Effect of γ-cyclodextrin as a lyoprotectant for freeze-dried actinidin.

Actinidin (ATD) is a cysteine protease found in kiwifruit. It is used to tenderize meat and to enhance the digestion of proteins in the small intestine. However, ATD is unstable during freeze-drying, which alters its bioactivity. It is well known that sugars have the ability to protect proteins from the stress of freeze-drying. In this study, we investigated the protective effect of various saccharides on the stability of ATD during freeze-drying. The ATD activities of the samples containing γ-cyclodextrin (CyD) showed only a small decrease, and compared with trehalose and sucrose, γ-CyD was a more effective stabilizer for ATD. Secondary structural changes in freeze-dried ATD were observed by circular dichroism spectroscopy and compared with the changes in stabilized samples. There was a close relationship between the α-helix content and the stabilization. The sugars stabilized the protein by suppressing the changes in the α-helix. Fourier transform infrared spectroscopy measurement showed that the amide I band of ATD with γ-CyD was shifted to a lower wavenumber compared with other sugars. Therefore, stronger hydrogen bonds may be formed between ATD and γ-CyD than between ATD and other sugars. The suppression of changes in the protein secondary structure accompanying the formation of hydrogen bonding between the protein and the sugar also contributed to the protective effect of the sugars.

Serum levels of IL-6 and IL-1ß can predict the efficacy of gemcitabine in patients with advanced pancreatic cancer.

BACKGROUND: With this study, we sought to characterise the impact of pro-inflammatory cytokines on the outcomes of gemcitabine monotherapy (GEM) in patients with pancreatic cancer (PC). METHODS: Treatment-naive patients with advanced PC and no obvious infections were eligible for enrolment. All of the patients were scheduled to undergo systemic chemotherapy. Serum pro-inflammatory cytokines were measured using an electro-chemiluminescence assay method before chemotherapy. High cytokine levels were defined as values greater than the median. Clinical data were collected prospectively. RESULTS: Sixty patients who received GEM were included in the analysis. High IL-6 and IL-1ß levels were poor prognostic factors for overall survival in a multivariate analysis (P=0.011 and P=0.048, respectively). Patients with both a high IL-6 level and a high IL-1ß level exhibited shortened overall and progression-free survival, a reduction in the tumour control rate, and a high dose intensity of GEM compared with patients with low levels of both IL-6 and IL-1ß. CONCLUSION: The serum levels of IL-6 and IL-1ß predict the efficacy of GEM in patients with advanced PC.

Effects of dietary gamma-cyclodextrin on voluntary activity and muscle strength in mice.

Gamma-cyclodextrin (γCD) is a cyclic oligosaccharide consisting of eight α-(1,4)-linked glucopyranose subunits, which is often used in the food and pharmaceutical industries. However, little is known regarding the metabolic activity of "empty" γCD per se. Therefore, in the present study young C57BL/6 male mice received a control diet (CON) or an experimental diet that was supplemented with 12.88% γCD exchanged against corn starch. After 6 weeks of treatment, the voluntary wheel running activity was monitored and the muscle strength of mice was measured by employing Kondziela's inverted screen test and forelimb grip strength assay. The γCD-treated mice covered a significantly larger distance per night (CON 8.6 km, γCD 12.4 km) and were significantly longer active (CON 340 min, γCD 437 min). Moreover, γCD-treated mice significantly performed better at the inverted screen test indicated by an enhanced Kondziela score (CON 3.10, γCD 4.63). These data suggest that dietary γCD leads to an increased endurance. We also found a slightly anti-glycemic effect of γCD during oral glucose tolerance test. However, our mice from the γCD group exhibited no difference in terms of GLUT2 protein level in ileum tissue nor increased muscle glycogen storage. Furthermore, γCD exhibited no DPP-4 inhibitory activity in vitro. By analysing candidate muscle genes and proteins related to endurance and muscle performance we did not observe any differences in terms of Sirt1, Pgc1α, Cpt1b, Mef2c, Myh1 and Myh2 gene expression levels as well as total oxidative phosphorylation (OXPHOS), mtTFA and GLUT4 protein expression levels in skeletal muscle in response to γCD. We could not fully establish the exact underlying molecular mechanisms of the fitness improvement by dietary γCD which warrants further investigations.

