Detachment of Brain Pericytes from the Basal Lamina is Involved in Disruption of the Blood–Brain Barrier Caused by Lipopolysaccharide-Induced Sepsis in Mice

The blood–brain barrier (BBB) is highly restrictive of the transport of substances between blood and the central nervous system. Brain pericytes are one of the important cellular constituents of the BBB and are multifunctional, polymorphic cells that lie within the microvessel basal lamina. The present study aimed to evaluate the role of pericytes in the mediation of BBB disruption using a lipopolysaccharide (LPS)-induced model of septic encephalopathy in mice. ICR mice were injected intraperitoneally with LPS or saline and were sacrificed at 1, 3, 6, and 24 h after injection. Sodium fluorescein accumulated with time in the hippocampus after LPS injection; this hyperpermeability was supported by detecting the extravasation of fibrinogen. Microglia were activated and the number of microglia increased with time after LPS injection. LPS-treated mice exhibited a broken basal lamina and pericyte detachment from the basal lamina at 6–24 h after LPS injection. The disorganization in the pericyte and basal lamina unit was well correlated with increased microglial activation and increased cerebrovascular permeability in LPS-treated mice. These findings suggest that pericyte detachment and microglial activation may be involved in the mediation of BBB disruption due to inflammatory responses in the damaged brain.     

Paternity analysis-based inference of pollen dispersal patterns, male fecundity variation, and influence of flowering tree density and general flowering magnitude in two dipterocarp species

Background and Aims

Knowledge of pollen dispersal patterns and variation of fecundity is essential to understanding plant evolutionary processes and to formulating strategies to conserve forest genetic resources. Nevertheless, the pollen dispersal pattern of dipterocarp, main canopy tree species in palaeo-tropical forest remains unclear, and flowering intensity variation in the field suggests heterogeneity of fecundity.

Methods

Pollen dispersal patterns and male fecundity variation of Shorea leprosula and Shorea parvifolia ssp. parvifolia on Peninsular Malaysian were investigated during two general flowering seasons (2001 and 2002), using a neighbourhood model modified by including terms accounting for variation in male fecundity among individual trees to express heterogeneity in flowering.

Key Results

The pollen dispersal patterns of the two dipterocarp species were affected by differences in conspecific tree flowering density, and reductions in conspecific tree flowering density led to an increased selfing rate. Active pollen dispersal and a larger number of effective paternal parents were observed for both species in the season of greater magnitude of general flowering (2002).

Conclusions

The magnitude of general flowering, male fecundity variation, and distance between pollen donors and mother trees should be taken into account when attempting to predict the effects of management practices on the self-fertilization and genetic structure of key tree species in tropical forest, and also the sustainability of possible management strategies, especially selective logging regimes.     

Autophagy Plays a Role in Chloroplast Degradation during Senescence in Individually Darkened Leaves

