Protein engineering of chymosin; modification of the optimum pH of enzyme catalysis

The aspartic proteinase chymosin exhibits a local network ofhydrogen bonds involving the active site aspartates and surroundingresidues which may have an influence on the rate and optimalpH of substrate cleavage. We have introduced into chymosin Bthe following substitutions: Asp304 to Ala (D304A), Thr218 toAla (T218A) and Gly244 to Asp (G244D, chymosin A), using oligonucleotide-directedmutagenesis. Kinetic analysis of these active mutants showsshifts in their pH optima to 4.4 D304A, 4.2 T218A and 4.0 G244Dcompared with 3.8 for chymosin B using a synthetic octapeptidesubstrate. The upward shift of the D304A and T218A may be dueto the loss of hydrogen bond interactions indirectly affectingthe catalytic aspartates 32 and 215. The G244D mutation whichis in a flexible loop on the surface of the enzyme may alterthe conformation of the specificity pockets on the prime sideof the scissile bond.     

Fresnel transform phase retrieval from magnitude

This report presents a generalized projection method for recovering the phase of a finite support, two-dimensional signal from knowledge of its magnitude in the spatial position and Fresnel transform domains. We establish the uniqueness of sampled monochromatic scalar field phase given Fresnel transform magnitude and finite region of support constraints for complex signals. We derive an optimally relaxed version of the algorithm resulting in a significant reduction in the number of iterations needed to obtain useful results. An advantage of using the Fresnel transform (as opposed to Fourier) for measurement is that the shift-invariance of the transform operator implies retention of object location information in the transformed image magnitude. As a practical application in the context of ultrasound beam measurement we discuss the determination of small optical phase shifts from near field optical intensity distributions. Experimental data are used to reconstruct the phase shape of an optical field immediately after propagating through a wide bandwidth ultrasonic pulse. The phase of each point on the optical wavefront is proportional to the ray sum of pressure through the ultrasound pulse (assuming low ultrasonic intensity). An entire pressure field was reconstructed in three dimensions and compared with a calibrated hydrophone measurement. The comparison is excellent, demonstrating that the phase retrieval is quantitative.     

Modulation of ovarian cytochrome P450 17 alpha-hydroxylase and cytochrome aromatase messenger ribonucleic acid by prolactin in the domestic turkey

The effect of exogenous ovine prolactin (oPRL) on preovulatory follicle P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA abundance was investigated in turkeys. Ovine PRL (124 IU/hen per day) was injected i.m. into four sets (n = 8) of laying turkeys for 2, 4, 8, or 14 days. Vehicle was injected into control hens for 8 days (n = 8). Blood samples were collected and serum was assayed for LH, progesterone (P), testosterone (T), and estradiol (E). Theca layers from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest preovulatory follicles and from small white follicles (SWF) were examined for C17 and ARO mRNA contents. The number of atretic follicles increased from 0 (vehicle-injected controls) to 9 (14-day-oPRL-injected hens). Serum E, T, and LH levels decreased, while P levels remained unchanged. There was a transient increase in theca C17 mRNA abundance of 2- and 4-day-oPRL-treated hen follicles. Cytochrome P450 ARO mRNA levels were reduced in SWF and F7 in response to oPRL. Thecal C17 and ARO mRNA content was reduced during follicular maturation in laying hens. ARO mRNA was not detectable in granulosa cells. The progressive decline in C17 and ARO mRNA content associated with follicular maturation as well as the absence of ARO mRNA in granulosa cells is consistent with the secretory activity of P, T, and E in preovulatory follicles. These findings suggest that reduced circulating E may be a consequence of suppressed ARO gene expression whereas the oPRL suppression of T secretion may not be coupled to C17 gene expression.     

Approximation to M/D/1 for ATM CAC, buffer dimensioning and cellloss performance

The authors derive a new, accurate, closed form approximation for the M/D/I queue. This can be arranged to yield expressions for the cell loss probability, the admissible load and the buffer length. These expressions have a direct application to cell scale queueing in ATM networks     

Tissue plasminogen activator, plasminogen activator inhibitor-1, and fibrin as indexes of clinical course in cardiac allograft recipients. An immunocytochemical study

