Purification of carbonic anhydrase isozyme VI (CA-VI) from swine saliva.

Salivary or secreted carbonic anhydrase (CA), which constitutes a new class of CA, designated CA-VI, was isolated. Swine CA-VI purified from swine saliva by inhibitor-affinity chromatography and ion exchange chromatography had a specific activity of 5,468 units/mg. The molecular weight was 250,000, as determined by gel filtration under non-denaturing conditions, and the subunit molecular weight was found to be 37,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that swine CA-VI consists of 7 subunits. The treatment of the enzyme with endo-N-acetylglucosaminidase F reduced its subunit molecular weight from 37,000 to 35,000 and 32,000. We raised a rabbit antibody against purfied swine CA-VI. Double immunodiffusion showed that anti-swine CA-VI serum reacted with swine CA-VI and swine saliva, but not with hemolysate (containing CA-I and CA-Il) or muscle extracts (containing CA-III). The concentration of CA-VI in swine saliva, measured using single radial immunodiffusion, was 0.027 +/- 0.017 mg/mg total protein.     

Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.

Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.     

Functional properties of oat globulin modified by a calcium-independent microbial transglutaminase

Oat globulin was modified by a calcium-independent microbial transglutaminase (TG). The TG-polymerized protein had higher solubility than the control at acidic pH and had improved water- and fat-binding properties. Incubation of 10% (w/v) oat globulin dispersions in the presence of TG at 37 degrees C led to the formation of a well-developed viscoelastic gel network with a microstructure characterized by thick strands and large clusters. The TG-induced gels had higher modulus values, lower loss tangent values, and lower frequency dependency than the heat-induced gels. The TG-induced gel system has the characteristics of classical polymer gel with permanent "chemical" cross-links, whereas the heat-denatured system has the characteristics of a temporary "physical" gel with breakable cross-links. Fourier transform infrared spectroscopy showed marked shift and intensity changes in several major bands, suggesting pronounced changes in protein conformation during TG-induced gelation. Aggregation of protein molecules was also indicated by the progressive increases in two infrared bands (1679-1682 and 1622-1625 cm(-)(1)) associated with the formation of intermolecular beta-sheets and strands. Results suggest that new food polymers with unique functionality can be produced from oat globulin treated with TG and that elastic gels can be formed near neutral pH, instead of the alkaline pH required for thermally induced oat globulin gels.     

Depth perception from image defocus in a jumping spider

The principal eyes of jumping spiders have a unique retina with four tiered photoreceptor layers, on each of which light of different wavelengths is focused by a lens with appreciable chromatic aberration. We found that all photoreceptors in both the deepest and second-deepest layers contain a green-sensitive visual pigment, although green light is only focused on the deepest layer. This mismatch indicates that the second-deepest layer always receives defocused images, which contain depth information of the scene in optical theory. Behavioral experiments revealed that depth perception in the spider was affected by the wavelength of the illuminating light, which affects the amount of defocus in the images resulting from chromatic aberration. Therefore, we propose a depth perception mechanism based on how much the retinal image is defocused.     

Low-magnetic-field control of electric polarization vector in a helimagnet

The mutual control of the electric and magnetic properties of a solid is currently of great interest because of the possible application for novel electronic devices. We report on the low-magnetic-field (for example, B values of +/-30 milliteslas) control of the polarization (P) vector in a hexaferrite, Ba2Mg2Fe12O22, which shows the helimagnetic spin structure with the propagation vector k0 parallel to [001]. The B-induced transverse conical spin structure carries the P vector directing perpendicular to both B and k0, in accord with the recently proposed spin-current model. Then, the oscillating or multidirectionally rotating B produces the cyclic displacement current via the flexible handling of the magnetic cone axis.     

Differences in lectin-binding properties between the common mucosal epithelium and follicle-associated epithelium in the rabbit small intestine

Differences in sugar distribution between the villous epithelium and follicle-associated epithelium (FAE) were compared using lectins in the rabbit small intestine. In every portion, villous columnar epithelial cells primarily exhibited a positive reaction to the GalNAc, GlcNAc, galactose, and oligosaccharide. In the ileal Peyer's patch (PP), whereas microvillous epithelial cells exhibited positive reactions, M cells tended to be negative. The villous epithelial reaction to the fucose group was negative, but M cells and microvillous epithelial cells showed a positive to the fucose. No epithelium had a positive reaction to the mannose and glucose. The variety of lectin-binding properties of villous epithelial cells and M cells may reflect specificity for the recognizing luminal substances such as antigenic molecules and bacterial elements.     

