Novel and simple loading procedure of cisplatin into liposomes and targeting tumor endothelial cells.

Although intravenous administration of high levels of cisplatin (CDDP) are limited due to its severe side effects, efficient delivery of CDDP directly to the tumor should improve the therapeutic response while potentially by-passing significant side effects. High loading of CDDP into liposomes is one technique that could be used as a potential drug delivery system. Since cis-diamminedinitratoplatinum (CDDP3) is highly soluble in water and converts to CDDP in the presence of chloride ions, we encapsulated CDDP3 into liposomes in the absence of chloride ions and supplemented chloride ions to prepare CDDP-encapsulated liposomes (CDDP-Lip) resulting in a significantly improved loading efficiency of CDDP. We further conjugated the CDDP-Lip with Sialyl Lewis(X) (CDDP-SLX-Lip) because we previously demonstrated Sialyl Lewis(X) enhanced efficient accumulation of liposomes into tumors in vivo. CDDP-SLX-Lip treated mice showed a survival rate of 75% at 14 days even if a lethal level of CDDP was injected into mice. Loss of body weight was negligible and no histological abnormality was found in a variety of normal tissues. Accumulation of CDDP-SLX-Lip was about 6 times more than that of CDDP-Lip or CDDP. As the result, there was better antitumor activity of CDDP-SLX-Lip than that of CDDP-Lip with significantly less toxic effects in normal tissues.

No association found between the type 1 sigma receptor gene polymorphisms and methamphetamine abuse in the Japanese population: a collaborative study by the Japanese Genetics Initiative for Drug Abuse.

It has been suggested that individual genetic factors are involved in susceptibility to drug dependence and the manifestation of drug-induced psychosis. The aim of this study was to examine the relation between methamphetamine abusers/psychosis and the type 1 sigma receptor gene polymorphisms. Subjects comprised 143 MAP abusers and 181 healthy controls. Two polymorphisms in the type 1 sigma receptor gene, GC-241-240TT and A61C (Gln2Pro), were examined in the present study. No significant differences were observed in either polymorphism between healthy controls and MAP abusers/psychosis. In the subgroup analyses, the rate of CC genotype of A61C tended to be higher in MAP patients who had experienced spontaneous relapse without MAP use than in those who had not (P = .06, OR = 3.02 95%CI = 0.92-9.92). However, the level of this significant trend did not remain after the Bonferroni's multiple correction. This study suggests that type 1 sigma receptor gene is unlikely to play a major role in substance abuse liability and/or the development of MAP psychosis.

Sustained swimming increases erythrocyte MCT1 during erythropoiesis and ability to regulate pH homeostasis in rat.

We investigated the effect of sustained swimming exercise on the increase in monocarboxylate transporter 1 (MCT1) concentration and its ability to regulate pH homeostasis in rat erythrocytes. Male Sprague-Dawley rats aged 9 weeks were divided into sedentary and swimming groups for both 1- and 3-week experiments. The exercise group swam for 30 - 60 min/day, 5 days/week. Before and 1 and 3 weeks after initiation of the exercise, blood was collected for lactate concentration measurement during pre-exercise rest and post-exercise recovery periods. On the last day of each experiment, venous blood and erythroid cells in bone marrow were collected to assay the capacity for erythropoiesis and MCT1 concentration. In the swimming group at 0 weeks (p < 0.05), 1 week (p < 0.01) and 3 weeks (p < 0.001), the blood lactate concentration post-exercise was significantly higher than at rest. The ratio of young erythrocytes to total erythrocytes was significantly higher in the 3-week swimming group than in the sedentary group (p < 0.05). The MCT1 concentration in erythrocytes was higher in the 3-week swimming group than in the sedentary group (18 %, p < 0.05), which was found in young erythrocytes (22 %, p < 0.05) when total erythrocytes were separated into young and old fractions. The MCT1 concentration in erythroid cells was higher in both the 1-week and 3-week swimming groups than in either of the sedentary groups (27 and 28 %, respectively, p < 0.05). The pH recovery of erythrocyte suspensions at 10, 15 and 20 seconds after addition of lactate to the suspension medium was significantly faster in the 3-week swimming group than in the sedentary group (p < 0.001). These findings suggest that erythrocyte MCT1 is increased during erythropoiesis in bone marrow and that the increase of the transporter facilitates, at least partly, lactate/proton co-transport due to sustained swimming exercise in rats.

