Leptin and high glucose stimulate cell proliferation in MCF-7 human breast cancer cells: reciprocal involvement of PKC-alpha and PPAR expression

Glucose concentration may be an important factor in breast cancer cell proliferation, and the prevalence of breast cancer is high in diabetic patients. Leptin may also be an important factor since plasma levels of leptin correlated with TNM staging for breast cancer patients. The effects of glucose and leptin on breast cancer cell proliferation were evaluated by examining cell doubling time, DNA synthesis, levels of cell cycle related proteins, protein kinase C (PKC) isozyme expression, and peroxisome proliferator-activated receptor (PPAR) subtypes were determined following glucose exposure at normal (5.5 mM) and high (25 mM) concentrations with/without leptin in MCF-7 human breast cancer cells. In MCF-7 cells, leptin and high glucose stimulated cell proliferation as demonstrated by the increases in DNA synthesis and expression of cdk2 and cyclin D1. PKC-alpha, PPARgamma, and PPARalpha protein levels were up-regulated following leptin and high glucose treatment in drug-sensitive MCF-7 cells. However, there was no significant effect of leptin and high glucose on cell proliferation, DNA synthesis, levels of cell cycle proteins, PKC isozymes, or PPAR subtypes in multidrug-resistant human breast cancer NCI/ADR-RES cells. These results suggested that hyperglycemia and hyperleptinemia increase breast cancer cell proliferation through accelerated cell cycle progression with up-regulation of cdk2 and cyclin D1 levels. This suggests the involvement of PKC-alpha, PPARalpha, and PPARgamma.     

Development and distribution of the early life stages of the longfin pearleye <Emphasis Type="Italic">Benthalbella linguidens</Emphasis> (Aulopiformes: Scopelarchidae) in the western North Pacific

The early life history and development of the scopelarchid Benthalbella linguidens was studied, based on 203 specimens (from 5.3 to 89.7mm in body length: BL) collected from Kuroshio, Oyashio waters and transition waters of the western North Pacific. The early life stages of B. linguidens are distinguished from those of other species of Benthalbella that inhabit the North Pacific by the characters of 62–64 myomeres in the larval stage and 26–28 anal fin rays in juvenile and transforming specimen. Larvae are elongate; notochord flexion begins at ca. 12mm BL and is completed at ca. 15mm BL. The fin ray complements are established at ca. 40mm BL. The single transforming specimen (89.7mm BL) that has peritoneal pigment was collected from transition waters. All larvae were collected from Kuroshio and transition waters from winter to early summer; however, the size of larvae in Kuroshio waters was apparently smaller than that in transition waters, with ranges of 5.3–32.4mm BL (mean 17.1) and 15.3–35.3mm BL (mean 27.5), respectively. Juveniles were distributed in transition and Oyashio waters and were absent in Kuroshio waters, where adults are commonly distributed. These occurrences of larvae and juveniles in the western North Pacific indicate that B. linguidens spawns in Kuroshio waters in winter and uses transition waters as nursery grounds.     

Identification and characterization of novel rat and human gonad-specific organic anion transporters

We have isolated three novel organic anion transporter cDNAs designated rat GST-1 (gonad-specific transporter), rat GST-2, and human GST, expressed at high levels in the testis. Rat GST-1, GST-2, and human GST consist of 748, 702, and 719 amino acids, respectively, and all molecules possess the 12 predicted transmembrane domains, which is a common structure of organic anion transporters. Northern blot analyses and in situ hybridization revealed that both of the rat molecules are highly expressed in the testis, especially in Sertoli cells, spermatogonia, and Leydig cells. Weak signals are also detected in the epididymis and ovary in adult rat. The exclusive expression of human GST mRNA in the testis was confirmed by RT-PCR. The pharmacological experiments of Xenopus laevis oocytes injected with the respective rat GST-1- and GST-2-cRNAs revealed that both rat GST-1 and GST-2 transport taurocholic acid, dehydroepiandrosterone sulfate, and T4 with Michaelis-Menten kinetics (taurocholic acid, Km = 8.9 and 2.5 microm, dehydroepiandrosterone sulfate, Km = 25.5 and 21.microm, and T4, Km = 6.4 and 5.8 for rat GST-1 and GST-2, respectively). T3 was also transported by rat GST-1 and GST-2. These data suggest that rat GST-1 and GST-2 might be one of the molecular entities responsible for transporting dehydroepiandrosterone sulfate and thyroid hormones involved in the regulation of sex steroid transportation and spermatogenesis in the gonad.     

Functional analysis of birch pollen allergen Bet v 1-specific regulatory T cells

Allergen-specific immunotherapy using peptides is an efficient treatment for allergic diseases. Recent studies suggest that the induction of CD4+ regulatory T (Treg) cells might be associated with the suppression of allergic responses in patients after allergen-specific immunotherapy. Our aim was to identify MHC class II promiscuous T cell epitopes for the birch pollen allergen Bet v 1 capable of stimulating Treg cells with the purpose of inhibiting allergic responses. Ag-reactive CD4+ T cell clones were generated from patients with birch pollen allergy and healthy volunteers by in vitro vaccination of PBMC using Bet v 1 synthetic peptides. Several CD4+ T cell clones were induced by using 2 synthetic peptides (Bet v 1(141-156) and Bet v 1(51-68)). Peptide-reactive CD4+ T cells recognized recombinant Bet v 1 protein, indicating that these peptides are produced by the MHC class II Ag processing pathway. Peptide Bet v 1(141-156) appears to be a highly MHC promiscuous epitope since T cell responses restricted by numerous MHC class II molecules (DR4, DR9, DR11, DR15, and DR53) were observed. Two of these clones functioned as typical Treg cells (expressed CD25, GITR, and Foxp3 and suppressed the proliferation and IL-2 secretion of other CD4+ T cells). Notably, the suppressive activity of these Treg cells required cell-cell contact and was not mediated through soluble IL-10 or TGF-beta. The identified promiscuous MHC class II epitope capable of inducing suppressive Treg responses may have important implication for the development of peptide-based Ag-specific immunotherapy to birch pollen allergy.     

