Application potency of engineered G159 mutants on P1 substrate pocket of subtilisin YaB as improved meat tenderizers

A serine protease, subtilisin YaB, produced by alkalophilic Bacillus YaB, shows promises as a potent meat tenderizer, because its substrate specificity is for small amino acids, which are found at high levels in meat connective tissue proteins. Substrate specificity engineering of the substrate binding pockets was used to generate more suitable meat-tenderizing mutants, G124A, G124V, G159A, and G159S, derived from recombinant wild subtilisin YaB and expressed in Bacillus subtilis DB104. The characteristics of these recombinant enzymes were studied to evaluate their usefulness as improved meat tenderizers. The proteolytic activities of recombinant subtilisin YaB, engineered subtilisin YaBs, and commercially available papain, bromelain, collagenase, and elastase were compared using elastin, collagen, casein, and myofibrillar proteins as substrates. Hydrolysis of beef proteins was evaluated using the myofibrillar fragmentation index and collagen solubility. The results demonstrated that recombinant mutant G159A was the most improved meat tenderizer and can be used in the meat pH range of 5.5-6.0 and the temperature range of 10-50 degrees C. Contrary to the result obtained from artificial substrate, mutant enzymes engineered on G124 residues did not exhibit better tenderizing ability when elastin, collagen, or meat was used as substrate, suggesting the necessity of evaluation by real substrate before protein-engineered enzymes are applied commercially.     

The dispersal of Culicoides brevitarsis in eastern New South Wales and associations with the occurrences of arbovirus infections in cattle

Distributions of the vector Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae) (determined from light trap data) and 2 arboviruses (determined from seroconversions in sentinel cattle) were studied in eastern New South Wales in 1993–94. C brevitarsis was recorded progressively from endemic areas on the north coast, to Nowra on the south coast, and westward to Scone, in the Hunter Valley. C brevitarsis also survived through winter at Paterson, in the Hunter Valley. Its apparently focal reappearance in this marginal area had no obvious effect on the broad pattern of its progression or the dispersal of Akabane and bluetongue viruses. These viruses were first recorded from foci near Coffs Harbour, on the mid-north coast. Their first occurrences at different locations were associated with those of C brevitarsis, but not with each other. The viruses were found only within the recorded limits of the vector's distribution. Delays between the initial occurrence of C brevitarsis and first evidence of virus transmissions at locations ranged from 2 to 7 months. The delays decreased away from the points of focus and were negatively associated with the time of initial occurrence of the vector. Seroconversions to the viruses were related to the presence of C brevitarsis. However, the densities of C brevitarsis had no apparent effect on the initial numbers of cattle seroconverting to either virus. The results support the conclusion that the progressions of C brevitarsis and Akabane and bluetongue viruses were the result of gradual movements by the vector.     

Interferon induction in dogs.

A synthetic complex of poly (I)-poly (C) with poly-L-lysine and carboxymethyl cellulose (poly ICLC), as well as UV-inactivated Newcastle disease virus, B-1 strain, was used to induce interferon production in dogs. Several criteria were used for interferon specificity. The interferon response depended on dosage and route of inoculation. Serum interferon concentrations usually reached a peak by 8 hours after inoculation (AI), rapidly declined thereafter, and were nondetectable in most instances at 24 hours AI. Dogs responded less to interferon inducers when reinoculated 2 days after primary induction. The interferon response was biphasic (2 and 8 hours AI) when reinoculated 1 week after primary inoculation. Reinoculation 2 weeks after primary inoculation simulated the first response. Although both inducers caused severe lymphopenia in dogs, the toxic side effects would limit clinical use (in dogs) of poly ICLC, but not of UV-inactivated Newcastle disease virus.     

Amantadine: neuromuscular blockade by suppression of ionic conductance of the acetylcholine receptor

Amantadine hydrochloride decreases the sensitivity of denervated mammalian muscle to iontophoretically applied acetylcholine. The drug depresses the amplitude of the end-plate current and reverses the slope of the relation between half-decay time and membrane potential suggesting that it alters the ionic conductance that is mediated by the acetylcholine receptor. Binding studies confirm that amantadine acts on the ion conductance modulator rather than the acetylcholine receptor.     

Tipburn of Chinese cabbage in relation to calcium nutrition and distribution

Heading Chinese cabbage (Brassica campestris ssp. pekinensis Rupr.) grown with low Ca or B developed dark tan lesions near the leaf margins. A synergistic effect of low Ca and low B accompanied the development of this symptom. Plants grown under humid conditions were free of tipburn. Plants grown under less humidity developed tipburn. Total chemical analysis revealed a steady decrease in total Ca from the outer to the inner leaves. Fractional chemical analysis showed 7 times more water-soluble Ca in the outer than in the inner leaves, and 2 times more 1 N NaCl-soluble Ca in the outer than in inner leaves. Threshold concentration of water-soluble forms of Ca for tipburn was around 3.0 mg/g dry weight. Data suggest that root pressure flow is required to move adequate amounts of water-soluble Ca to the wrapper leaves to prevent tipburn of Chinese cabbage.     

