Comparative effects of a recombinant and a mutein type of granulocyte colony stimulating factor on the growth of Meth-A fibrosarcoma with 5-fluorouracil chemotherapy

The present study was designed to evaluate the effects of a recombinant human G-CSF (rhG-CSF) and a mutein G-CSF(KW-2228) on leucopenia and tumor growth in mice treated with 5-fluorouracil (5-FU). In normal mice, the number of leucocytes (white blood cell, WBC) reached the peak 12 hours after a single injection of either type of G-CSF and decreased to the normal level after 24 hours. Daily administration induced a continuous increase in the WBC count, however, administrations at intervals did not. Meth-A fibrosarcoma was subcutaneously inoculated into the backs of syngeneic BALB/c mice. The mice were treated with 5-FU alone or with G-CSFs. Chemotherapy with 5-FU alone resulted in leucopenia and an insignificant inhibition of tumor growth. The conjunctive administration of G-CSFs with 5-FU resulted in a significantly augmented inhibition of tumor growth, and leukopenia was not seen. This augmenting effect was more prominent with KW-2228.These results suggest that in 5-FU chemotherapy G-CSFs may be beneficial in restoring the number of leucocytes from leucopenic state and in augmenting the tumor inhibitory effect. Furthermore, KW-2228 may be more beneficial than the natural type rhG-CSF.     

Automated system for high-throughput protein production using the dialysis cell-free method

High-throughput protein production systems have become an important issue, because protein production is one of the bottleneck steps in large-scale structural and functional analyses of proteins. We have developed a dialysis reactor and a fully automated system for protein production using the dialysis cell-free synthesis method, which we previously established to produce protein samples on a milligram scale in a high-throughput manner. The dialysis reactor was designed to be suitable for an automated system and has six dialysis cups attached to a flat dialysis membrane. The automated system is based on a Tecan Freedom EVO 200 workstation in a three-arm configuration, and is equipped with shaking incubators, a vacuum module, a robotic centrifuge, a plate heat sealer, and a custom-made tilting carrier for collection of reaction solutions from the flat-bottom cups with dialysis membranes. The consecutive process, from the dialysis cell-free protein synthesis to the partial purification by immobilized metal affinity chromatography on a 96-well filtration plate, was performed within ca. 14 h, including 8 h of cell-free protein synthesis. The proteins were eluted stepwise in a high concentration using EDTA by centrifugation, while the resin in the filtration plate was washed on the vacuum manifold. The system was validated to be able to simultaneously and automatically produce up to 96 proteins in yields of several milligrams with high well-to-well reliability, sufficient for structural and functional analyses of proteins. The protein samples produced by the automated system have been utilized for NMR screening to judge the protein foldedness and for structure determinations using heteronuclear multi-dimensional NMR spectroscopy. The automated high-throughput protein production system represents an important breakthrough in the structural and functional studies of proteins and has already contributed a massive amount of results in the structural genomics project at the RIKEN Structural Genomics/Proteomics Initiative (RSGI).     

Landscape of protein–small ligand binding modes

Elucidating the mechanisms of specific small‐molecule (ligand) recognition by proteins is a long‐standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein–ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all‐against‐all comparison of 20,040 protein–ligand complexes provided the landscape of the protein–ligand binding modes and its relationships with protein‐ and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R² = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein–ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them.     

Analysis of Genetic Relationships and Antimicrobial Susceptibility of Verotoxin-Producing Escherichia coli Strains Isolated in Nagasaki Prefecture,Japan in 1996

A total of 19 Escherichia coli O157 isolates were obtained in Nagasaki Prefecture, in the southwestern part of Japan, between 1990 and 1996. Pulsed-field gel electrophoresis (PFGE) and computer-assisted analysis were applied to determine genetic relationships among these strains. Fragment patterns of the isolates in Nagasaki, as determined by PFGE, were compared with those of isolates in other areas where large outbreaks and sporadic cases of E. coli O157 infection occurred. Similarity values of all the strains isolated in Nagasaki Prefecture were over 0.65 except for E. coli O26. Some strains were identical to the strains isolated from the areas where large outbreaks occurred. All strains were susceptible to ampicillin, fosfomycin, minocycline, amikacin, ofloxacin and sulfamethoxazole-trimethoprim.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

Brevibacterium insectiphilium KY 3446 (Steinhous, Breed AHU 1401) was found to accumulate IMP from hypoxanthine and UMP from uracil, respectively. This strain is thus considered to present the fourth example in salvage-type fermentation, in addition to Micrococcus sodonensis, Arthrobacter citreus and Brevibacterium ammoniagenes reported previously.

IMP from adenine and UMP from cytosine were also produced by KY 3446, respectively. Further, the addition of inosine and adenosine instead of the bases also caused IMP accumulation.

