Nation-wide litter fall data from 21 forests of the Monitoring Sites 1000 Project in Japan

This data paper reports litter fall data collected in a network of 21 forest sites in Japan. This is the largest litter fall data set freely available in Japan to date. The network is a part of the Monitoring Sites 1000 Project launched by the Ministry of the Environment, Japan. It covers subarctic to subtropical climate zones and the four major forest types in Japan. Twenty-three permanent plots in which usually 25 litter traps were installed were established in old-growth or secondary natural forests. Litter falls were collected monthly from 2004, and sorted into leaves, branches, reproductive structures and miscellaneous. The data provide seasonal patterns and inter-annual dynamics of litter falls, and their geographical patterns, and offer good opportunities for meta-analyses and comparative studies among forests.     

Collagen XVIII and corneal reinnervation following keratectomy

The significance of collagen XVIII in the regulation of corneal reinnervation remains largely unknown. We used whole-mount immunoconfocal microscopy to localize collagen XVIII to the nerve basement membrane of wild-type (WT) mouse corneas. Transmission electron microscopy showed corneal nerve disorganization in collagen XVIII knockout mice (col18a1(-/-)). Antibody 2H3-specific neurofilament colocalized with collagens XVIII and IV and laminin-2 in WT mouse corneas, but did not colocalize with collagen IV and laminin-2 in col18a1(-/-) mouse corneas. Following keratectomy, col18a1(-/-) mice displayed decreased corneal neurite extension compared to WT mice. Our data indicate that collagen XVIII may play an important role in corneal reinnervation after wounding.     

Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels

Vasculogenesis, the in-situ assembly of angioblast or endothelial progenitor cells (EPCs), may persist into adult life, contributing to new blood vessel formation. However, EPCs are scattered throughout newly developed blood vessels and cannot be solely responsible for vascularization. Here, we identify an endothelial progenitor/stem-like population located at the inner surface of preexisting blood vessels using the Hoechst method in which stem cell populations are identified as side populations. This population is dormant in the steady state but possesses colony-forming ability, produces large numbers of endothelial cells (ECs) and when transplanted into ischaemic lesions, restores blood flow completely and reconstitutes de-novo long-term surviving blood vessels. Moreover, although surface markers of this population are very similar to conventional ECs, and they reside in the capillary endothelium sub-population, the gene expression profile is completely different. Our results suggest that this heterogeneity of stem-like ECs will lead to the identification of new targets for vascular regeneration therapy.     

Spatial distribution of canopy and subcanopy species along a sloping topography in a cool-temperate conifer-hardwood forest in the snowy region of Japan

The spatial distributions of canopy and subcanopy species (50cm stem length) were investigated within a plot extending from the top of a ridge to the valley bottom in a cool-temperate old-growth mixed forest, dominated by Cryptomeria japonica and Fagus crenata, in the snowy region of Japan. Based on the longitudinal profile of the slope, the study slope was divided into the relatively gentle upper slope position (US), the steep lower slope position (LS), the flat valley bottom (VB) and the boundary zone between the upper and lower slopes (BS). Spatial dispersal and the association patterns of species were analyzed in upperstory (10cm d.b.h) and understory (<10cm d.b.h) layers. Dominant species in the upperstory layer abruptly changed from Cryptomeria to Fagus at the BS site. In contrast, the understory trees of many species, including shade-intolerant and evergreen species, were independent of the location of conspecific upperstory trees or canopy gaps and extended their distributions on and around the BS site. Significant, diverse canopy and subcanopy species occurred at this site in both upperstory and understory layers. On the BS site, which is the lower margin of Cryptomeria-dominated vegetation, there were many medium-sized C.japonica that were killed by uprooting or breaking of the stems as a result of heavy snow pressure. It is suggested that the snow pressure gradient along a slope has a strong influence on community structure and the maintenance of diverse canopy and subcanopy species in this snowy mixed forest.     

Secondary alcohol dehydrogenase from a vinyl alcohol oligomer-degrading Geotrichum fermentans; stabilization with Triton X-100 and activity toward polymers with polymerization degrees less than 20

An NAD+-dependent secondary alcohol dehydrogenase has been found in the cells of Geotrichum fermentans WF9101 grown on 2,4-pentanediol (the constitutional unit of vinyl alcohol polymers). Although the enzyme was unstable and lost its activity very rapidly, we found that it could be purified after stabilization in a buffer containing 0.01% Triton X-100. The molecular weight of the purified enzyme was 88,000 with subunit size of molecular weight 42,000. The enzyme, which was specific for the (S)-(+)-enantiomers, was active on various secondary mono-ols and diols, notably, 2-hexanol. The K_m values at 30 °C, pH 8.0, were 240 m for 2-hexanol and 48.7 m for NAD⁺. This enzyme showed activity toward vinyl alcohol polymers with average degrees of polymerization of less than 20, and was assumed to contribute on the degradation of poly(vinyl alcohol) in synergism with other microorganisms.     

Suppression and regression of choroidal neovascularization in mice by a novel CCR2 antagonist, INCB3344

Purpose

To investigate the effect of an intravitreally administered CCR2 antagonist, INCB3344, on a mouse model of choroidal neovascularization (CNV).