Adenovirus serotype 35 vector-mediated transduction following direct administration into organs of nonhuman primates.

Adenovirus (Ad) serotype 35 (Ad35) vectors have attracted remarkable attention as alternatives to conventional Ad serotype 5 (Ad5) vectors. In a previous study, we showed that intravenously administered Ad35 vectors exhibited a safer profile than Ad5 vectors in cynomolgus monkeys, which ubiquitously express CD46, an Ad35 receptor, in a pattern similar to that in humans. However, the Ad35 vectors poorly transduced the organs. In this study, we examined the transduction properties of Ad35 vectors after local administration into organs of cynomolgus monkeys. The vectors transduced different types of cells depending on the organ. Hepatocytes and microglia were mainly transduced after the vectors were injected into the liver and cerebrum, respectively. Injection of the vectors into the femoral muscle resulted in the transduction of cells that appeared to be fibroblasts and/or macrophages. Conjunctival epithelial cells showed transgene expression following infusion into the vitreous body of the eyeball. Transgene expression was limited to areas around the injection points in most of the organs. In contrast, Ad35 vector-mediated transgene expression was not detected in any of the organs not injected with Ad35 vectors. These results suggest that Ad35 vectors are suitable for gene delivery by direct administration to organs.

Natural killer type 2 bias in remission of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease characterized by clinical relapse and remission. Because of the potential role of natural killer (NK) cells in the regulation of autoimmunity, we have examined cytokine profile and surface phenotype of NK cells in the peripheral blood of MS. Here we demonstrate that NK cells in the remission of MS are characterized by a remarkable elevation of IL-5 mRNA and a decreased expression of IL-12Rbeta2 mRNA, as well as a higher expression of CD95. Moreover, the NK cells from MS in remission produced much larger amounts of IL-5 than did those from controls after stimulation with phorbol myristate acetate (PMA) and ionomycin. These features are reminiscent of those of NK type 2 (NK2) cells that can be induced in a condition favoring functional deviation of T cells toward Th2. Remarkably, the NK cells lose the NK2-like property when relapse of MS occurs, but regain it after recovery. We also found that NK2 cells induced in vitro inhibit induction of Th1 cells, suggesting that the NK2-like cells in vivo may also prohibit autoimmune effector T cells. Taken together, it is possible that NK cells play an active role in maintaining the remission of MS.

Neuronal specificity of alpha-synuclein toxicity and effect of Parkin co-expression in primates.

Recombinant adeno-associated viral (rAAV) vector-mediated overexpression of alpha-synuclein (alphaSyn) protein has been shown to cause neurodegeneration of the nigrostriatal dopaminergic pathway in rodents and primates. Using serotype-2 rAAV vectors, we recently reported the protective effect of Parkin on alphaSyn-induced nigral dopaminergic neurodegeneration in a rat model. Here we investigated the neuronal specificity of alphaSyn toxicity and the effect of Parkin co-expression in a primate model. We used another serotype (type-1) of AAV vector that was confirmed to deliver genes of interest anterogradely and retrogradely to neurons in rats. The serotype-1 rAAV (rAAV1) carrying alphaSyn cDNA (rAAV1-alphaSyn), and a cocktail of rAAV1-alphaSyn and rAAV1 carrying parkin cDNA (rAAV1-parkin) were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral GABAergic and nigrostriatal dopaminergic neurons. Injection of rAAV1-alphaSyn alone decreased tyrosine hydroxylase immunoreactivity in the striatum compared with the contralateral side injected with a cocktail of rAAV1-alphaSyn and rAAV1-parkin. Immunostaining of striatonigral GABAergic neurons was similar on both sides. Overexpression of Parkin in GABAergic neurons was associated with less accumulation of alphaSyn protein and/or phosphorylation at Ser129 residue. Our results suggest that the toxicity of accumulated alphaSyn is not induced in non-dopaminergic neurons and that the alphaSyn-ablating effect of Parkin is exerted in virtually all neurons in primates.

Inhibitory effect of fumaric acid on 3-methyl-4'-(dimethylamino)-azobenzene-induced hepatocarcinogenesis in rats.

