Relationships between Micro-Vascular and Iodine-Staining Patterns in the Vicinity of the Tumor Front of Superficial Esophageal Squamous Carcinoma

Objective

The aim of the present study was to clarify differences between micro-vascular and iodine-staining patterns in the vicinity of the tumor fronts of superficial esophageal squamous cell carcinomas (ESCCs).

Methods

Ten consecutive patients with ESCCs who were treated by endoscopic submucosal dissection (ESD) were enrolled. At the edge of the iodine-unstained area, we observed 183 sites in total using image-enhanced magnifying endoscopy. We classified the micro-vascular and iodine-staining patterns into three types: Type A, in which the line of vascular change matched the border of the iodine-unstained area; Type B, in which the border of the iodine-unstained area extended beyond the line of vascular change; Type C, in which the line of vascular change extended beyond the border of the iodine-unstained area. Then, by examining histopathological sections, we compared the diameter of intra-papillary capillary loops (IPCLs) in cancerous areas and normal squamous epithelium.

Results

We investigated 160 sites that the adequate quality of pictures were obtained. There was no case in which the line of vascular change completely matched the whole circumference of the border of an iodine-unstained area. Among the 160 sites, type A was recognized at 76 sites (47.5%), type B at 79 sites (49.4%), and type C at 5 sites (3.1%). Histological examination showed that the mean diameter of the IPCLs in normal squamous epithelium was 16.2±3.7μm, whereas that of IPCLs in cancerous lesions was 21.0±4.4μm.

Conclusions

The development of iodine-unstained areas tends to precede any changes in the vascularity of the esophageal surface epithelium.     

Repetitive Glucose Spikes Accelerate Atherosclerotic Lesion Formation in C57BL/6 Mice

Background

A number of epidemiological studies demonstrated that postprandial hyperglycemia is a risk factor for cardiovascular disease in individuals with impaired glucose tolerance. Although several laboratory studies have addressed the plausible causal role of postprandial acute hyperglycemia (glucose spikes) in the development of atherosclerosis, there is little convincing evidence in vivo whether the atherosclerotic lesion formation can be accelerated solely by glucose spikes. Here, we assessed the effect of repetitive glucose spikes on atherosclerotic lesion formation in mice.

Methods

Female C57BL/6 mice were fed an atherogenic diet from 8 to 28 weeks of age. During the atherogenic diet feeding period, the mice orally received a glucose solution (50 mg glucose/mouse; G group) or water (W group) twice daily, 6 days a week. Atherosclerotic lesion formation in the aortic sinus was quantitatively analyzed in serial cross-sections by oil red O staining.

Results

G group mice showed transient increases in blood glucose level (~5 mmol/L above W group), and the levels returned to levels similar to those in W group mice within 60 min. No significant differences in glucose tolerance, insulin sensitivity, and plasma lipid profiles were observed after the 20-week repetitive administration between the 2 groups. G group mice showed an approximately 4-fold greater atherosclerotic lesion size in the aortic sinus than W group mice. Gene expression levels of Cd68 and Icam1 in the thoracic aorta were higher in G group mice than in W group mice.

Conclusions

These results indicate that glucose spikes can accelerate atherosclerotic lesion formation, with little influence on other metabolic disorders. Repetitive glucose administration in wild-type mice may serve as a simple and useful approach to better understanding the causal role of glycemic spikes in the development of atherosclerosis.     

LC3B is indispensable for selective autophagy of p62 but not basal autophagy

Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.     

Observation of Small Cluster Formation in Concentrated Monoclonal Antibody Solutions and Its Implications to Solution Viscosity

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals. It is hypothesized that some concentrated mAb solutions exhibit formation of a solution phase consisting of reversibly self-associated aggregates (or reversible clusters), which is speculated to be responsible for their distinct solution properties. Here, we report direct observation of reversible clusters in concentrated solutions of mAbs using neutron spin echo. Specifically, a stable mAb solution is studied across a transition from dispersed monomers in dilute solution to clustered states at more concentrated conditions, where clusters of a preferred size are observed. Once mAb clusters have formed, their size, in contrast to that observed in typical globular protein solutions, is observed to remain nearly constant over a wide range of concentrations. Our results not only conclusively establish a clear relationship between the undesirable high viscosity of some mAb solutions and the formation of reversible clusters with extended open structures, but also directly observe self-assembled mAb protein clusters of preferred small finite size similar to that in micelle formation that dominate the properties of concentrated mAb solutions.     

Identification of the linkage group of the Z sex chromosomes of the sand lizard (Lacerta agilis,Lacertidae) and elucidation of karyotype evolution in lacertid lizards

The sand lizard (Lacerta agilis, Lacertidae) has a chromosome number of 2n?=?38, with 17 pairs of acrocentric chromosomes, one pair of microchromosomes, a large acrocentric Z chromosome, and a micro-W chromosome. To investigate the process of karyotype evolution in L. agilis, we performed chromosome banding and fluorescent in situ hybridization for gene mapping and constructed a cytogenetic map with 86 functional genes. Chromosome banding revealed that the Z chromosome is the fifth largest chromosome. The cytogenetic map revealed homology of the L. agilis Z chromosome with chicken chromosomes 6 and 9. Comparison of the L. agilis cytogenetic map with those of four Toxicofera species with many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) showed highly conserved linkage homology of L. agilis chromosomes (LAG) 1, 2, 3, 4, 5(Z), 7, 8, 9, and 10 with macrochromosomes and/or macrochromosome segments of the four Toxicofera species. Most of the genes located on the microchromosomes of Toxicofera were localized to LAG6, small acrocentric chromosomes (LAG11–18), and a microchromosome (LAG19) in L. agilis. These results suggest that the L. agilis karyotype resulted from frequent fusions of microchromosomes, which occurred in the ancestral karyotype of Toxicofera and led to the disappearance of microchromosomes and the appearance of many small macrochromosomes.     