Effect of meropenem on disposition kinetics of valproate and its metabolites in rabbits.

PURPOSE: We investigated the effect of meropenem (MEPM) on the disposition kinetics of valproate (VPA) and its metabolites in rabbits. METHODS: Rabbits were given 75 mg/kg VPA intravenously with or without 300 mg/kg MEPM. RESULTS: The plamsa total clearance of VPA was significantly increased to about 1.5 times the control (6.09 mL/min/kg vs. 4.28 mL/min/kg) by MEPM (P < 0.05). The values of the area under the plasma concentration-time curve (AUC) of 2-en-VPA, a product of beta-oxidation, and VPA-glucuronide (VPA-G) were significantly decreased to about 55% and 78% of the control, respectively (P < 0.05). The cumulative urinary excretions of VPA in the control and MEPM-treated groups were 0.54% and 0.62% of the dose, respectively, whereas those of VPA-G were 45.6% and 62.5%, respectively. The urinary excretion of VPA-G was significantly increased by MEPM (P < 0.05). Further, in the case of 33.8 mg/kg VPA-G administered intravenously the AUC value of VPA-G was unchanged by MEPM, whereas that of the generated VPA was significantly decreased to about half of the control. CONCLUSIONS: The increase of the total clearance of VPA caused by MEPM appears to be a consequence of increased renal clearance of VPA-G, as well as suppression of VPA-G hydrolysis in the liver.

Identification of two down-regulated genes in rat liver allografts by mRNA differential display.

Total RNA differential display (DD) using random primers was performed for rat orthotopic liver transplantation (OLT) models. DA (RT1a) donor livers were transplanted into DA, PVG (RT1c), and LEW (RT1l) recipients: (1) syngeneic OLT (DA-DA): no rejection occurs; (2) allogeneic OLT (DA-PVG): rejection occurs, but is naturally overcome without immunosuppression; (3) allogeneic OLT (DA-LEW): animals die of acute rejection within 14 days. cDNA was isolated from selected bands, re-amplified for sequencing, and confirmed by Northern blots. Two down-regulated genes were observed in day-7 allogeneic OLT livers (DA-PVG, DA-LEW), while they were consistently expressed in day-7 syngeneic OLT (DA-DA) livers. These two genes were identified as alpha-glutathione sulfotransferase (alpha-GST) Ya gene and estrogen sulfotransferase (EST), respectively. Northern blots confirmed that their expression was down-regulated in OLT (DA-PVG) livers on days 7-26 and gradually restored. The mRNA expression of GST and EST may be good markers to predict rejection or induction of tolerance.

Gene organization of bovine BCNT that contains a portion corresponding to an endonuclease domain derived from an RTE-1 (Bov-B LINE), non-LTR retrotransposable element: duplication of an intramolecular repeat unit downstream of the truncated RTE-1.