Chloroplasts contain approximately 80% of total leaf nitrogen and represent a major source of recycled nitrogen during leaf senescence. While bulk degradation of the cytosol and organelles in plants is mediated by autophagy, its role in chloroplast catabolism is largely unknown. We investigated the effects of autophagy disruption on the number and size of chloroplasts during senescence. When leaves were individually darkened, senescence was promoted similarly in both wild-type Arabidopsis (Arabidopsis thaliana) and in an autophagy-defective mutant, atg4a4b-1. The number and size of chloroplasts decreased in darkened leaves of wild type, while the number remained constant and the size decrease was suppressed in atg4a4b-1. When leaves of transgenic plants expressing stroma-targeted DsRed were individually darkened, a large accumulation of fluorescence in the vacuolar lumen was observed. Chloroplasts exhibiting chlorophyll fluorescence, as well as Rubisco-containing bodies, were also observed in the vacuole. No accumulation of stroma-targeted DsRed, chloroplasts, or Rubisco-containing bodies was observed in the vacuoles of the autophagy-defective mutant. We have succeeded in demonstrating chloroplast autophagy in living cells and provide direct evidence of chloroplast transportation into the vacuole.Chloroplasts contain 75% to 80% of total leaf nitrogen mainly as proteins (Makino and Osmond, 1991). During leaf senescence, chloroplast proteins are gradually degraded as a major source of nitrogen for new growth (Wittenbach, 1978; Friedrich and Huffaker, 1980; Mae et al., 1984), correlating with a decline in photosynthetic activity, while chloroplasts gradually shrink and transform into gerontoplasts, characterized by the disintegration of the thylakoid membranes and accumulation of plastoglobuli (for a recent review, see Krupinska, 2006). Concomitantly, a decline in the cellular population of chloroplasts is also evident in many cases, for example, during natural (Kura-Hotta et al., 1990; Inada et al., 1998), dark-induced (Wittenbach et al., 1982), and nutrient-limited senescence (Mae et al., 1984; Ono et al., 1995), suggesting the existence of a whole chloroplast degradation system. Some electron microscopic studies have shown whole chloroplasts in the central vacuole, which is rich in lytic hydrolases (Wittenbach et al., 1982; Minamikawa et al., 2001). However, there is no direct evidence of chloroplasts moving into the vacuole in living cells and the mechanism of transport is not yet understood (Hörtensteiner and Feller, 2002; Krupinska, 2006).The most abundant chloroplast protein is Rubisco (EC 4.1.1.39), comprising approximately 50% of the soluble protein (Wittenbach, 1978). The amount of Rubisco decreases rapidly in the early phase of leaf senescence, although more slowly in the later phase (Friedrich and Huffaker, 1980; Mae et al., 1984). In contrast, the chloroplast number remains relatively constant, making it impossible to explain Rubisco loss solely by whole chloroplast degradation. However, the mechanism of intrachloroplastic Rubisco degradation is still unknown (for review, see Feller et al., 2008). Using immunoelectron microscopy, we previously demonstrated in naturally senescing wheat (Triticum aestivum) leaves that Rubisco is released from chloroplasts into the cytoplasm and transported to the vacuole for subsequent degradation in small spherical bodies, named Rubisco-containing bodies (RCBs; Chiba et al., 2003). Similar chloroplast-derived structures were also subsequently confirmed in senescent leaves of soybean (Glycine max) and/or Arabidopsis (Arabidopsis thaliana) by electron microscopy (Otegui et al., 2005), and recently in tobacco (Nicotiana tabacum) leaves by immunoelectron microscopy, although the authors gave them a different name, Rubisco vesicular bodies (Prins et al., 2008). RCBs have double membranes, which seem to be derived from the chloroplast envelope; thus, the RCB-mediated degradation of stromal proteins represents a potential mechanism for chloroplast shrinkage during senescence. We recently demonstrated that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole by ATG-dependent autophagy via RCBs, using leaves treated with concanamycin A, a vacuolar H⁺-ATPase inhibitor (Ishida et al., 2008). To investigate further, we wished to observe chloroplast autophagy and degradation directly in living cells to determine whether autophagy is responsible for chloroplast shrinkage and whether it is involved in the vacuolar degradation of whole chloroplasts during leaf senescence.Autophagy is known to be a major system for the bulk degradation of intracellular proteins and organelles in the vacuole in yeast and plants, or the lysosome in animals (for detailed mechanisms, see reviews by Ohsumi, 2001; Levine and Klionsky, 2004; Thompson and Vierstra, 2005; Bassham et al., 2006). In those systems, a portion of the cytoplasm, including entire organelles, is engulfed in membrane-bound vesicles and delivered to the vacuole/lysosome. A recent genome-wide search confirmed that Arabidopsis has many genes homologous to the yeast autophagy genes (ATGs; Doelling et al., 2002; Hanaoka et al., 2002; for detailed functions of ATGs, see the reviews noted above). Using knockout mutants of ATGs and a monitoring system with an autophagy marker, GFP-ATG8, numerous studies have demonstrated the presence of the autophagy system in plants and its importance in several biological processes (Yoshimoto et al., 2004; Liu et al., 2005; Suzuki et al., 2005; Thompson et al., 2005; Xiong et al., 2005, 2007; Fujiki et al., 2007; Phillips et al., 2008). These articles suggest that autophagy plays an important role in nutrient recycling during senescence, especially in nutrient-starved plants. The atg mutants exhibited an accelerated loss of some chloroplast proteins, but not all, under nutrient-starved conditions and during senescence, suggesting that autophagy is not the sole mechanism for the degradation of chloroplast proteins; other, as yet unidentified systems must be responsible for the degradation of chloroplast contents when the ATG system is compromised (Levine and Klionsky, 2004; Bassham et al., 2006). However, it still remains likely that autophagy is responsible for the vacuolar degradation of chloroplasts in wild-type plants.Prolonged observation is generally required to follow leaf senescence events in naturally aging leaves and senescence-associated processes tend to become chaotic over time. To observe chloroplast degradation over a short period, and to draw clear conclusions, a suitable experimental model of leaf senescence is required. Weaver and Amasino (2001) reported that senescence is rapidly induced in individually darkened leaves (IDLs) of Arabidopsis, but retarded in plants subjected to full darkness. In addition, Keech et al. (2007) observed a significant decrease of both the number and size of chloroplasts in IDLs within 6 d.In this study, using IDLs as a senescence model, we aimed to investigate the involvement of autophagy in chloroplast degradation. We show direct evidence for the transport of whole chloroplasts and RCBs to the vacuole by autophagy.     

