Enzymatic cyclization of a potent bowman-birk protease inhibitor,sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide

The most potent known naturally occurring Bowman-Birk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine beta-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI-1[6,5] is approximately 9:1 regardless of whether trypsin is incubated with SFTI-1[6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[6,5] revealed high heterogeneity, and multiple species of SFTI-1[6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a beta-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an iso-aspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.     

Epr analysis reveals three tissues responding to endotoxin by increased formation of reactive oxygen and nitrogen species

The excessive formation of reactive oxygen and nitrogen species (RONS) in tissue has been implicated in the development of various diseases. In this study we adopted ex vivo low temperature EPR spectroscopy combined with spin trapping technique to measure local RONS levels in frozen tissue samples. CP-H (1-hydroxy-3-carboxy-pyrrolidine), a new nontoxic spin probe, was used to analyze RONS in vivo. In addition, nitrosyl complexes of hemoglobin were determined to trace nitric oxide released into blood. By this technique we found that RONS formation in tissue of control animals increased in the following order: liver < heart < brain < cerebellum < lung < muscle < blood < ileum < kidney < duodenum < jejunum. We also found that endotoxin challenge, which represents the most common model of septic shock, increased the formation of RONS in rat liver, heart, lung, and blood, but decreased RONS formation in jejunum. We did not find changes in RONS levels in other parts of gut, brain, skeletal muscles, and kidney. Scavenging of RONS by CP-H was accompanied by an increase in blood pressure, indicating that LPS-induced vasodilatation may be due to RONS, but not due to nitric oxide. Experiments with tissue homogenates incubated in vitro with CP-H showed that ONOO⁻ and O₂^•−, as well as other not identified RONS, are detectable by CP-H in tissue. In summary, low-temperature EPR combined with CP-H infusion allowed detection of local RONS formation in tissues. Increased formation of RONS in response to endotoxin challenge is organ specific.     

The discovery of acylated beta-amino acids as potent and orally bioavailable VLA-4 antagonists

Acylated beta-amino acids are described as potent, specific and orally bioavailable antagonists of VLA-4. The initial lead was identified from a combinatorial library. Subsequent optimization using a traditional medicinal chemistry approach led to significant improvement in potency (up to 8-fold) while maintaining good pharmacokinetic properties.     

Effect of binding of Cd2+ on bacterial reaction center mutants: proton-transfer uses interdependent pathways

In bacterial reaction center of Rhodobacter sphaeroides, Cd2+ binds in stoichiometric amount to the protein. In the wild type, this results into a notable decrease of the rates of electron-transfer between the two quinone acceptors after the first (kAB(1)) and second flash (kAB(2)). We have studied these effects in two single mutants, L209PY and L209PF. L209Pro is situated in a protein region rich in hydrogen-bond networks involving water molecules. We show that (1) the combined effects of Cd2+ binding and point mutations have a cumulative consequence in the two mutants, decreasing very substantially the observed rates of electron-transfer. Interestingly, the [Cd2+] titration curves of kAB(2) in the L209PY and L209PF mutants are nearly superimposable to those previously reported for the M17DN and L210DN mutants (Paddock, M. L., Feher, G., and Okamura, M. Y. (2000) Proc. Natl. Acad. Sci U.S.A. 97, 1548-1553). These observations suggest a common effect of all of these mutations (L209, M17, L210) on the protonation state of the histidine cluster to which Cd2+ binds; (2) in the L209PY mutant, the pH titration curves of kAB(1), kAB(2), and k(H)(+), the proton-transfer rate at the second flash, are systematically downshifted by 1.5-2 pH units in the presence of 300 microM Cd2+, similarly to the wild type RCs (Gerencser, L., and Maroti, P. (2001) Biochemistry 40, 1850-1860). We propose that Cd2+ binding influences the electrostatics of interdependent ways of proton penetration within the protein, involving at least, directly or indirectly, L209P, L210D, and M17D, probably in conjunction with hydrogen-bonded connected water molecules.     