Methods

CNV was induced by laser photocoagulation on Day 0 in wild type mice. INCB3344 or vehicle was administered intravitreally immediately after laser application. On Day 14, CNV areas were measured on retinal pigment epithelium (RPE)-choroid flat mounts and histopathologic examination was performed on 7 µm-thick sections. Macrophage infiltration was evaluated by immunohistochemistry on RPE-choroid flat mounts and quantified by flow cytometry on Day 3. Expression of vascular endothelial growth factor (VEGF) protein in RPE-choroid tissue was examined by immunohistochemistry and ELISA, VEGF mRNA in sorted macrophages in RPE-choroid tissue was examine by real-time PCR and expression of phosphorylated extracellular signal-regulated kinase (p-ERK 1/2) in RPE-choroid tissue was measured by Western blot analysis on Day 3. We also evaluated the efficacy of intravitreal INCB3344 to spontaneous CNV detected in Cu, Zn-superoxide dismutase (SOD1) deficient mice. Changes in CNV size were assessed between pre- and 1week post-INCB3344 or vehicle administration in fundus photography and fluorescence angiography (FA).

Results

The mean CNV area in INCB3344-treated mice decreased by 42.4% compared with the vehicle-treated control mice (p<0.001). INCB3344 treatment significantly inhibited macrophage infiltration into the laser-irradiated area (p<0.001), and suppressed the expression of VEGF protein (p = 0.012), VEGF mRNA in infiltrating macrophages (p<0.001) and the phosphorylation of ERK1/2 (p<0.001). The area of spontaneous CNV in Sod1 ^−/− mice regressed by 70.35% in INCB3344-treated animals while no change was detected in vehicle-treated control mice (p<0.001).

Conclusions

INCB3344 both inhibits newly forming CNV and regresses established CNV. Controlling inflammation by suppressing macrophage infiltration and angiogenic ability via the CCR-2/MCP-1 signal may be a useful therapeutic strategy for treating CNV associated with age-related macular degeneration.     

Induction of an incomplete autophagic response by cancer-preventive geranylgeranoic acid (GGA) in a human hepatoma-derived cell line

GGA (geranylgeranoic acid) is a natural polyprenoic acid, derivatives of which has been shown to prevent second primary hepatoma. GGA induces mitochondria-mediated PCD (programmed cell death), which may be relevant to cancer prevention. To gain further insights into GGA-induced PCD, autophagy processes were examined in human hepatoma-derived HuH-7 cells. Treatment of HuH-7/GFP (green fluorescent protein)-LC3 cells with GGA induced green fluorescent puncta in the cytoplasm within 30 min and their massive accumulation at 24 h. After 15 min of GGA treatment, a burst of mitochondrial superoxide production occurred and LC3β-I was appreciably converted into LC3β-II. GGA-induced early stages of autophagy were unequivocally confirmed by electron-microscopic observation of early/initial autophagic vacuoles. On the other hand, LC3β-II as well as p62/SQSTM1 (sequestosome 1) continuously accumulated and co-localized in the cytoplasmic puncta after GGA treatment. Furthermore, GGA treatment of HuH-7/mRFP (monomeric red fluorescent protein)-GFP-LC3 cells showed yellow fluorescent puncta, whereas glucose deprivation of the cells gave red fluorescent puncta. These results strongly suggest that GGA induces the initial phase of autophagy, but blocks the maturation process of autolysosomes or late stages of autophagy, insomuch that GGA provides substantial accumulation of autophagosomes under serum-starvation conditions in human hepatoma cells.     

A case of insulin resistant diabetes with possible antibodies to insulin receptors.

A case of a 19-year-old, non-obese female with insulin resistant diabetes mellitus and polycystic ovary syndrome was reported. The maximal insulin requirement attained 360 units per day, but a satisfactory control of diabetes did not follow. The patient's serum contained not only anti-insulin antibodies, but also possible anti-insulin receptor antibodies which were demonstrated by the 125I-insulin binding test using insulin receptors derived from human placental plasma membrane. The insulin resistance in this case was assumed to be caused primarily by possible blocking antibodies to insulin receptors and partly by anti-insulin antibodies because of the following observations. First, high serum free insulin (165 microunits/ml) without hypoglycemia indicates the presence of insulin resistance due to other factors than antiinsulin antibodies. Second, the titer of 125I-insulin binding capacity of serum was not unusually higher than those seen in chronically insulin-treated diabetics. Third, immunologically heterospecies insulin (fish insulin) was also ineffective. The clinical features such as absence of ketoacidosis and association with polycystic ovary syndrome resemble those of an unique diabetic syndrome reported previously though acanthosis nigricans and endogenous hyperinsulinemia were not found in this case. Her insulin resistance remitted spontaneously and over the next 18 months' observation, her diabetes remained regulated without insulin therapy.     

2-Methoxycinnamaldehyde inhibits tumor angiogenesis by suppressing Tie2 activation

Blood vessels are mainly composed of intraluminal endothelial cells (ECs) and mural cells adhering to the ECs on their basal side. Immature blood vessels lacking mural cells are leaky; thus, the process of mural cell adhesion to ECs is indispensable for stability of the vessels during physiological angiogenesis. However, in the tumor microenvironment, although some blood vessels are well-matured, the majority is immature. Because mural cell adhesion to ECs also has a marked anti-apoptotic effect, angiogenesis inhibitors that destroy immature blood vessels may not affect mature vessels showing more resistance to apoptosis. Activation of Tie2 receptor tyrosine kinase expressed in ECs mediates pro-angiogenic effects via the induction of EC migration but also facilitates vessel maturation via the promotion of cell adhesion between mural cells and ECs. Therefore, inhibition of Tie2 has the advantage of completely inhibiting angiogenesis. Here, we isolated a novel small molecule Tie2 kinase inhibitor, identified as 2-methoxycinnamaldehyde (2-MCA). We found that 2-MCA inhibits both sprouting angiogenesis and maturation of blood vessels, resulting in inhibition of tumor growth. Our results suggest a potent clinical benefit of disrupting these two using Tie2 inhibitors.