BCNT (a protein named after Bucentaur or craniofacial development protein 1) has a unique structure in Ruminantia. Bovine BCNT contains a region of the endonuclease domain derived from a truncated RTE-1 (previously called Bov-B LINE), a non-LTR retrotransposable repetitive element, and two repeat units (intramolecular repeat, IR) each with 40 amino acids in the C-terminal region. In contrast the human and mouse BCNT proteins contain one repeat unit and lack the RTE-1-derived portion. The 3' UTR of bovine bcnt cDNA also contains an approximately 300-bp portion homologous to the 3'-part of RTE-1. We examined the bovine bcnt genomic DNA sequence to understand how the bovine bcnt gene has been organized. The sequence of 3' UTR homologous portion was found to more closely resemble the Art2 element than the bovine RTE-1. By PCR screening a bovine/hamster hybrid somatic cell panel, the bovine bcnt gene was mapped to chromosome 18, syntenic human chromosome 16q on which human BCNT is located. The bcnt genomic DNA sequence corresponding to the cDNA downstream of a RTE-1 derived portion reveals that each IR unit is flanked by both 5'-side and 3'-side introns and that 3'-UTR consists of one exon. The alignment of the above sequence with a bovine RTE-1 did not show any significant homology downstream of the endonuclease domain. On the other hand, the alignment of the intron sequences with each other revealed that the six sequential homologous segments ranging in size from 40 to 453 bp existed over a 1 kb long sequence between both the 5'- and 3'-side introns flanking each bovine IR unit. In addition, both the 174-bp of 5'-side intron and 80-bp of 3'-side intron neighboring each 120-bp IR exon are significantly homologous among the two bovine IRs, human IR and mouse IR. These results suggest that a truncated bovine RTE-1 was inserted into the intron upstream of an IR unit of an ancestor bcnt gene and that a duplication of a relatively long region that includes IR occurred in the bovine genome.

Yak1p, a DYRK family kinase, translocates to the nucleus and phosphorylates yeast Pop2p in response to a glucose signal.

POP2 protein of Saccharomyces cerevisiae is a component of a protein complex that regulates the transcription of many genes. We found that the 97th threonine residue (Thr 97) of Pop2p was phosphorylated upon glucose limitation. The Thr 97 phosphorylation occurred within 2 min after removing glucose and was reversed within 1 min after the readdition of glucose. The effects of hexokinase mutations and glucose analogs indicate that this phosphorylation is dependent on glucose phosphorylating activity. We purified a protein kinase that phosphorylates a peptide containing Thr 97 of Pop2p and identified it as Yak1p, a DYRK family kinase. Phosphorylation of Pop2p was barely detectable in a yak1Delta strain. We found that Yak1p interacted with Bmh1p and Bmh2p only in the presence of glucose. A GFP-Yak1p fusion protein shuttled rapidly between the nucleus and the cytoplasm in response to glucose. A strain with alanine substituted for Thr 97 in Pop2p showed overgrowth in the postdiauxic transition and failed to stop the cell cycle at G(1) phase in response to glucose deprivation. Thus, Yak1p and Pop2p are part of a novel glucose-sensing system in yeast that is involved in growth control in response to glucose availability.

Cloning and characterization of a novel serine/threonine protein kinase gene expressed predominantly in developing brain.

We have isolated a rat gene, sbk, that encodes a novel serine/threonine protein kinase possessing a consensus sequence for an SH3-binding domain from developing rat brain. Rat SBK comprises 417 amino-acid residues consisting of a serine/threonine protein kinase consensus sequence followed by a C-terminal proline-rich region. Sequence comparison with other known kinases revealed that sbk belongs to a novel family of serine/threonine protein kinases structurally related to a Xenopus gastrula-specific protein kinase, Pk9.7. An in vitro kinase assay demonstrated that the SBK protein autophosphorylates at serine/threonine residues. Transcripts of sbk were strongly detected in brain, and the distribution shows an association with neurons but not glial cells. A marked increase in sbk transcripts was observed in developing brain in the late embryonic stage when dramatic neuronal proliferation, migration, and maturation occur. Fluorescence in situ hybridization analysis was used to map sbk to mouse chromosome 7F1-F3 and rat chromosome 1q21. These data suggest a role for SBK in signal-transduction pathways related to the control of brain development.

Insulin increases blood flow rate in the microvasculature of cremaster muscle of the anesthetized rats.