Highly sensitive chromatographic assay for dopamine determination during in vivo cerebral microdialysis in the rat

A highly sensitive, yet simple, isocratic high-performance liquid chromatographic (HPLC) assay with electrochemical detection (ED) for the determination of extracellular dopamine (DA) in brain microdialysates is presented. The method makes possible the detection of less than 100 pM (less than 1 fmol on column) and the quantitation of 200 pM (2 fmol on column) of DA with the use of a narrow-bore rather than capillary or microbore column. Analysis is feasible within an 11-min run-time, and thus is suitable for the relatively short sampling intervals used in microdialysis experiments. In the calibration range of 0.2 to 10 nM, the method has excellent linearity and precision, with intra-day relative standard deviations (RSD) of 0.5-2.4% and between-day RSD of 2.1-4.3%.     

Transglutaminase-Catalyzed Protein Cross-Linking in the Molecular Program of Apoptosis and Its Relationship to Neuronal Processes

1. One type of transglutaminase is usually accumulated in various forms of naturally occurring cell death and apoptosis. The accumulated enzyme is activated during the death process, leading to the formation of cross-linked protein structures. Degradation of the cross-linked apoptotic bodies results in the elevation of the (-glutamyl)lysine isodipeptide concentration in body fluids, which may provide a diagnostic tool to monitor the apoptosis rate in various tissues under normal and pathologic conditions.2. Extensive protein cross-linking may be directly related to the act of killing in some cells. In others, the effect of protein cross-linking is palliative, preventing leakage of macromolecules and enhancing phagocytosis of the dead cells.3. Tissue transglutaminase has been implicated in some physiologic functions of the nervous system.4. The molecular machinery of apoptosis is present and easily evoked in neuronal cells.5. Effector elements of the apoptosis process have been associated with the pathogenesis of neurologic disorders. Tissue transglutaminase, representing one of the effector elements of apoptosis, may be induced and activated in cells following ischemia. It may also participate in the formation of abnormal cell inclusions and A deposits in amyloid plaques.     

Effect of amino acid analogs on the development of thermotolerance and on thermotolerant cells

Exposure of HA-1 Chinese hamster fibroblasts to amino acid analogs has been shown to have a heat-sensitizing effect as well as inducing the heat shock response (Li and Laszlo, 1985a). In this study, we have examined the effect of amino acid analogs on the development of thermotolerance after a brief heat shock or exposure to sodium arsenite and the effect of amino acid analogs on cells that are already thermotolerant. Exposure of HA-1 cells to amino acid analogs inhibited the development of thermotolerance following a mild heat shock or treatment with sodium arsenite. However, cells that were already thermotolerant were resistant to the sensitizing action of amino acid analogs. The refractoriness of thermotolerant cells to amino acid analog treatment developed in parallel with thermotolerance. The uptake of the arginine analog, canavanine, and its incorporation into proteins was not altered in the thermotolerant cells. Furthermore, another biological consequence of exposure to amino acid analogs, sensitization to ionizing radiation, also was not altered in the thermotolerant cells. The inhibition of the development of thermotolerance by amino acid analogs and the refractoriness of thermotolerant cells to the heat-sensitizing action of amino acid analogs lend further support the role of heat-shock proteins in the phenomenon of thermotolerance. © 1993 Wiley-Liss, Inc.     

Sodium binding sites of gramicidin A: sodium-23 nuclear magnetic resonance study.

In ethanol-water mixtures (90:10), the gramicidin dimer binds Na+ cations at well-defined sites, with a binding constant K = 4 M-1. Partial desolvation of Na+ occurs upon binding, as judged from the magnitude of the quadrupolar coupling constant (1.7 MHz) for bound sodium. The binding sites are identified with the outer sites flanking the channel entrances. The rate constants for binding and release are k+ less than or equal to 2.2 X 10(9) M-1 s-1 and k- less than or equal to 5.5 X 10(8) s-1, respectively.     

Epstein-Barr virus encoded nuclear protein EBNA-3 binds a novel human uridine kinase/uracil phosphoribosyltransferase

Background  

Epstein-Barr virus (EBV) infects resting B-lymphocytes and transforms them into immortal proliferating lymphoblastoid cell lines (LCLs) in vitro. The transformed immunoblasts may grow up as immunoblastic lymphomas in immuno-suppressed hosts.