Increase in bioluminescence intensity of firefly luciferase using genetic modification

Firefly luciferase is widely used for enzymatic measurement of ATP, and its gene is used as a reporter for gene expression experiments. From our mutant library, we selected novel mutations in Photinus pyralis luciferase with higher luminescence intensity. These included mutations at Ile423, Asp436, and Leu530. Luciferase is structurally composed of a large N-terminal active site domain (residues 1-436), a flexible linker (residues 436-440) peptide, and a small C-terminal domain (residues 440-550) facing the N domain. Thus, the mutations are located at the junction of the N-terminal domain and the flexible linker, in the flexible linker peptide, and in the tip of the C-terminal domain, respectively. Substitution of Asp436 with a nonbulky amino acid such as Gly remarkably increased the substrate affinity for ATP and d-luciferin. Substitution of Ile423 with a hydrophobic amino acid such as Leu and that of Leu530 with a positively charged amino acid such as Arg increased the substrate affinity and the turnover rate. Combining these mutations, we obtained luciferases that generate more than 10-fold higher luminescence intensity than the wild-type enzyme.     

Temperature and zooplankton size structure: climate control and basin‐scale comparison in the North Pacific

The global distribution of zooplankton community structure is known to follow latitudinal temperature gradients: larger species in cooler, higher latitudinal regions. However, interspecific relationships between temperature and size in zooplankton communities have not been fully examined in terms of temporal variation. To re‐examine the relationship on a temporal scale and the effects of climate control thereon, we investigated the variation in copepod size structure in the eastern and western subarctic North Pacific in 2000–2011. This report presents the first basin‐scale comparison of zooplankton community changes in the North Pacific based on a fully standardized data set obtained from the Continuous Plankton Recorder (CPR) survey. We found an increase in copepod community size (CCS) after 2006–2007 in the both regions because of the increased dominance of large cold‐water species. Sea surface temperature varied in an east–west dipole manner, showing the typical Pacific Decadal Oscillation pattern: cooling in the east and warming in the west after 2006–2007. The observed positive correlation between CCS and sea surface temperature in the western North Pacific was inconsistent with the conventional interspecific temperature–size relationship. We explained this discrepancy by the geographical shift of the upper boundary of the thermal niche, the 9°C isotherm, of large cold‐water species. In the eastern North Pacific, the boundary stretched northeast, to cover a large part of the sampling area after 2006–2007. In contrast, in the western North Pacific, the isotherm location hardly changed and the sampling area remained within its thermal niche throughout the study period, despite the warming that occurred. Our study suggests that while a climate‐induced basin‐scale cool–warm cycle can alter copepod community size and might subsequently impact the functions of the marine ecosystem in the North Pacific, the interspecific temperature–size relationship is not invariant and that understanding region‐specific processes linking climate and ecosystem is indispensable.     

Use of a fluorescence lifetime imaging microscope in an apoptosis assay of Ewing's sarcoma cells with a vital fluorescent probe

A fluorescence lifetime imaging microscope (FLIM) was applied to study early-stage apoptotic cells stained with a SYTO13 dye. The fluorescence lifetime of SYTO13 in healthy cells was 3.8+/-0.3ns but was reduced to 2.4+/-0.4 and 1.9+/-0.2ns after a 3-h period of incubation with SYTO13 when doxorubicin, a known inducer of apoptosis, was added to human Ewing's family tumor cells at final concentrations of 250 and 500nM, respectively, in a dose-dependent experiment. On the other hand, in a time-dependent experiment, the fluorescence lifetime decreased to 2.5+/-0.5 and 1.7+/-0.4ns at a doxorubicin concentration of 750nM after 2 and 4h, respectively. A possible explanation for these results is self-quenching induced by a change in interprobe distance that arises from the condensation of DNA during apoptosis. In this study, the FLIM system was employed to investigate early-stage apoptosis that involves only small morphological changes, suggesting the potential advantage of this method for evaluating small biological effects in living cells.     

Demonstration of the third antigenically distinct outer membrane lipoprotein (OmlA) in Actinobacillus pleuropneumoniae serotype 7

The gene encoding an outer membrane lipoprotein (OmlA) of Actinobacillus pleuropneumoniae strain WF83 (serotype 7 reference strain), designated omlA₇, was sequenced. The amino acid sequence of OmlA₇ showed 64.5 and 71.6% identity to that of OmlA from serotypes 1 (OmlA₁) and 5 (OmlA₅), respectively. The first 134 amino acids of OmlA₇ were identical to those of OmlA₅. A Southern blot analysis revealed the presence of a gene highly homologous to the omlA₇ in the reference strains of serotypes 3, 4, 6, and 7. A Western blot analysis using a specific antiserum against a recombinant OmlA₇ detected expression of the homologous proteins in the serotypes 4, 6, and 7 reference strains and a serotype 3 field strain, but not in a serotype 3 reference strain. The data demonstrate the third antigenically distinct OmlA is expressed in A. pleuropneumoniae.