This strain grew well on sucrose medium, and produced IMP and UMP in higher yields on sucrose than on glucose medium.

Excessive amounts of Mn²⁺ stimulated growth, but markedly inhibited IMP production. The optimal concentration of Mn²⁺ for IMP accumulation induced morphogenetic alterations from normal and small to abnormal and large cells.     

A Low-Dose β1-Blocker in Combination with Milrinone Improves Intracellular Ca2+ Handling in Failing Cardiomyocytes by Inhibition of Milrinone-Induced Diastolic Ca2+ Leakage from the Sarcoplasmic Reticulum

Objectives

The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism.

Background

The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca²⁺ handling in heart failure remains unclear.

Methods

We investigated the effect of milrinone plus landiolol on intracellular Ca²⁺ transient (CaT), cell shortening (CS), the frequency of diastolic Ca²⁺ sparks (CaSF), and sarcoplasmic reticulum Ca²⁺ concentration ({Ca²⁺}_SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB).

Results

In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca²⁺}_SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca²⁺}_SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes.

Conclusion

A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca²⁺ leak.     

The role of K ATP channels on ischemia-reperfusion injury in the rat testis

AimsTo investigate the participation of K_ATP channels on the ischemia-reperfusion (IR)-induced apoptosis in the rat testis.Main methodsEight-week-old male Sprague–Dawley rats were divided into three groups: control and IR rats without or with cromakalim (300 μg/kg intraperitoneally), 30 min before the induction of ischemia. The right testicular artery and vein were clamped to induce ischemia in the testis. Sixty minutes after the ischemia, a 24 h period of reperfusion followed. Then, expressions of K_IR6.1, K_IR6.2, caspase-3, PARP, Fas, FasL, and K_IR6.1 and K_IR6.2 mRNAs were investigated by Western blot analyses and real-time PCR methods, respectively. Furthermore, testicular tissues were processed for histological evaluation and TUNEL staining.Key findingsExpressions of K_IR6.1 protein and mRNA were more than 10-fold of those of K_IR6.2 protein and mRNA in the testis. IR significantly increased the expressions of K_IR6.1 protein and mRNA as well as K_IR6.2 mRNA, caspase-3, and TUNEL index in the testis compared to the control. PARP expressions were significantly lower in the IR group than those of the control. Histologically, severe acute germ cell damage was observed in the IR testis. Treatment with cromakalim ameliorated these parameters compared to the non-treated IR group. There were no significant differences on Fas, FasL and protein level of K_IR6.2 expressions between any of the groups.SignificanceTreatment with cromakalim has a protective effect against IR-induced testicular damage via activating K_ATP channels. This is the first study to give evidence for the advantageous effect of cromakalim in the germ cell-specific apoptosis induced by testicular IR.     

Persistent High Numbers of Clostridium perfringens in the Intestines of Japanese Aged Adults

TSN agar was applicable for enumeration of Clostridium perfringens in fecal samples of adults but not in those of infants. It was demonstrated using TSN agar that some healthy aged adults had persistently carried C. perfringens at levels ranging from 10⁷ to 10⁹, while some others ranged from 10³ to 10⁶ per ml volume of fecal sample although all of these adults had the same diets. In the test for agglutinability of isolates of C. perfringens collected from two elderly adults, a younger adult and a baby, it was demonstrated that most of the isolates obtained from an aged adult of high levels for 19 months belonged the same serotype, while rapid alteration of serotypes could be observed in three other persons with high or low levels. In spite of as many as 10⁹ C. perfringens per ml of feces, no trace of α-toxin could be detected in the fecal samples. In in vitro tests, fecal suspension suppressed the production of α-toxin although it allowed the organism to grow sufficiently.     

Fluorescence Polarization of αs1, κ-Caseins and αs1-κ-Casein Complex

Conjugates of α^s1-,κ-caseins and α^s1-,κ-casein complex were prepared with dimethylaminonaphthalenesulfonate and pyrenebutyrate. Their fluorescence lifetimes and the rotational relaxation times were measured by single photon counting technique and fluorescence depolarization technique, respectively. Both dimethylaminonaphthalenesulfonate and pyrenebutyrate conjugates had more than two lifetimes and the longer lifetime of pyrenebutyrate conjugates was near 140 nsec.

The rotational relaxation time of pyrenebutyrate α^s1-,κ-casein complex was smaller than that of pyrenebutyrate κ-casein polymer, which suggested that the complex formation of α^s1- and κ-casein polymers led to dissociation of the κ-casein polymer.

Changes of the rotational relaxation time as a function of weight ratio of α^s1- and κ-casein polymers (α^s1/κ) showed the specific variation and it was suggested that 4 moles of α^s1-κ-casein complex were formed from one mole of κ-casein polymer.