No abstract

SD-OCT accurately represented retinal lamination and photoreceptor loss and recovery during light-induced damage and subsequent regeneration. SD-OCT was less accurate at detecting the inner nuclear layer in ouabain-damaged retinas, but accurately detected the undamaged outer nuclear layer. Thus, SD-OCT provides a noninvasive and quantitative method to assess the morphology and the extent of damage and repair in the zebrafish retina.

19F NMR relaxation studies have been carried out on a fluorotryptophan-labeled E. coli periplasmic glucose/galactose receptor (GGR). The protein was derived from E. coli grown on a medium containing a 50:50 mixture of 5-fluorotryptophan and [2,4,6,7-2H4]-5-fluorotryptophan. As a result of the large gamma-isotope shift, the two labels give rise to separate resonances, allowing relaxation contributions of the substituted indole protons to be selectively monitored. Spin-lattice relaxation rates were determined at field strengths of 11.75 T and 8.5 T, and the results were analyzed using a model-free formalism. In order to evaluate the contributions of chemical shift anisotropy to the observed relaxation parameters, solid-state NMR studies were performed on [2,4,6,7-2H4]-5-fluorotryptophan. Analysis of the observed 19F powder pattern lineshape resulted in anisotropy and asymmetry parameters of delta sigma = -93.5 ppm and eta = 0.24. Theoretical analyses of the relaxation parameters are consistent with internal motion of the fluorotryptophan residues characterized by order parameters S2 of approximately 1, and by correlation times for internal motion approximately 10(-11)s. Simultaneous least squares fitting of the spin-lattice relaxation and line-width data with tau i set at 10 ps yielded a molecular correlation time of 20 ns for the glucose-complexed GGR, and a mean order parameter S2 = 0.89 for fluorotryptophan residues 183, 127, 133, and 195. By contrast, the calculated order parameter for FTrp284, located on the surface of the protein, was 0.77. Significant differences among the spin-lattice relaxation rates of the five fluorotryptophan residues of glucose-complexed GGR were also observed, with the order of relaxation rates given by: R1F183 > R1F127 approximately R1F133 approximately R1F195 > R1F284. Although such differences may reflect motional variations among these residues, the effects are largely predicted by differences in the distribution of nearby hydrogen nuclei, derived from crystal structure data. In the absence of glucose, spin-lattice relaxation rates for fluorotryptophan residues 183, 127, 133, and 195 were found to decrease by a mean of 13%, while the value for residue 284 exhibits an increase of similar magnitude relative to the liganded molecule. These changes are interpreted in terms of a slower overall correlation time for molecular motion, as well as a change in the internal mobility of FTrp284, located in the hinge region of the receptor.

The presented method, initially developed for human OCT, has been adapted for mice, with the potential to be adapted for other animals as well. Quantitative in vivo assessment of the retina in mice allows changes to be measured longitudinally, decreasing the need for them.

Kinetic measurements on a fluorescent peptide analog of the p17/p24 cleavage site of the Gag polyprotein demonstrate the conformational selectivity of human immunodeficiency virus, type 1 protease for the trans conformation of the Tyr-Pro bond. A mean cis/trans ratio of 0.3, and a cis 3 trans isomerization rate constant of 0.022 s ؊1 are determined at T ‫؍‬ 22°C. This rate is in excellent agreement with that predicted by 19 F NMR studies of structurally analogous peptides containing a fluorine/hydroxyl substitution on the tyrosyl residue. Addition of recombinant human cyclophilin resulted in a significant enhancement of this rate, and it is proposed that this enzyme, which has been shown to be associated with the Gag protein, functions as an auxiliary enzyme for the protease during cleavage in the virion.Three of the eight consensus sequences cleaved by HIV-1 1 protease involve Araa-Pro bonds, where Araa corresponds to the aromatic amino acids tyrosine or phenylalanine (1). Since imide bonds are known to exhibit conformational cis-trans heterogeneity, the existence of such cleavage sites leads to questions concerning the conformational specificity of the protease. It has been demonstrated that cleavage of Xaa-Pro imide bonds by prolidase, aminopeptidase P, and carboxypeptidase P is specific for the trans conformation of Xaa-Pro dipeptides (2-5) and further that proteases can be conformationally selective even for nearby, non-scissile peptide bonds (6 -9). Indeed, this conformational specificity of chymotryptic cleavage for the P 2 -P 3 bond has provided the basis for assays used to demonstrate the existence of peptidyl proline isomerases (10). Several lines of evidence suggest that HIV protease might be specific for the trans imide bond conformation: 1) crystallographic studies of inhibitors containing proline (11) or thiazolidine (12) indicate a trans conformation; 2) the ability of the protease to cleave ordinary amide bonds as well as imide bonds suggests a capability for cleaving trans imide bonds (1). It is important to emphasize, however, that neither of these observations provides unambiguous proof. For example, it is well established that inhibitors that exhibit close structural relationships to natural substrates can nevertheless bind to enzymes in dramatically different conformations (13).There are several motivations for understanding the conformational selectivity of HIV protease. 1) Many of the test peptides used to assay the protease contain imide Xaa-Pro bonds. In the event of conformational selectivity, the overall kinetic characterization of these assays should be generalized to include a combination of cleavage and isomerization rates, with the specific mix determined by the physical conditions such as temperature, buffer, etc. 2) Conformational selectivity by the protease could have significant implications for the kinetics of the cleavage process in vivo, closely analogous to the effect of cis-trans imide bond isomerism on protein folding and unfolding. The recent discovery of a close associa...