Relationship between the ultrasonographic findings of suspected superficial digital flexor tendon injury and the prevalence of subsequent severe superficial digital flexor tendon injuries in Thoroughbred horses: a retrospective study

The onset of severe injury to the superficial digital flexor tendon (SDFT) is extremely difficult to predict from slight changes in ultrasonographic findings in cases with no apparent clinical signs. This study investigated the relationship between an increased cross-sectional area (CSA) or edema in the subcutaneous tissue around the tendon and the subsequent onset of severe SDFT injury in Thoroughbred racehorses. Horses were classified into three groups based on ultrasound diagnosis (USD) findings: Group A included cases with enlarged tendons; Group B included cases with tendons of normal size but with prominent edema in the peritendinous tissue; and Group C (control group) included cases with no abnormal USD findings. The incidence of subsequent severe tendon injury was significantly higher in the horses in Groups A (25.7%, 28/101) and B (28.3%, 65/212) than in those in Group C (4.9%, 2/41). There were no significant differences in the median period and the median number of races from the first examination to the subsequent tendon injury between Groups A (140 days, 1 race) and B (120 days, 1 race). The results of this study revealed that horses with increased CSA and peritendinous edema are likely to suffer a subsequent severe tendon injury. Also, these two USD findings, i.e., increased CSA and peritendinous edema, indicate the risk of onset of severe SDFT injury.     

Novel fibrinolytic enzyme in fermented shrimp paste, a traditional asian fermented seasoning

A novel fibrinolytic enzyme was purified from fermented shrimp paste, a popular seasoning used in Asian countries. The enzyme is a monomer with an apparent molecular weight of 18 kDa, and it is composed primarily of beta-sheet and random coils. The N-terminal amino acid sequence was determined to be DPYEEPGPCENLQVA. It is a neutral protease with an optimal activity from pH 3 to 7. No inhibition was observed with PMSF, Pepstatin A, E64, and 1,10-phenanthroline, but the enzyme was slightly inhibited by EDTA and Cu(2+). It was relatively specific to fibrin or fibrinogen as a protein substrate, yet it hydrolyzed none of the plasma proteins in the studies. In vitro, the enzyme was resistant to pepsin and trypsin digestion. It also had an anticoagulant activity measured with activated partial thrombin time and prothrombin time tests. The novel fibrinolytic enzyme derived from traditional Asian foods is useful for thrombolytic therapy. In addition, this enzyme has a significant potential for food fortification and nutraceutical applications, such that its use could effectively prevent cardiovascular diseases.     

Antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme

This work was aimed at the isolation, purification, and characterization of novel antimicrobial peptides from chicken egg white lysozyme hydrolysate, obtained by peptic digestion and subsequent tryptic digestion. The hydrolysate was composed of over 20 small peptides of less than 1000 Da, and had no enzymatic activity. The water-soluble peptide mixture showed bacteriostatic activity against Gram-positive bacteria (Staphylococcus aureus 23-394) and Gram-negative bacteria (Escherichia coli K-12). Two bacteriostatic peptides were purified and sequenced. One peptide, with the sequence Ile-Val-Ser-Asp-Gly-Asp-Gly-Met-Asn-Ala-Trp, inhibited Gram-negative bacteria E. coli K-12 and corresponded to amino acid residues 98-108, which are located in the middle part of the helix-loop-helix. Another novel antimicrobial peptide inhibited S. aureus 23-394 and was determined to have the sequence His-Gly-Leu-Asp-Asn-Tyr-Arg, corresponding to amino acid residues 15-21 of lysozyme. These peptides broadened the antimicrobial activity of lysozyme to include Gram-negative bacteria. The results obtained in this study indicate that lysozyme possesses nonenzymatic bacteriostatic domains in its primary sequence and they are released by proteolytic hydrolysis.     

Isolation and measurement of carbonic anhydrase isoenzymes in erythrocytes of dogs

OBJECTIVE: To purify canine carbonic anhydrase (CA) isoenzymes CA-I and CA-II and to determine concentrations of CA-I and CA-II in erythrocytes of Beagles and dogs native to Japan. SAMPLE POPULATION: Blood samples from 116 Beagles, including 24 pregnant Beagles, and blood samples from 29 dogs native to Japan. PROCEDURE: Canine CA-I and CA-II were purified by use of column chromatography. Concentrations of CA-I and CA-II in erythrocytes of dogs were determined, using an ELISA. RESULTS: Mean (+/- SD) concentrations of CA-I and CA-II in erythrocytes of Beagles were 3.21+/-0.86 and 1.63+/-0.39 mg/g of Hb, respectively. Mean concentration of CA-I was greater in male Beagles than female Beagles. In contrast, mean concentration of CA-II was greater in female Beagles than male Beagles. Furthermore, concentration of CA-II was greater in pregnant female Beagles than male or nonpregnant female Beagles. Mean concentrations of CA-I and CA-II in erythrocytes of dogs native to Japan were 11.03+/-4.39 and 3.29+/-0.91 mg/g of Hb, respectively. Mean concentration of CA-I was greater in male dogs from Japan than female dogs from Japan. CONCLUSIONS AND CLINICAL RELEVANCE: The ELISA used in this study proved to be precise and sensitive for determining CA-I and CA-II concentrations in dogs. The ELISA may enable study of changes in isoenzymes associated with hereditary or metabolic disorders of blood or other body fluids, using only a small sample. Measurement of the concentrations of CA isoenzymes in dogs may be of diagnostic value.