Reversible covalent binding of peptide nitriles to papain

The dissociation constants for reversible covalent binding of twelve peptide nitrile inhibitors to the active site of papain have been measured by means of fluorescence titration. The binding constants generally parallel the kinetic specificity constants (kcat/Km) for related papain substrates, supporting earlier suggestions that peptide nitriles behave as transition state analog inhibitors of papain. In ten cases the temperature dependence of binding was analyzed to determine the enthalpic and entropic contributions to the binding energy. A compensation plot of delta H vs. T delta S resulted in two parallel lines, one for 'specific' nitriles (i.e., N-Ac-L-aa-NHCH2CN; aa = Phe, Leu, Met) and the other for 'non-specific' nitriles (e.g., N-Ac-D-Phe-NHCH2CN, PhCH2CH2CONHCH2CN hippurylnitrile, etc.). For both specific and nonspecific nitriles representing an 1800-fold range of Kd values (0.27 microM-490 microM), the solvent deuterium isotope effect on binding (Kd(H2O)/Kd(D2O) = DKd) was very close to 2.0. This isotope effect could be accounted for entirely by the simple protonic change which occurs upon the reversible addition of the active site sulfhydryl of papain to the nitrile group of the peptide derivative to form a covalent thioimidate linkage. In contrast, six closely related non-nitrile ligands containing identical peptide side chains but having C-terminal groups incapable of binding covalently to papain had unmeasureably high dissociation constants. Collectively, these results indicate that strong binding of peptide nitrile substrate analogs to papain requires a combination of (1) hydrophobic interaction (especially at the P2 position), (2) specific intermolecular hydrogen bonding and (3) covalent interaction of the nitrile with the active site sulfhydryl group.     

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.     

Purification and properties of D-myo-inositol 1,4,5-trisphosphate 3-kinase from rat brain. Susceptibility to calpain

A new, rapid method for purification of inositol(1,4,5)P3 3-kinase in high yield from rat brain is described. Purified enzyme exhibited a polypeptide of Mr = 53,000 on sodium dodecyl sulfate-polyacrylamide gel and a specific activity of 29 mumol/min/mg at 37 degrees C in the absence of calmodulin. Inclusion of calpain inhibitors was critical for obtaining the 53-kDa protein as the major product and 0.1% of the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylamino]-2-propanesulfonate, was necessary to stabilize enzyme activity. In the absence of calpain inhibitors, the 53-kDa protein degraded progressively during purification and yielded a mixture containing polypeptides of various sizes. Relative intensity of these degradation products on sodium dodecyl sulfate-polyacrylamide gel varied from one preparation to another. However, broad band(s) at the 42-45 kDa region and a band at 35 kDa were always weak, while bands of 53, 51, 40 (sometimes doublets), 33, and 32 KDa were usually strong. The fact that all of these polypeptides including the weak bands of 42-45 and 35 kDa were derived from the 53 kDa form was confirmed by their immunocross-reactivity with monoclonal antibodies to the 53 kDa form. When the 51, 40, and a mixture of the 33 and 32 kDa forms were obtained separately and nearly free from other forms, each of them exhibited catalytic activity. Nevertheless, calmodulin binds to polypeptides larger than 35,000 but not to the 33 and 32 kDa forms. Incubation of the purified 53 kDa form with calpain generated a fragmentation pattern nearly identical to that generated during purification in the absence of calpain inhibitors. Incubation with five other endoproteases produced proteolytic fragments slightly different from those by calpain. However, the general fragmentation patterns generated by the proteases were similar, suggesting that inositol(1,4,5)P3 3-kinase contains several motifs susceptible to a variety of proteases.     

Review of the East Asian species of the genera Hybrizon Fallén and Ghilaromma Tobias (Hymenoptera: Ichneumonidae: Hybrizontinae)

East Asian species of the genera Hybrizon and Ghilaromma are reviewed. Four species of Hybrizon, H. buccatus (Brébisson 1825), H. ghilarovi Tobias, 1988, H. juncoi (Ceballos 1957) and H. flavofacialis Tobias, 1988 and two species of Ghilaromma, G. orientalis Tobias, 1988 and G. ussuriensis Tobias, 1988, were recognized. H. ghilarovi was recorded from Korea, Japan and China, while H. juncoi was recorded from Korea, for the first time. The specimens recorded from Japan as G. fuliginosi (Wilkinson, 1930) by Watanabe (1984) are referred to G. orientalis herein. This species is newly recorded from Korea and Japan. Keys to East Asian species of Hybrizon and the world species of Ghilaromma are also provided.     

Fast and sensitive delineation of brain tumor with clinically compatible moxifloxacin labeling and confocal microscopy

Delineation of brain tumor margins during surgery is critical to maximize tumor removal while preserving normal brain tissue to obtain optimal clinical outcomes. Although various imaging methods have been developed, they have limitations to be used in clinical practice. We developed a high‐speed cellular imaging method by using clinically compatible moxifloxacin and confocal microscopy for sensitive brain tumor detection and delineation. Moxifloxacin is a Food and Drug Administration (FDA) approved antibiotic and was used as a cell labeling agent through topical administration. Its strong fluorescence at short visible excitation wavelengths allowed video‐rate cellular imaging. Moxifloxacin‐based confocal microscopy (MBCM) was characterized in normal mouse brain specimens and visualized their cytoarchitecture clearly. Then, MBCM was applied to both brain tumor murine models and two malignant human brain tumors of glioblastoma and metastatic cancer. MBCM detected tumors in all the specimens by visualizing dense and irregular cell distributions, and tumor margins were easily delineated based on the cytoarchitecture. An image analysis method was developed for automated detection and delineation. MBCM demonstrated sensitive delineation of brain tumors through cytoarchitecture visualization and would have potentials for human applications, such as a surgery‐guiding method for tumor removal.      

Cytotoxic T lymphocyte response against multiple simian immunodeficiency virusA (SIV) proteins in SIV-infected macaques.

To identify the target proteins of CD8+ T lymphocytes we have explored the cytolytic immune responses of 12 rhesus macaques experimentally infected with the simian immunodeficiency virus (SIVmac). Target cells were autologous B cell lines presenting SIVmac proteins after infection with recombinant vaccinia viruses. The eight following proteins were studied: ENV, POL, GAG, NEF, VIF, REV, TAT, and VPX. Macaque PBMC stimulated with Con A and expanded in T cell growth factor-containing medium produced cell lines with cytolytic activity in the majority of infected animals (9/12). The structural proteins ENV, POL, and GAG were recognized by cell lines derived from nine, eight, and six macaques, respectively. The small regulatory proteins also represented efficient CTL targets, a specific activity being detected against NEF (8/12), REV (7/12), VPX (7/12), TAT (6/12), and VIF (5/12). Most cytotoxic responses (except those directed against ENV) were mediated by CD8 cells and were MHC class I restricted. Limiting dilution analysis allowed us to quantify the frequency of CTL precursors and confirmed the high immunogenicity of multiple SIV proteins. Three different patterns of response could be defined: six animals were able to recognize at least six of the eight tested target proteins, two of them reacting with all eight target proteins. The other three responder macaques reacted only against a few SIV proteins, whereas no cytotoxic activity was detected in the three remaining infected macaques and in the nine negative controls. The six animals responding against multiple proteins were still healthy 12 to 22 mo after infection with two of them presenting a decrease in circulating CD4 cells concurrently to the disappearance of the CTL response. Conversely, three nonresponder or low responder macaques developed an overt disease after 4 to 12 mo, and two other presented a very low level of CD4 cells, suggesting that the pattern of response may be of prognostic value.