglutinin (40 to 56%), a lectin that binds GlcNAc. The soluble oligomersN,N′-diacetylchitobiose orN,N′,N"-triacetylchitotriose caused an inhibition of 14 to 23%. Sarkosyl-insoluble MPs able to bind chitin particles were isolated and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; two of these peptides (molecular sizes, 36 and 53 kDa) specifically bind GlcNAc.