BACKGROUND: Tissue-type plasminogen activator (TPA) is the principal activator of plasminogen. Since hemostasis in the microcirculation of allografts is a well-recognized complication of transplantation, we asked (1) whether the distribution and amount of cellular TPA in biopsies of transplanted human hearts are associated with fibrin deposits in and around the microcirculation, (2) whether such changes involve the physiological inhibitors of TPA and plasmin, and (3) whether the presence of these activators and inhibitors of fibrinolysis in tissue is correlated with clinical outcome. METHODS AND RESULTS: We immunocytochemically quantified the presence of fibrin, plasmin, TPA, and the TPA inhibitor PAI-1 in 938 biopsies from 68 consecutive cardiac allografts over a 54-month period. The localization, distribution, and quantification of TPA in arteriolar smooth muscle cells revealed that 35 of the 68 allografts maintained vascular TPA reactivity consistent with time-zero biopsies of autologous donor hearts: this was designated as the normal TPA group. In contrast, 33 of the 68 allografts significantly lost vascular TPA reactivity compared with time-zero biopsies of autologous donor hearts: this was designated as the depleted TPA group. Analysis of sequential biopsies from both groups during 54 months revealed that the mean cumulative quantitative TPA value for the normal TPA group was 1.0 +/- 0.01, whereas the depleted TPA group value was 1.9 +/- 0.02 (P = .0001), and the mean cumulative quantitative fibrin value for the normal TPA group was 1.0 +/- 0.01, whereas the depleted TPA group value was 1.5 +/- 0.05 (P = .0001). Biopsies of allografts in the depleted TPA group contained endothelial reactivity for TPA-PAI-1 complexes, whereas biopsies from the normal TPA group did not. Plasmin-associated molecules were rarely identified in biopsies of the normal TPA group but were present in the depleted TPA group, and the fibrin-to-plasmin ratio in the normal TPA group always was less than the fibrin-to-plasmin ratio in biopsies from the depleted TPA group. Analysis of demographic and risk factors revealed no significant differences between patients in the normal and depleted TPA groups, but none of the 35 patients in the normal TPA group died or were retransplanted, and 13 of the 33 patients in the depleted TPA group died or required retransplantation (P = .0001). CONCLUSIONS: Time-zero hearts (n = 68) and 34 of 38 stable allografts contained immunocytochemically detectable TPA only in vascular smooth muscle cells. Twenty-nine of 30 patients with normal TPA in their time-zero biopsies who subsequently developed a poor clinical outcome were found to have depleted TPA in biopsies evaluated during their first postoperative month and remained depleted throughout the study. Of 33 patients with depleted TPA, 39% died or required retransplantation. Depleted arteriolar TPA associated significantly with vascular and interstitial deposits of fibrin, plasmin, and endothelial TPA-PAI-1 complexes. These findings indicate that hemostatic and fibrinolytic pathways are activated in falling allografts, and they reveal evidence of depleted TPA before clinical or histopathological signs of failure. Patients with such allografts were found to be at high risk of death independently of other widely used clinical/laboratory parameters of prediction.     

Experimental Considerations when Characterizing Materials Friction with Atomic Force Microscopy

Operational details and experimental methodology regarding the use of atomic force microscopy for the measurement of tribological data are discussed. Data are presented that highlight both the concerns associated with instrumental alignment and the benefits of comprehensive friction–load data collected at microscopic length scales.     

Exercise, dietary obesity, and growth in the rat

Four regimens: high-fat diet, exercised (I); chow, exercised (II); high-fat sedentary (III); and chow, sedentary (IV) were initiated in 35-day-old male rats. Growth was exponential in I and II and exponential progressing to rectilinear in III and IV. The exponential model predicted the decreasing rank order in asymptotic weight to be: III, IV, I, II. Body composition data (9 components) showed rank order in masses of fat and the fat-free body mass compartment (FFBM) to be the same as for asymptotic live weight. The rectilinear growth mode probably reflected fat accretion. High-fat diet increased and treadmill exercise decreased FFBM, the latter being reversible. These effects depended on regimen initiations by the 5-7th wk of age. During growth, masses of H2O, muscle, and skin increased as functions of body size; bone as a function of age; and heart, liver, gut, testevity, and diet. Growth in body size was expressed more precisely with FFBM, instead of live weight, as the index of size.     

Compositional analysis by g.l.c. of monomer feed in copolymerization — errors due to adsorption of monomer to copolymers

It is shown that the relative errors in determining the monomer feed composition by gas-liquid chromatography (g.l.c.) are not always independent of the degree of conversion to copolymer. In view of the sensitivity of the integral copolymerization equation to errors of the magnitude likely to be associated with g.l.c. analysis, the determination of small changes in the value of copolymerization parameters by this method can be very unreliable. Data showing the extent of adsorption of monomers to copolymers are presented.