Fumaric acid (FA) was examined for its effect on hepatocarcinogenesis in rats fed 3-methyl-4'-(dimethylamino)azobenzene (3-Me-DAB). Male DONRYU rats were given approximately 0.5 g 3-Me-DAB by being fed a diet containing 0.06% 3-Me-DAB for 50 days; they were then given a diet containing 1% FA and drinking water containing 0.025% FA for 51 weeks. The administration of FA effectively suppressed the development of hepatocellular carcinoma, hyperplastic nodules, and hyperplastic areas in the livers of rats fed 3-Me-DAB.

Inhibitory effect of fumaric acid on hepatocarcinogenesis by thioacetamide in rats.

An inhibitory effect of fumaric acid (FA) on hepatocarcinogenesis was examined in rats fed thioacetamide (TAA). A group of male DONRYU rats were fed TAA at a level of 0.035% in the diet for 40 weeks and then fed a basal diet for 40 weeks. Hepatic carcinomas developed in 9 of 41 animals of this group fed TAA alone. The effect of FA on the carcinogenesis was examined in 2 groups fed both TAA and FA; one group of rats were fed FA at 1% in a basal diet after ingestion of TAA, and another group of rats were fed TAA plus a supplement of 1% FA in the diet. The inhibitory effect of FA on TAA carcinogenesis was so marked that no hepatic carcinomas were found in both groups fed FA in combination with TAA.

Screening of the Men1 gene and discovery of germ-line and somatic mutations in apparently sporadic parathyroid tumors.

Hyperparathyroidism is the first manifestation in a majority of multiple endocrine neoplasia (MEN1) patients. To discriminate between sporadic and hereditary parathyroid tumors and characterize MEN1 somatic mutations, we examined MEN1 gene mutations in patients who had undergone surgery for sporadic parathyroid tumors. DNA was extracted from fresh frozen parathyroid tumor specimens from 112 patients as well as from peripheral blood leukocytes from 64 of the 112 patients. Sequence analysis was performed to examine exons 2-10 of the MEN1 gene for mutations. Loss of heterozygosity (LOH) was also examined by an analysis of codon 418 and 541, which lie within a polymorphic region of MEN1. Somatic MEN1 mutations were found in 25 of the 112 patients (22%). Two patients had two point mutations (508del33 and Y341X and 363insT and 1767delT, respectively). A total of 27 mutations were characterized, 20 of which have not been reported previously. There were 7 nonsense mutations, 10 frameshift mutations, 2 splice site deletions, 5 missense mutations, and 3 in-frame mutations. Nineteen mutations (70%) predicted truncation of the menin protein. Germ-line MEN1 mutations were found in 3 of 64 patients (5%) who had no family history of endocrine tumors associated with MEN1, and these patients were identified as MEN1 gene probands. LOH at the MEN1 locus was detected in three parathyroid tumors showing germ-line mutation. LOH was significantly frequent in parathyroid tumors with somatic MEN1 mutations (15 of 22 tumors, 68%) but not in those without germ-line or somatic MEN1 mutations (14 of 51 tumors, 28%; P = 0.0011). Our findings suggest that alterations of both alleles of the MEN1 gene may be associated not only with endocrine tumors of affected MEN1 patients but also with sporadic parathyroid tumors. Germ-line MEN1 gene analysis can distinguish heritable from nonheritable parathyroid tumors, and MEN1 gene evaluation of patients with apparently sporadic parathyroid tumor is recommended before parathyroid surgery.

A Pharmacokinetic/Pharmacodynamic Drug-Drug Interaction Study of Tofogliflozin (a New SGLT2 Inhibitor) and Selected Anti-Type 2 Diabetes Mellitus Drugs.

OBJECTIVE: Tofogliflozin is an oral hypoglycemic agent with a novel mechanism of action that reduces blood glucose levels by promoting glucose excretion in urine, achieved by selectively inhibiting sodium-glucose co-transporter 2 (SGLT2). We evaluated the effects of several selected anti-type 2 diabetes mellitus (T2DM) drugs-glimepiride, metformin, sitagliptin, pioglitazone, miglitol, nateglinide, and voglibose-on the pharmacokinetics and pharmacodynamics of tofogliflozin, and the effects of tofogliflozin on the pharmacokinetics of these anti-T2DM drugs in healthy male volunteers. METHODS: A single dose of either tofogliflozin alone, one of the anti-T2DM drugs alone, or co-administration of tofogliflozin and the anti-T2DM drug was administered to 108 healthy men. Cmax, AUCinf, and cumulative urine glucose excretion after co-administration of tofogliflozin and each of the anti-T2DM drugs was evaluated relative to the values of those parameters after administration of each drug alone. RESULTS: None of the anti-T2DM drugs had any effect on tofogliflozin exposure. Tofogliflozin had no or little effect on the exposure of any anti-T2DM drug. No anti-T2DM drug had any major effect on the cumulative urine glucose excretion induced by tofogliflozin. There were no safety concerns evident after administration of any drug alone or in co-administration. CONCLUSIONS: Neither the pharmacokinetics nor the pharmacodynamics of tofogliflozin was affected by any of the anti-T2DM drugs evaluated in this study, nor was the pharmacokinetics of any of the anti-T2DM drugs affected by tofogliflozin in healthy male volunteers.