The hemodynamic actions of insulin in skeletal muscle microvasculature are not yet well elucidated. In the present study, we investigated the effects of systemic insulin injection on arteriole and capillary diameter and blood flow rate in rat cremaster muscle, using intravital real-time confocal laser-scanning microscope system in combination with selective fluorescent labeling. Subcutaneous insulin injecbon (1 U/kg) significantly increased serum insulin levels at 15 minutes as compared with saline injection. At 15 and 30 minutes after insulin injection, blood glucose levels were significantly lower compared to saline injected controls. Arteriolar diameter was significantly increased at 15 and 30 minutes by insulin. Arteriolar erythrocyte flow velocity was significantly increased at 15 and 30 minutes. In addition, capillary erythrocyte flow velocity was increased at 15 and 30 minutes. These results demonstrated that calculated blood flow rates in capillary and arteriole increased after insulin injection.

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts).

There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BCtheta) of perfringolysin O (theta-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BCtheta on a sucrose gradient. BCtheta was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCtheta binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BCtheta binding to FLDF. The staining patterns of BCtheta and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCtheta binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BCtheta binding does not cause any damage to cell membranes, indicating that BCtheta is a useful probe for the detection of membrane rafts in living cells.

VNTR sequence on human chromosome 11p15 that affects transcriptional activity.

We found a variable number of tandem repeat (VNTR) sequence with 38-bp repetitive units in the promoter region of a gene of unknown function on human chromosome 11p15. Polymerase chain reaction amplification of this VNTR sequence using genomic DNA from 80 unrelated individuals revealed two common alleles, one with 10 (79% allelic frequency) and the other with 14 (14% allelic frequency) repetitive units, and two rare alleles with 22 (3%) or 30 (3%) repetitive units. We investigated whether differences in the length of this VNTR sequence would affect transcriptional activity of a heterologous promoter by transient transfection to NEC8, embryonal carcinoma cells derived from testis. The activity of the promoter was suppressed significantly when the VNTR region was cloned upstream, in a manner dependent on the number of repeats present in the VNTR sequence. The results implied that this polymorphic VNTR sequence might function as transcriptional regulator in 11p15, with differences in the number of repetitive units influencing efficiency of transcription of the gene lying downstream.

Leptin injection into white adipose tissue elevates renal sympathetic nerve activity dose-dependently through the afferent nerves pathway in rats.

Recent studies suggested that leptin in white adipose tissue (WAT) affected the sympathetic out flow to several tissues. We examined whether elevations of renal sympathetic nerve activity (RSNA) and blood pressure (BP) could be observed by leptin injection into WAT in rats. Injections of leptin (10 and 100 ng/ml per kg) into WAT evoked the activation of RSNA dose-dependently. Circulating sympathetic nerve activators, such as leptin, insulin, glucose and lactate, were unchanged by any doses of leptin. In addition, BP was not affected by leptin injections during a 90 min experimental period. These data suggested that leptin activated the afferent nerves through the sensors in WAT, resulting in elevation of RSNA.

Transformation of rat fibroblasts by phospholipase C-gamma1 overexpression is accompanied by tyrosine dephosphorylation of paxillin.

We previously have shown that the overexpression of phospholipase C-gamma1 (PLC-gamma1) in rat 3Y1 fibroblasts results in malignant transformation (Chang, J.-S., Noh, D.Y., Park, I.A., Kim, M;.J., Song, H., Ryu, S.H. and Suh, P.-G. (1997) Cancer Res. 57, 5465-5468). The transformed cells, which initially are in an elongated and flat form after seeding in plastic dishes, become rounded during continued culture. We found that tyrosine dephosphorylation of paxillin accompanies this morphological change of the transformed cells and that PLC-gamma1 co-immunoprecipitates together with paxillin and vice versa, but not after the cells have become round. Transformed cells growing on fibronectin-pre-coated dishes regain their flat morphology and this is accompanied by paxillin tyrosine phosphorylation. Furthermore, immunoprecipitation analysis showed that paxillin forms a heteromeric complex with PLC-gamma1 in cells grown on fibronectin. These results suggest that a complex formation between paxillin and PLC-gamma1 may play a role in cell-substrate adhesion.

