Renal cell carcinoma tumors induce T cell apoptosis through receptor-dependent and receptor-independent pathways

Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.     

Transducible form of p47phox and p67phox compensate for defective NADPH oxidase activity in neutrophils of patients with chronic granulomatous disease

Protein delivery to primary cells by protein transduction domain (PTD) serves as a novel measure for manipulation of the cells for biological study and for the treatment of various human conditions. Although the method has been employed to modulate cellular function in vitro, only limited reports are available on its application in the replacement of deficient signaling molecules into primary cells. We examined the potential of recombinant proteins to compensate for defective cytosolic components of the NADPH oxidase complex in chronic granulomatous disease (CGD) neutrophils in both p47(phox) and p67(phox) deficiency. The p47(phox) or p67(phox) protein linked to Hph-1 PTD was effectively expressed in soluble form and transduced into human neutrophils efficiently without eliciting unwanted signal transduction or apoptosis. The delivered protein was stable for more than 24h, expressed in the cytoplasm, translocated to the membrane fraction upon activation, and, most importantly able to restored reactive oxygen species (ROS) production. Although research on human primary neutrophils using the protein delivery system is still limited, our data show that the protein transduction approach for neutrophils may be applicable to the control of local infections in CGD patients by direct delivery of the protein product.     

Effects of aerobic exercise on metabolic syndrome improvement in response to weight reduction

Objective: The objective was to test effects of aerobic exercise training on metabolic syndrome (MetSyn) improvement in response to weight reduction. Research Methods and Procedures: A total of 459 overweight and obese women (age, 49 ± 9 years; BMI, 28 ± 3 kg/m²) were recruited for a baseline examination to test the relationship between cardiorespiratory fitness and metabolic syndrome prevalence; among these, 67 subjects with MetSyn were treated with 14‐week weight‐loss programs, which included low‐calorie diet and aerobic exercise. The MetSyn was defined according to the Examination Committee of Criteria for “Metabolic Syndrome” in Japan. Maximal oxygen uptake (V?o _2max) during a maximal cycling test was measured as an index of cardiorespiratory fitness at baseline and after the intervention. Results: In the baseline examination, age‐ and BMI‐adjusted odds ratios for MetSyn prevalence in the low, middle, and upper thirds of V?o _2max were 1.0 (referent), 0.50 (95% confidence interval, 0.26 to 0.95), and 0.39 (95% confidence interval, 0.14 to 0.96), respectively (linear trend, p = 0.02). The adjusted odds ratios for MetSyn improvement in the two interventions with diet alone and diet plus exercise were 1.0 and 3.68 (95% confidence interval, 1.02 to 17.6; p = 0.04), respectively. Discussion: These results suggest that adding aerobic exercise training to a dietary weight‐reduction program further improves MetSyn (adjusted odds ratio, 3.68) in obese women, compared with diet alone. Further studies on an association between V?o _2max change and MetSyn improvement are needed.     

Fatty acid composition in fetal, neonatal, and cultured cardiomyocytes in rats

Reconstructed myocardial tissue still does not have enough pulsatile contraction. It is well known that fetal and mature neonatal cardiomyocytes utilize glucose and lipid, respectively, as their energy substrates, and that cultured ones mainly use glucose in spite of their age comparable to neonate ones, probably due to insufficient supply of lipids from culture medium. In the present study, we compared 7 saturated, 6 monounsaturated, and 11 polyunsaturated fatty acid contents in cultured cardiomyocytes (Cul group) with those in fetal (Fet group, approximately 17 d after impregnation) and neonatal (Neo group, 9 d old) rats, where the age of the Cul cells were set nearly equal to the Neo ones. Saturated fatty acid contents in the Cul group were generally lower than those in the Fet group and were close to those in the Neo group, except for C12:0 of which content was highest in the Neo group. Monounsaturated fatty acid contents in the Cul group were generally lower than those in the Fet group but similar to or higher than those in the Neo group, except for C24:1n-9 of which content was again highest in the Neo group. In contrast, most of polyunsaturated fatty acid (PUFA) contents in the Cul group appeared lower than those in both the Fet and Neo groups, and differences in 5 of 10 detected PUFAs were significant between the Cul and Neo groups. The results suggest that PUFA contents in cultured cardiomyocytes might be insufficient to exert enough contractile ability. In conclusion, it could be necessary for cultured cardiomyocytes to uptake more lipid; PUFAs in particular.     

