Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin.

We have previously shown that replacing the P1-site residue (Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lys results in the acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively. However, the inhibitory activities thus induced are not strong. In the present study, we introduced additional amino acid replacements around the reactive site to try to make the P1-site mutants more effective inhibitors of chymotrypsin or trypsin. The amino acid replacement Asp-->Tyr at the P2' site of OMCHI3(P1Met) resulted in conversion to a 35000-fold more effective inhibitor of chymotrypsin with an inhibitor constant (K(i)) of 1. 17x10(-11) M. The K(i) value of OMCHI3(P1Met, P2'Ala) indicated that the effect on the interaction with chymotrypsin of removing a negative charge from the P2' site was greater than that of introducing an aromatic ring. Similarly, enhanced inhibition of trypsin was observed when the Asp-->Tyr replacement was introduced into the P2' site of OMCHI3(P1Lys). Two additional replacements, Asp-->Ala at the P4 site and Arg-->Ala at the P3' site, made the mutant a more effective inhibitor of trypsin with a K(i) value of 1. 44x10(-9) M. By contrast, Arg-->Ala replacement at the P3' site of OMCHI3(P1Met, P2'Tyr) resulted in a greatly reduced inhibition of chymotrypsin, and Asp-->Ala replacement at the P4 site produced only a small change when compared with a natural variant of OMCHI3. These results clearly indicate that not only the P1-site residue but also the characteristics, particularly the electrostatic properties, of the amino acid residues around the reactive site of the protease inhibitor determine the strength of its interactions with proteases. Furthermore, amino acids with different characteristics are required around the reactive site for strong inhibition of chymotrypsin and trypsin.     

Physiological responses of exercised-fatigued individuals exposed to wet-cold conditions.

Thirteen healthy and fit men [age = 27 +/- 8 (SD) yr, height = 177 +/- 5 cm, mass = 75 +/- 7 kg, body fat = 14 +/- 5%, maximal O2 consumption = 51 +/- 4 ml. kg-1. min-1] participated in an experiment designed to test their thermoregulatory response to a challenging cold exposure after 5 h of demanding mixed exercise during which only water was consumed. Subjects expended 7,314 +/- 741 kJ on cycling, rowing, and treadmill-walking machines, performed 8,403 +/- 1,401 kg. m of mechanical work during resistance exercises, and completed 120 inclined sit-ups. Subjects then assumed a seated position in a 10 degrees C air environment while wearing shorts, T-shirt, rain hat, and neoprene gloves and boots. After 30 min the subjects were showered continuously with cold water ( approximately 920 ml/min at 10 degrees C) on their backs accompanied by a 6 km/h wind for up to 4 h. Blood samples were taken from the nondominant arm every 30 min during the exposure and assayed for energy metabolites, hormones, indexes of hydration, and neurotransmitters. Counterbalanced control trials without prior exercise were also conducted. Blood insulin was higher during the control trial, whereas values of glycerol, nonesterified fatty acids, beta-hydroxybutyrate, lactate, cortisol, free triiodothyronine, and thyroxine were lower. Three subjects lasted the maximum duration of 4.5 h for control and fatigue trials, with final rectal temperatures of 36.43 +/- 0.21 and 36.08 +/- 0.49 degrees C, respectively. Overall, the duration of 172 +/- 68 (SD) min for the fatigue trial was not significantly different from that of the control trial (197 +/- 72 min) and, therefore, was not affected by the preexposure exercise. Although duration was positively correlated to body fatness and shivering intensity, the latter was not correlated to any physical characteristic or the fitness level of the individual.     

The role of the cysteine-rich region of the β2 integrin subunit in the leukocyte function-associated antigen-1 (LFA-1, αLβ2, CD11a/CD18) heterodimer formation and ligand binding

The cysteine-rich region (CRR) of the β2 integrin subunit was replaced by that of β1 to give the chimera β2NV1. β2NV1 can combine with αL to form a variant leukocyte-function-associated antigen (LFA)-1 on COS cell surface, suggesting that the specificity of the β2 interaction with αL does not lie in the CRR. Unlike those expressing wild-type LFA-1, COS cells expressing αLβ2NV1 are constitutively active in intercellular adhesion molecule (ICAM)-1 adhesion. These results suggest that activation of LFA-1 involves the release of an intramolecular constraint, which is maintained, in part, by the authentic β2 CRR.     

A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc-5 interacts genetically with mig-2, which encodes Rho GTPase. These results suggest that EXC-5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.     

Effect of various methods of immobilization on the stability of a microbial biosensor for surfactants based onPseudomonas rathonis T

The operating and storage stability of a receptor element of an amperometric biosensor based on thePseudomonas rathonis strain T capable of degrading surfactants was tested. Microbial cells were immobilized by incorporation in gels (agar, agarose, and calcium-alginate), polyvinyl alcohol membrane, adhesion to Chromatographic paper GF/A, or by cross-linking induced by glutaric aldehyde. Incorporation of microbial cells in agar gel provides long-standing conservation of their activity and viability during measurements of high concentrations of surfactants and allows the receptor element of the biosensor to be rapidly recovered after measurements.     

Identification of the structure of large-scale MHD perturbations in a tokamak from Mirnov signals

An algorithm is proposed that allows one to identify the MHD mode structure in toroidal plasmas by processing signals from Mirnov probes measuring plasma MHD activity. The algorithm differs fundamentally from the diagnostic methods presently used in tokamaks, being simpler and more efficient. The algorithm is based on constructing an analytic signal using the Hilbert transformation of the Mirnov signals at a given instant. The phase and amplitude dependences obtained take into account the toroidal effects and allow one to determine the number and amplitude of the excited MHD mode. The algorithm was approbated with both test signals and actual signals from MHD diagnostics in the T-10 tokamak. It is demonstrated that the algorithm can be used to analyze single-mode MHD instabilities in toroidal plasmas.     

Interrelation of the changes in the blood rheological properties and the microcirculatory disorders in the early and late stages of experimental hyperlipoproteinemia

Experiments were made on rabbit fed an atherogenic diet (0.5 g/kg cholesterol) singly for 15 and 24 h and repeatedly for 3, 9 and 30 days. At early stages of lipid metabolism distress the interrelationship was established between blood rheological disorders and microcirculatory abnormalities. The dependence of the initial reaction of some rheological characteristics on their initial level was marked.     

Nitrogen fertigation of greenhouse-grown cucumber

Summary This greenhouse study investigated the response of trickle-irrigated cucumber (Cucumis sativa cv. ‘Petita’) to three N levels applied with every irrigation via the irrigation stream. The plants were grown in pots filled with 12 kg of soil. Water containing 5.8, 11.8, or 17.8 mmol N/l, and uniformly supplied with 2.0 and 3.9 mmol/l of P and K, respectively, was applied two to three times daily. In all treatments of 0.3 leaching fraction was allowed. The resulting total N applications were 15.7, 31., and 47.2 g N/plant. The total amount of water applied was 1851/plant. Total N and NO₃-N, in lajinae and petioles, increased with increasing N level whereas P and K in generated decreased. Although different NO₃/NH₄ ratios in the treatments may have influeced the response to N, it could be concluded that the highest yield was obtained with 11.8 mmol N/1 due to increased number of fruit. In the root volume of this treatment the NO₃-N concentration in the soil solution was aroun 7 mmol/1 for most of the growing season. The dry matter concentration of fruits was not affected by the N levels. It was concluded that 11.8 mmol N/1 applied with every irrigation via the irrigation stream is adequate to cover the needs of greenhous-grown cucumber for higher yield (9.42 kg/plant over a harvesting period of 93 days).