Partial nuclear localization of a bovine phosphoprotein, BCNT, that includes a region derived from a LINE repetitive sequence in Ruminantia.

BCNT, named after Bucentaur, is a protein that contains a 324-amino-acid region derived from part of a long interspersed DNA sequence element (LINE) in Ruminantia. However, the unique portion is completely missing in human and mouse BCNTs. Since no significant information on their function has been obtained by homology search, we at first examined cellular localization and biochemical characteristics of bovine BCNT to get a hint on its function. Subcellular fractionation and immunohistochemical analyses using a normal bovine epithelial cell line and bovine brain revealed that a significant amount of bovine BCNT is localized in the nuclei, while the major portion is present in the cytosol. Furthermore, it was shown that bovine BCNT is a phosphoprotein and that both bovine and human BCNTs are phosphorylated by casein kinase II in vitro. These results show that BCNTs consist of a unique family, probably a substrate of casein kinase II, which may contribute further to the understanding of gene evolution.

Effects of high-fat diet intake on glucose uptake in central and peripheral tissues of non-obese rats.

We previously demonstrated that plasma glucose concentration was higher while plasma insulin concentration was lower in rats fed a high-fat diet. In the present study, we examined the effects of high-fat diet on glucose uptake in central and peripheral tissues in non-obese rats. Forty male Sprague-Dawley rats were fed high- or low-fat diets for 4 wk. Body weight and body fat accumulation were not different between the two diet groups after 4 wk. Glucose uptake in the skeletal muscles and adipose tissues, estimated by the 2-deoxy-D-glucose method, was lower in the rats fed the high-fat diet than that in the rats fed the low-fat diet, whereas uptake in the liver and pancreas did not differ between the two groups. Glucose uptake in the hypothalamus and cortex was higher in the high-fat diet group as compared with that in the low-fat diet group. These results suggest that increased plasma glucose levels in rats fed the high-fat diet were caused by a decrease in glucose uptake in the skeletal muscles and adipose tissues. Reduced plasma insulin level in the high fat diet group with no difference in glucose uptake in the pancreas may be due to increased sympathetic activity in the pancreas resulting from the increased glucose uptake in the brain regions involved in autonomic functions.

Existence of a bovine LINE repetitive insert that appears in the cDNA of bovine protein BCNT in ruminant, but not in human, genomes.

A novel protein, BCNT, originally isolated from bovine brain and named after Bucentaur, contains an internal portion that is translated from part of bovine LINE repetitive sequence (Bov-B LINE). Human cDNA highly homologous to the bovine bcnt (bbcnt) cDNA has been isolated but does not contain a sequence similar to the Bov-B LINE insert (Nobukuni, T., Kobayashi, M., Omori, A., Ichinose, S., Iwanaga, T., Takahashi, I., Hashimoto, K., Hattori, S., Kaibuchi, K., Miyata, Y., Masui, T., Iwashita, S., 1997. An Alu-linked repetitive sequence corresponding to 280 amino acids is expressed in a novel bovine protein, but not in its human homologue. J. Biol. Chem. 272, 2801-2807). In this study, we conducted a polymerase chain reaction analysis to investigate whether such a Bov-B LINE insert is present in bcnt orthologs in other animals and in the genomic sequence of the human BCNT (hBCNT) gene. The results indicate that the Bov-B LINE insert is present in the genomic sequences of bcnt orthologs from sheep, goats, axis deer, and mouse deer (chevrotain), that is in Ruminantia, but not in pigs or human. Analysis of the bbcnt genomic sequence around the Bov-B LINE insert revealed a large part of the inserted Bov-B LINE sequence to be included in an exon; this is followed by a 54-nucleotide sequence that is highly homologous to Bov-B LINE in the 3'-side intron. The hBCNT gene was isolated and found to consist of seven exons and six introns, among which the intron corresponding to the Bov-B LINE insertion site in the bbcnt genome is 16.5kb in length with no sequence similar to Bov-B LINE. Based on these results, it seems likely that the Bov-B LINE insert is derived from a long Bov-B LINE repetitive sequence transposed to an ancestral bcnt gene in Ruminantia and reformed as a new exon through new splicing sites in the transposed sequence.