Maturation-related variations in prostaglandin and fatty acid content of ovary in the kuruma prawn (Marsupenaeus japonicus)

Total lipid, fatty acids and prostaglandins (PGF(2 alpha) and PGE(2)) in the ovary of kuruma prawns (Marsupenaeus japonicus) were measured during ovarian development. The level of ovarian total lipid increased with an increase in the gonad-somatic index (GSI). No significant difference was found in fatty acid composition among different stages of ovarian development. However, the content of arachidonic acid (precursor of PG(2)), but not eicosapentanoic acid (precursor of PG(3)), was significantly lower at stages I and II than at stage V (P<0.01). When ovarian PGF(2 alpha) and PGE(2) levels were plotted against GSI, no correlation was found in either PG. However, in terms of ovarian developmental stages, the level of ovarian PGs was high (approx. 20 pg/mg) at stage I, followed by marked decreases at stages IV and V (PGF(2 alpha), P<0.01) and stage IV (PGE(2), P<0.01). These results suggest that ovarian PGs and arachidonic acid are deeply involved in ovarian maturation in kuruma prawns.     

The effect of long-range interactions on the secondary structure formation of proteins

The influence of long-range residue interactions on defining secondary structure in a protein has long been discussed and is often cited as the current limitation to accurate secondary structure prediction. There are several experimental examples where a local sequence alone is not sufficient to determine its secondary structure, but a comprehensive survey on a large data set has not yet been done. Interestingly, some earlier studies denied the negative effect of long-range interactions on secondary structure prediction accuracy. Here, we have introduced the residue contact order (RCO), which directly indicates the separation of contacting residues in terms of the position in the sequence, and examined the relationship between the RCO and the prediction accuracy. A large data set of 2777 nonhomologous proteins was used in our analysis. Unlike previous studies, we do find that prediction accuracy drops as residues have contacts with more distant residues. Moreover, this negative correlation between the RCO and the prediction accuracy was found not only for beta-strands, but also for alpha-helices. The prediction accuracy of beta-strands is lower if residues have a high RCO or a low RCO, which corresponds to the situation that a beta-sheet is formed by beta-strands from different chains in a protein complex. The reason why the current study draws the opposite conclusion from the previous studies is examined. The implication for protein folding is also discussed.     

Purification Process for the Preparation and Characterizations of Hen Egg White Ovalbumin,Lysozyme, Ovotransferrin,and Ovomucoid

Abstract

In this study, four major egg white proteins were purified by two step ion exchange chromatography followed by precipitation. Lysozyme and ovalbumin were separated with Q Sepharose Fast Flow anion exchange chromatography in the first step, resulting in two peaks of lysozyme and three peaks of ovalbumin with 87% and 70% purity by HPLC, respectively. Ovotransferrin was separated with CM-Toyopearl 650 M cation exchange chromatography in the second step, giving 80% purity. Ovomucoid was precipitated from the partial purified protein fraction from the first two steps, and concentrated in the final step to yield 90% purity. Protein recoveries were estimated to be 55, 21, 54, and 21% for lysozyme, ovotransferrin, ovalbumin, and ovomuciod, respectively. Lysozyme was identified from the purified peaks using zymogram refolding gel, whereas ovalbumin was identified by western blotting. Purified ovotransferrin and ovomucoid was identified by mass spectrometry. The results indicate that this purification process is adequate for preparation of lysozyme, ovalbumin, ovotransferrin, and ovomucoid, at least on a laboratory scale.     

Studies on Mold Dextranases

Nine strains capable of producing dextranase were isolated from soil. Among them, a strain belonging to the genus Aspergillus was chosen as the best producer of the enzyme. The mold produced greater amounts of dextranase than those found in some strains in the genus Penicillium, when grown aerobically at 28°C for 5 to 6 days in medium containing 1% dextran, 1% NaNO₃ or polypeptone, 0.2% yeast extracts, 0.4% K₂HPO₄ and small amounts of inorganic salts, pH 8.5. From the comparative taxonomic experiments, the mold used here was identified to be a strain belonging to Aspergillus carneus.