Evaluation of head-out constant volume body plethysmography for measurement of specific airway resistance in conscious, sedated sheep

OBJECTIVE: To evaluate the use of a modified whole body plethysmograph in awake sheep. ANIMALS: 10 healthy adult sheep. PROCEDURE: Concurrent measurements of specific airway resistance (sR(aw)) and pulmonary resistance (R(L)) were obtained using a novel noninvasive head-out constant-volume plethysmograph and esophageal balloon-pneumotachography, respectively. All data were collected before and after external resistive loading with 1 and 5.6 cm H2O/L/s. Functional residual capacity (FRC) was measured by helium dilution for computation of airway resistance (R(aw)) preloading (R(aw) = sR(aw)/FRC). RESULTS: The sR(aw) and R(L) were closely correlated in 10 adult sheep. Additionally, sR(aw), and R(L) accurately reflected the magnitude of added resistance. The mean FRC was 52 mL/kg and used to calculate R(aw). At baseline, the values for R(aw) were significantly correlated with sR(aw) and R(L). CONCLUSIONS AND CLINICAL RELEVANCE: Precise measurements of sR(aw) and R(aw) at baseline and sR(aw) after external resistive loading were obtained by use of this novel noninvasive plethysmographic technology. This method should have application to veterinary patients or animals used in research in which noninvasive rapid or serial measurements of sR(aw) in the conscious state are required.     

The embryonic vertebrate heart tube is a dynamic suction pump

The embryonic vertebrate heart begins pumping blood long before the development of discernable chambers and valves. At these early stages, the heart tube has been described as a peristaltic pump. Recent advances in confocal laser scanning microscopy and four-dimensional visualization have warranted another look at early cardiac structure and function. We examined the movement of cells in the embryonic zebrafish heart tube and the flow of blood through the heart and obtained results that contradict peristalsis as a pumping mechanism in the embryonic heart. We propose a more likely explanation of early cardiac dynamics in which the pumping action results from suction due to elastic wave propagation in the heart tube.     

Phenotypic and molecular characterization of isolates of Ornithobacterium rhinotracheale from chickens and pigeons in Taiwan

Forty Ornithobacterium rhinotracheale (ORT) strains were isolated from 28 chickens and 12 pigeons for the first time in Taiwan. All isolates reacted positively in the p-nitrophenyl-beta-D-galactopyranoside (PNPG) and oxidase tests, showing an API 20NE identification system biocode 0-0-2-0-0-0-4. All the pigeon isolates and 85.7% (24 of 28) of the chicken isolates belonged to serotype A. Compared to the ORT ATCC 51464 strain, 14.3% (4 of 28) of chicken isolates and 58.3% (7 of 12) of pigeon isolates showed smaller colonies after 72 hr incubation. Most of the chicken isolates (22 of 28), but none of the pigeon isolates, could agglutinate chicken and pigeon red blood cells. There appears to be a correlation that ORT isolates with a larger colony size tend to be more able to agglutinate red blood cells than the ORT isolates with a smaller colony size. A majority of isolates was sensitive to amoxicillin, ampicillin, ceftiofur, penicillin, and oxytetracycline. The 16S ribosomal RNA (rRNA) sequences of 23 Taiwanese ORT isolates showed high identity (98%-100%) to sequences in GenBank. Phylogenetic analysis of these sequences showed that pigeon isolates formed a distinctive cluster, while chicken isolates and all other 16S rRNA sequences obtained from GenBank belonged to another two clusters. The results indicate that pigeon ORT isolates are different from most chicken isolates in regard to a number of phenotypic and molecular traits.     

Earthquake in a maze: compressional rupture branching during the 2012 M(w) 8.6 Sumatra earthquake

Seismological observations of the 2012 moment magnitude 8.6 Sumatra earthquake reveal unprecedented complexity of dynamic rupture. The surprisingly large magnitude results from the combination of deep extent, high stress drop, and rupture of multiple faults. Back-projection source imaging indicates that the rupture occurred on distinct planes in an orthogonal conjugate fault system, with relatively slow rupture speed. The east-southeast-west-northwest ruptures add a new dimension to the seismotectonics of the Wharton Basin, which was previously thought to be controlled by north-south strike-slip faulting. The rupture turned twice into the compressive quadrant, against the preferred branching direction predicted by dynamic Coulomb stress calculations. Orthogonal faulting and compressional branching indicate that rupture was controlled by a pressure-insensitive strength of the deep oceanic lithosphere.