An Alu-linked repetitive sequence corresponding to 280 amino acids is expressed in a novel bovine protein, but not in its human homologue.

A novel protein harboring a 280-amino acid region from an Alu-linked repetitive sequence (bovine Alu-like dimer-driven family) was isolated from a bovine brain S-100 fraction using monoclonal antibodies against a rat GTPase-activating protein that shares the same epitope. The protein has an apparent molecular mass of 97 kDa (p97). Western blot analysis using extracts prepared from various tissues showed p97 to be predominantly detected in brain and moderately in liver and lung. From sequence analysis of the cDNA encoding p97, it was found that the 840-base pair sequence homologous to a part of the bovine Alu-like dimer-driven family, which has never been shown to be expressed, occurs in the middle of the protein coding region. The protein also contains a pair of intramolecular repeats composed of 40 highly hydrophilic amino acids at the C terminus. Human cDNA homologous to p97 was cloned, and its nucleotide sequence demonstrates that the 840-base pair repetitive sequence and one of the intramolecular repeats are missing. We named p97 bovine BCNT after Bucentaur. These results show that bovine BCNT is a unique molecule and suggest that an analysis of the relationship between bovine bcnt and its human homologue may help further the understanding of gene organization and evolution.

Human rasGTPase-activating protein (human counterpart of GAP1m): sequence of the cDNA, primary structure of the protein, production and chromosomal localization.

When cDNA encoding rat rasGTPase-activating protein (rat GAP1m) was used as a probe, two partial cDNA clones of a human counterpart of rat GAP1m were isolated from a cDNA library derived from growth-arrested normal human ectocervical epithelial cells. One clone was found to be a cDNA of premature mRNA with two introns. A complete cDNA of human GAP1m was constructed by a series of reverse transcription-polymerase chain reaction (RT-PCR) using total RNA from human epidermoid carcinoma A431 cells. Human GAP1m shows 87.7% nucleotide identity to rat GAP1m in open reading frame and encodes an 850-amino acid protein that shows 89.2% identity to rat GAP1m. A 100-kDa protein was detected in A431 cells by Western blotting with anti-rat GAP1m antibody. The human GAP1m gene was mapped to chromosome 3q24-q26.

[Responses of cytokines, acute phase proteins, and polymorphonuclear cell elastase to surgical stress in the patients with esophageal cancer].

The serum levels of cytokines (interleukin-1 beta; IL-1 beta, interleukin-6; IL-6, tumor necrosis factor alpha; TNF alpha), and acute phase proteins (CRP, alpha 1-antitrypsin; alpha 1-AT, alpha 1-acid glycoprotein; alpha 1-AG, fibrinogen; FBG, pancreatic secretory trypsin inhibitor; PSTI), and the plasma concentration of polymorphonuclear cell elastase; PMN-E and white blood cell counts were measured in 18 patients with esophageal cancer who underwent radical esophagectomy through right thoracotomy and reconstruction with gastric tube. Peripheral venous blood samples were obtained before and just after operation, and on the 1st, 2nd, 3rd, 7th and 14th post-operative day. The serum concentrations of IL-6 just after operation were significantly correlated with volume of blood loss during operation and duration of thoracotomy. Plasma PMN-E levels just after operation seemed to be correlated with those factors, but its correlation was not statistically significant. Serum IL-6 levels began to increase markedly just after operation, and reached the maximum by the 1st post-operative day. This elevation preceded that of acute phase proteins, indicating that IL-6 may induce the production of acute phase proteins in vivo. Furthermore, peak serum values of IL-6 after operation were correlated with volume of blood loss and duration of thoracotomy. These results suggest that elevation of IL-6 and PMN-E levels may reflect the degree of surgical stress, and the measurement of IL-6 and PMN-E is useful for the early detection of an inflammatory response.