Identification of proteolytic products as indicators of quality in ground and whole meat

This study was devoted to the identification of specific peptides and proteins which can serve as indicators of spoilage in meat. Samples of ground and whole meat were subjected to storage at 4C, at intervals of 0, 2, 4, 8, 12 and 16 days, samples were analyzed for pH and microbial populations and subjected to extraction and separation of individual sarcoplasmic and myofibrillar peptides and proteins by SDS (sodium dodecyl sulfate) and native electrophoresis and by RP-HPLC. Sarcoplasmic protein and peptide fractions from RP-HPLC were collected and identified by (electrospray ionization mass spectrometry) ESI-MS. The results demonstrated substantial differences in microbial population and pH between ground and whole meat during storage. Separation by SDS-electro-phoresis showed substantial changes in myofibrillar protein of ground meat after 12 days and of whole meat after 16 days of storage. Separation and identification of sarcoplasmic proteins by SDS-electrophoresis and by RP-HPLC followed by ESI-MS revealed the disappearance of a protein fraction band of MW 36 kDa after 8 days of storage in ground and whole meat

Sequence-specific cleavage of RNA by a hybrid ribonuclease H

AbstractSite-specific cleavage of the 22-, 132- and 534-base RNAs by the DNA/protein hybrid R Nase H were examined. The 22-base RNA was chemically synthesized, and 132- and 534-base RNAs were prepared by run-off transcription. The hybrid enzyme cleaves these RNAs, which contain a single target sequence, primarily at the unique phosphodiester bond within the target sequence. The hybrid enzyme performs multiple turnovers, and at a substrate/enzyme ratio of 10:1 the RNAs are almost completely cleaved by the hybrid enzyme at 37°C within 1 h. We propose that hybrid RNase H molecules with various oligodeoxyribonucleotides function as RNA restriction enzymes and are useful for structural and functional studies of RNA

Bifunctional thrombin inhibitors based on the sequence of hirudin45-65.

The interaction of alpha-thrombin with the hirudin (HV1) fragment N alpha-acetyl desulfo hirudin45-65 (P51) was investigated. Kinetic analysis revealed that P51 inhibits the proteolysis of a tripeptidyl substrate with Ki = 0.72 +/- 0.13 and 0.11 +/- 0.03 microM for bovine and human alpha-thrombins, respectively. The inhibition was partially competitive, affecting substrate binding to the enzyme-inhibitor complex by a factor alpha = 2 (bovine) and alpha = 4 (human) characteristic of hyperbolic inhibitors. P51 also inhibited thrombin-induced fibrin clot formation with IC50 values of 0.94 +/- 0.20 and 0.058 +/- 0.006 microM for bovine and human alpha-thrombins, respectively. The enhanced antithrombin activity for human thrombin could be attributed to species variations in the putative auxiliary "anion" exosite since N alpha-acetyl desulfo hirudin55-65 displayed the same rank order of potency shift in a clotting assay without inhibiting the amidolytic activity of either enzyme. From these observations, a potent thrombin inhibitor was designed having modified residues corresponding to the P1 and P3 recognition sites. N alpha-Acetyl[D-Phe45, Arg47] hirudin45-65 (P53) emerged as a pure competitive inhibitor with a Ki = 2.8 +/- 0.9 nM and IC50 = 4.0 +/- 0.8 nM (human alpha-thrombin) and is designated as a "bifunctional" inhibitor. Its enhanced potency could be explained by a cooperative intramolecular interaction between the COOH-terminal domain of the inhibitor and the auxiliary exosite of thrombin on the one hand, and the modified NH2-terminal residues with the catalytic site on the other

Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

ATM and ATR protein kinases play a crucial role in cellular DNA damage responses. The inhibition of ATM and ATR can lead to the abolition of the function of cell cycle checkpoints. In this regard, it is expected that checkpoint inhibitors can serve as sensitizing agents for anti-cancer chemo/radiotherapy. Although several ATM inhibitors have been reported, there are no ATR-specific inhibitors currently available. Here, we report the inhibitory effect of schisandrin B (SchB), an active ingredient of Fructus schisandrae, on ATR activity in DNA damage response. SchB treatment significantly decreased the viability of A549 adenocarcinoma cells after UV exposure. Importantly, SchB treatment inhibited both the phosphorylation levels of ATM and ATR substrates, as well as the activity of the G2/M checkpoint in UV-exposed cells. The protein kinase activity of immunoaffinity-purified ATR was dose-dependently decreased by SchB in vitro (IC50: 7.25 μM), but the inhibitory effect was not observed in ATM, Chk1, PI3K, DNA-PK, and mTOR. The extent of UV-induced phosphorylation of p53 and Chk1 was markedly reduced by SchB in ATM-deficient but not siATR-treated cells. Taken together, our demonstration of the ability of SchB to inhibit ATR protein kinase activity following DNA damage in cells has clinical implications in anti-cancer therapy