Identification of protein kinase disruptions as suppressors of the calcium sensitivity of S. cerevisiae Δptp2 Δmsg5 protein phosphatase double disruptant

The double disruptant of the S. cerevisiae protein phosphatase (PPase) genes, PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes calcium-sensitive growth (Ca^s). Previous study using Fluorescent-activated cell sorting (FACS) analysis showed that this growth defect with calcium occurs at G1–S transition in the cell cycle. We discovered that six non-essential protein kinase (PKase) disruptions (Δbck1, Δmkk1, Δslt2/Δmpk1, Δmck1, Δssk2 and Δyak1) suppressed the Ca^s-phenotype of the Δptp2 Δmsg5 double disruptant. Bck1p, Mkk1p and Slt2p are components of the mitogen-activated protein kinase (MAPK) cascade of cell wall integrity pathway (Slt2 pathway), and Mck1p is its down regulator. Ssk2p is the MAPK kinase kinase of the high-osmolarity glycerol (HOG) pathway, while Yak1p is a negative regulator for the cAMP-dependent PKA pathway. FACS analysis revealed that only the disruption of Δssk2 and Δyak1 but not Δbck1, Δmkk1, Δslt2 and Δmck1 was able to suppress the delayed G1–S transition, suggesting that suppression of the growth defect is not always accompanied by suppression of the G1–S transition delay. The discovery of these PKases as suppressors revealed that in addition to the previously anticipated Slt2 pathway, HOG, Yak1p and Mck1p regulatory pathways may also be involved in the calcium sensitivity of the Δptp2 Δmsg5 double disruptant.     

Strategy for comprehensive identification of human N‐myristoylated proteins using an insect cell‐free protein synthesis system

To establish a strategy for the comprehensive identification of human N‐myristoylated proteins, the susceptibility of human cDNA clones to protein N‐myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an insect cell‐free protein synthesis system. One‐hundred‐and‐forty‐one cDNA clones with N‐terminal Met‐Gly motifs were selected as potential candidates from ～2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N‐myristoylation was evaluated using fusion proteins, in which the N‐terminal ten amino acid residues were fused to an epitope‐tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N‐myristoylated. The metabolic labeling experiments both in an insect cell‐free protein synthesis system and in the transfected COS‐1 cells using full‐length cDNA revealed that 27 out of 29 proteins were in fact N‐myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N‐myristoylated proteins that have not been reported previously to be N‐myristoylated, indicating that this strategy is useful for the comprehensive identification of human N‐myristoylated proteins from human cDNA resources.     

Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat (Prionailurus bengalensis euptilurus)

Although the Tsushima leopard cat (Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17β and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17β concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy.     

Purification and characterization of a stable oxygen-evolving Photosystem II complex from a marine centric diatom, Chaetoceros gracilis

Oxygen-evolving Photosystem II particles (crude PSII) retaining a high oxygen-evolving activity have been prepared from a marine centric diatom, Chaetoceros gracilis (Nagao et al., 2007). The crude PSII, however, contained a large amount of fucoxanthin chlorophyll a/c-binding proteins (FCP). In this study, a purified PSII complex which was deprived of major components of FCP was isolated by one step of anion exchange chromatography from the crude PSII treated with Triton X-100. The purified PSII was still associated with the five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31 and PsbU, and showed a high oxygen-evolving activity of 2135 μmol O₂ (mg Chl a)^− 1 h^− 1 in the presence of phenyl-p-benzoquinone which was virtually independent of the addition of CaCl₂. This activity is more than 2.5-fold higher than the activity of the crude PSII. The activity was completely inhibited by 3-(3,4)-dichlorophenyl-(1,1)-dimethylurea (DCMU). The purified PSII contained 42 molecules of Chl a, 2 molecules of diadinoxanthin and 2 molecules of Chl c on the basis of two molecules of pheophytin a, and showed typical absorption and fluorescence spectra similar to those of purified PSIIs from the other organisms. In this study, we also found that the crude PSII was significantly labile, as a significant inactivation of oxygen evolution, chlorophyll bleaching and degradation of PSII subunits were observed during incubation at 25 °C in the dark. In contrast, these inactivation, bleaching and degradation were scarcely detected in the purified PSII. Thus, we succeeded for the first time in preparation of a stable PSII from diatom cells.     

Unique features of the rice blast resistance <Emphasis Type="Italic">Pish</Emphasis> locus revealed by large scale retrotransposon-tagging

Background  

R gene-mediated resistance is one of the most effective mechanisms of immunity against pathogens in plants. To date some components that regulate the primary steps of plant immunity have been isolated, however, the molecular dissection of defense signaling downstream of the R proteins remains to be completed. In addition, R genes are known to be highly variable, however, the molecular mechanisms responsible for this variability remain obscure.