Injury of mouse lymphocytes caused by exogenous methyl linoleate hydroperoxides in vitro.

The incubation with methyl linoleate hydroperoxides (MLHPO), a model of lipid peroxides, depressed DNA, RNA and protein syntheses of mouse thymic lymphocytes and increased the amount of thiobarbituric acid-reactive substances in lymphocytes. These phenomena were also found in the splenic lymphoblasts in the DNA synthetic phase (S-phase) obtained by mitogen. Prior culturing with all-rac-alpha-tocopherol increased DNA synthesis in splenic lymphoblasts. Electron microscopically, cytoplasmic micro-organelles of splenic lymphoblasts in the S- and G2-phases were markedly destroyed as compared with nuclei. No discernible changes were observed in not-blastotransformed lymphocytes under these experimental conditions. These findings indicate that thymic lymphocytes and splenic lymphoblasts are affected by exogenous lipid peroxides, and cytoplasmic micro-organelles of splenic lymphoblasts might be markedly damaged by exogenous lipid peroxides as compared to their nuclei.

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.

Cross-linking of L5 protein to 5 S RNA in rat liver 60-S subunits by ultraviolet irradiation.

After rat liver 60-S ribosomal subunits were irradiated with ultraviolet light at 254 nm, they were treated with EDTA and then subjected to sucrose density-gradient centrifugation to isolate 5 S RNA-protein complex. When 5 S RNA-protein was analyzed by SDS-acrylamide gel electrophoresis which dissociated noncovalent 5 S RNA-protein, two protein bands were observed. The one showed a slower mobility than the protein band (L5) of 5 S RNA-protein from non-irradiated 60 S subunit and the other showed the same mobility as L5 protein. Since the former band was shown to be specific to ultraviolet-irradiation, it was considered as cross-linked 5 S RNA-protein. After the two protein bands were iodinated with 125 I, labeled protein was extracted and treated with RNAase. Thereafter, it was analyzed by two-dimensional acrylamide gel electrophoresis, followed by autoradiography. The results indicate that the protein component of cross-linked 5 S RNA-protein is L5 protein (ribosomal protein; these proteins are designated according to the proposed uniform nomenclature. The correlation between that and our nomenclature was reported by McConkey et al. (1979) Mol. Gen. Genet. 169, 1-6. They also confirm the results previously reported (Terao, K., Takahashi, Y. and O(gata, K. (1975) Biochim. Biophys. Acta 402, 230-237).

Differences between the protein moieties of active subunits and EDTA-treated subunits of rat liver ribosomes with specific references to a 5 S rRNA - protein complex.

When active 40 S subunits of rat liver ribosomes were treated with EDTA, the conversion of 40 S subunits to 30 S subunits occurred with partial release of 13 kinds of proteins out of 29 kinds of 40 S proteins. Buy contrast, 60 S subunits completely lost one protein (L3) by EDTA-treatment with concomitant release of the fraction sedimenting at about 7 S (7 S fraction). It was found that the 7 S fraction contained 5 S ribosomal RNA as well as L3 protein having a molecular weight of 38,000. Buoyant density in CsCl of the 7 S fraction was 1.60 g/cm3, suggesting that this fraction consisted of RNA and protein at an approximately equal ratio on a weight basis. These findings, taken together with the molecular weights of 5 S rRNA (40,000) and L3 protein, may indicate that the 5 S ribosomal RNA - protein complex from rat liver 60 S subunits consists of one molecule of 5S ribosomal RNA and one molecule of L3 protein.

Identification of the protein cross-linked to 3'-terminus of 5 S RNA in rat liver ribosomal 60 S subunits.