Chlortetracycline and Demeclocycline Inhibit Calpains and Protect Mouse Neurons against Glutamate Toxicity and Cerebral Ischemia

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-D-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains

Optimal Robust PID control for first- and second-order plus dead-time processes

The present study proposes a new design method for a proportional-integral-derivative (PID) control system for first-order plus dead-time (FOPDT) and over-damped second-order plus dead-time (SOPDT) systems. What is presented is an optimal PID tuning constrained to robust stability. The optimal tuning is defined for each one of the two operation modes the control system may operate in: servo (reference tracking) and regulation (disturbance rejection). The optimization problem is stated for a normalized second-order plant that unifies FOPDT and SOPDT process models. Different robustness levels are considered and for each one of them, the set of optimal controller parameters is obtained. In a second step, suitable formulas are found that provide continuous values for the controller parameters. Finally, the effectiveness of the proposed method is confirmed through numerical examples

Isolation of cDNA Encoding 7H6-Reactive Polypeptide Defines a New Class of Protein with alpha-Helical Coiled-Coil Structure and DA-Box Similar to Yeast Chromosomal Segregation Proteins

7H6 monoclonal antibody was recently developed in our laboratory by im-munizing mice with a bile canaliculus-rich fraction of the rat liver. The anti-body reacted with a novel 155 Kd polypeptide designated 7H6 antigen that specifically localizes at tight junctions of various epithelia. Correlations of the paracellular barrier function of the tight junction with expression of the 7H6 antigen at the cell border have suggested important roles of this polypeptide for the maintenance of tight junctional functions. As the first step for the analysis of the antigen at the molecular level, we isolated a series of cDNA clones encod-ing 7H6-reactive polypeptides. Five clones were isolated by immunoscreening. Among them a clone designated RL5.3 which carries the largest 5.3Kb insert was characterized in this study. Both plaque screening and immunoblotting of the fusion protein produced by the RL5.3 clone with lysogen confirmed that the pro-tein specifically reacts with the 7H6 monoclonal antibody. Using DNA fragmentsof the RL5.3 clone, 21 clones were further identified. Studies with restriction enzymes and probe hybridization revealed that all the cDNA clones were derived from a single class of transcripts. A partial sequence identified one open reading frame with an α-helical coiled-coil structure and highly conserved aspartate (D)- alanine (A) residues with a helix-loop-helix structure corresponding to DA- box. Since this domain has been specifically found in yeast chromosomal segregation proteins (SMC1, CUT3 and CUT14), the polypeptide encoded by the RL5.3 clone provides the first rodent counterpart of these protein family. Yeast is known to be lethal when SMC and CUT proteins are deleted, suggesting essential roles of these proteins for cell cycle progression as a regulator for chromosomal segregation. Identification of a mammalian counterpart of this pro-tein family may give us some clues for a better understanding of fundamental regulatory mechanisms in the function of tigh junctions

ナンキョク カンソクセン シラセ デ エラレタ センジョウ ジュウリョク データ ノ セイビ

砕氷船「しらせ」船不での重力測定は,第27次南極地域観測隊以来,継続実施されており,およそ17年分のデータが蓄積されている.この内,第27次隊から第33次隊のデータについては,すでに処理されたMGD77フォーマットのデータが提供されていたが,第34次隊以降のデータについては,統一的なデータ処理がされないままの状態であった.そこで,今回,第34次隊から第46次隊の間に得られたデータについて新たにデータ処理を実施し,重力異常データを作成した.この際,特に長波長域での重力データの精度向不のため,第27次隊以降のすべてのデータについて衛星高度計による海域重力異常を基準としたドリフト補正を行った.ここでは,これらの処理の概要ならびに得られたデータセットの状況について報告する.A ship-borne gravity survey on board the icebreaker Shirase has been continuously conducted since JARE-27 (the 27th Japanese Antarctic Research Expedition); about 17 years of gravity data have been accumulated. Although data obtained from JARE-27 to JARE-33 have already been processed and stored in MGD77 format, data starting with JARE-34 have remained unprocessed. We newly processed the data from JARE-34 to JARE-46 to obtain free-air gravity anomalies. To attain better quality, especially in the long wavelength gravity signals, we also applied drift corrections for all the data sets since JARE-27 by employing satellite derived gravity anomalies as a reference gravity field. This report summarizes the data processing and the status of the surface ship gravity data