Fluorine nuclear magnetic resonance studies of the cleavage of peptides containing a 4-fluorophenylalanine (FPhe)-Pro bond have been performed in order to determine the conformational specificity of FPhe-Pro bond cleavage by pepsin. The peptides selected were substrates of HIV protease or of avian sarcoma virus protease, both of which have been reported to be cleaved specifically at X-Pro by pepsin as well as by the corresponding viral protease enzyme. By working at 0 degrees C, it was possible to separate kinetically cleavage and cis/trans isomerization. For the case of the protease substrate, Ser-Gln-Asn-FPhe-Pro-Ile-Val-Gln, cleavage was shown to be specific for the trans conformation. A value for the rate constant for hydrolysis of the trans peptide divided by the Michaelis constant, ktH/KMtrans = 0.3 min-1 mM-1 was obtained with this substrate, and the Michaelis constant appears to be considerably higher than the substrate concentration, 3.7 mM, used in the study. On a slower time scale, additional cleavages can readily be detected. For the avian leukemia virus protease substrate, Thr-Phe-Gln-Ala-FPhe-Pro-Leu-Arg-Glu-Ala, the cleavage was both slower and less specific. In addition to the primary cleavage at the FPhe-Pro site, cleavage also occurs at the Ala-FPhe bond on a somewhat slower time scale. In addition to the conformational specificity of the cleavage reaction, these results indicate that pepsin is a better model for HIV protease than for avian leukemia virus protease.

The broadening of the line spread function (LSF) in the drift direction with increasing drift distance in the kinestatic charge detector is substantially reduced when small amounts (less than 1%) of trimethylamine [(CH3)3 N] are added to the x-ray detection medium (krypton or xenon). The LSF of a mixture of Kr and 0.01% trimethylamine (TMA) was measured as a function of distance at 15, 25, and 35 atm absolute pressure. The full width at half-maximum (FWHM) of the LSF was reduced from about 1.0 mm to less than 0.5 mm at a drift distance of 4.0 mm for the three pressures. The LSF's of mixtures of xenon and TMA at concentrations ranging from 0.0004% to 0.4% in one run and 0.06% to 4.0% in a second run were measured at a constant pressure of 20 atm. The FWHM of the LSF was reduced from 0.6 to 0.4 mm at 4.0 mm for the xenon measurements. The optimum concentration of TMA in Xe was found to be in the neighborhood of 0.1%. The use of TMA reduced the drift distance-dependent LSF broadening to the level expected from ionic diffusion, space charge repulsion, and electric field nonuniformity, and it may be possible to reduce the 0.4-mm FWHM plateau through the use of an improved Frisch grid design. Observation of negative charge carriers showed that electron attachment increases with increasing TMA concentration, although this could be caused by impurities in the TMA. The implications of these results are discussed in terms of extending the maximum drift distance attainable in a kinestatic charge detector.

A prototype scanning imaging system which employs a kinestatic charge detector (KCD) and is under the control of a VAXstation II/GPX computer is described. The operating principles and advantages of the KCD method are reviewed. The detector is a 256-channel ionization drift chamber which creates a two-dimensional x-ray projection image by scanning the detector past the object of interest. The details of the drift chamber design, the signal collection electrodes (channels), and the Frisch grid geometry are given. Also described are the scanning gantry design, computer-controlled drive motor circuit, and safety features. The data acquisition system for the capture of a 1 M byte digital image is presented. This includes amplification, filtration, analog-to-digital conversion, data buffering, and transfer to the VAXstation II computer. The image processing and display techniques specific to the KCD are outlined and the first two-dimensional image taken with this system is presented.