To cross-link the 3'-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with 125I, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioautography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the alpha-chymotrypsin digest of this 125I-labeled protein-oligonucleotide complex was similar to that of the digest of 125I-labeled L5 protein. The results indicate that L5 protein binds to the 3'-terminal region of 5 S RNA in rat liver 60 S subunits.

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions.

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25 degrees C. Replacement of K+ with Na+ effectively prevented the lysis by SCN-.K+ salts of NO3-, Br- and I-, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN- than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN- is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na+ observed here was considered to be manifested by specific interactions of Na+ with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.

Presence of role of the 5SrRNA-L5 protein complex (5SRNP) in the threonyl- and histidyl-tRNA synthetase complex in rat liver cytosol.

A complex containing Thr-RS and His-RS was purified about 1000 to 2000-fold from rat liver cytosol by successive column chromatographies on Sephadex G-200, Phenyl-Sepharose CL-4B, and tRNA-Sepharose. The ratio of the specific activity of Thr-RS and His-RS was relatively constant throughout the purification steps, suggesting that the two synthetases were co-purified as a complex. Chromatographic analyses of the tRNA-Sepharose fraction by Sephadex G-150 column chromatography showed the presence of a hybrid form of the Thr-RS monomer and the His-RS monomer in addition to dimer forms of both enzymes from the pattern of activity of both enzymes. The monomer form of Thr-RS showed high activity comparable to the dimer form and the monomer form of His-RS showed definite activity. An association form of Thr-RS and His-RS dimers was detected by Sephadex G-200 chromatography of rat liver cytosol. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 55SrRNA blot analysis of the tRNA-Sepharose fraction using an antibody against ribosomal protein L5, showed the presence of ribosomal protein L5 in this fraction. These findings suggest that the presence of a 5SRNA-L5 protein complex (5SRNP) in the Thr-RS and His-RS complex. 5SRNP enhanced the activity of Thr-RS in a freshly prepared tRNA-Sepharose fraction. It also enhanced the activity of the rat liver cytosol for the attachment of [3H]threonine to endogenous tRNA. This activity was inhibited by an antibody against protein L5, and the inhibition was reversed by addition of 5SRNP. These results indicate that 5SRNP plays a role as a positive effector of Thr-RS in the complex.

Identification of neighbouring protein pairs in the rat liver 40-S ribosomal subunits cross-linked with dimethyl suberimidate.

(1) The 40-S ribosomal subunits of rat liver were treated with a bifunctional cross-linking reagent, dimethyl suberimidate. Cross-linked protein-protein dimers were separated by two-dimensional acrylamide gel electrophoresis. The stained cross-linked complexes within the gel were radioiodinated without the elution of proteins from the gel and were cloven into the original monomeric protein constituents by ammonolysis. The proteins in each dimer were finally identified by two-dimensional acrylamide gel electrophoresis of the cloven monomeric proteins, followed by radioautography of the stained gel. (2) The molecular weights of cross-linked complexes were determined by SDS-polyacrylamide gel electrophoresis and were compared with those of their constituent proteins. (3) The following dimers were proposed from these results: S3-S12 (S3 or S3a-S11), S4-S12 (S3b-S11, S5-S7 (S4-S6), S5-S22 (S4-S23 or S24), S6-S8 (S5-S7), S8-S16 (S7-S18), S17-S21 (S16--S19) and S22A-S22B (S23-S24), designated according to our numbering system [1]. The designations according to the proposed uniform nomenclature [2] are described in parentheses.

Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation.

We isolated highly intact and tightly coupled mitochondria from the rat ascites hepatoma cell line AH130 by disruption of the cell membrane by nitrogen cavitation. These isolated mitochondria were found to have essentially the same functional properties as rat liver mitochondria, but unlike the latter, hexokinase (HK) was bound to their membrane. Using the tumor mitochondrial preparation, we examined the source of ATP for phosphorylation of glucose by HK under conditions in which intra- and extramitochondrial ATP-generation systems operated separately or together. Results showed that the membrane-bound HK utilized ATP derived from the most efficiently operating ATP generation system, i.e., oxidative phosphorylation. However, when the rate of extramitochondrial ATP generation was much greater than that of oxidative phosphorylation, HK used ATP from the extramitochondrial ATP-generation system.