Soil fertility status under shifting cultivation in East Kalimantan with special reference to mineralization patterns of labile organic matter

We investigated soil fertility status under shifting cultivation in East Kalimantan with special reference to mineralization patterns of labile soil organic matter (SOM). The soils in this region were generally strongly acidic with high Al, low bases and low pH values. A 133-day incubation experiment using fresh soils revealed that NH₄ ⁺ often accumulated during the course of N mineralization, indicating a delay of nitrification relative to N mineralization in these soils. Principal component analysis followed by stepwise multiple linear regression showed the contribution of soil physicochemical properties to mineralization patterns of SOM. Those results indicated that the overall SOM level positively contributed to the amount of readily mineralizable C and N, NH₄ at day 133, and NO₃ at day 133. The results also showed that the factors relating to soil acidity and P and K depletion, as well as accumulation of readily mineralizable C, contributed to suppress nitrification and accelerate NH₄ ⁺ accumulation and possibly subsequent N immobilization. Our results suggest that it will be difficult to establish a cropping system without a long period of fallow unless very high amounts of fertilizer as well as liming are applied in these regions.     

Nav1.7 sodium channel-induced Ca influx decreases tau phosphorylation via glycogen synthase kinase-3β in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells expressing Na_v1.7 sodium channel isoform, veratridine increased Ser⁴⁷³-phosphorylation of Akt and Ser⁹-phosphorylation of glycogen synthase kinase-3β by 217 and 195%, while decreasing Ser³⁹⁶-phosphorylation of tau by 36% in a concentration (EC₅₀ = 2.1 μM)- and time (t_1/2 = 2.7 min)-dependent manner. These effects of veratridine were abolished by tetrodotoxin or extracellular Ca²⁺ removal. Veratridine (10 μM for 5 min) increased translocation of Ca²⁺-dependent conventional protein kinase C-α from cytoplasm to membranes by 47%; it was abolished by tetrodotoxin, extracellular Ca²⁺ removal, or Gö6976 (an inhibitor of protein kinase C-α), and partially attenuated by LY294002 (an inhibitor of phosphatidylinositol 3-kinase). LY294002 (but not Gö6976) abrogated veratridine-induced Akt phosphorylation. In contrast, either LY294002 or Gö6976 alone attenuated veratridine-induced glycogen synthase kinase-3β phosphorylation by 65 or 42%; however, LY294002 plus Gö6976 completely blocked it. Veratridine (10 μM for 5 min)-induced decrease of tau phosphorylation was partially attenuated by LY294002 or Gö6976, but completely blocked by LY294002 plus Gö6976; okadaic acid or cyclosporin A (inhibitors of protein phosphatases 1, 2A, and 2B) failed to alter tau phosphorylation. These results suggest that Na⁺ influx via Na_v1.7 sodium channel and the subsequent Ca²⁺ influx via voltage-dependent calcium channel activated (1) Ca²⁺/protein kinase C-α pathway, as well as (2) Ca²⁺/phosphatidylinositol 3-kinase/Akt and (3) Ca²⁺/phosphatidylinositol 3-kinase/protein kinase C-α pathways; these parallel pathways converged on inhibitory phosphorylation of glycogen synthase kinase-3β, decreasing tau phosphorylation.     

KSHV-infected PEL cell lines exhibit a distinct gene expression profile

We analyzed the gene expression profiles of lymphocyte-originated tumor cell lines - primary effusion lymphoma (PEL) cell lines, T-cell leukemia (TCL) cell lines, Burkitt lymphoma (BL) cell lines - and two sets of normal peripheral blood mononuclear cells (PBMCs) - in order to determine characteristic gene expression profiles for each of the former three groups. And we found that these cell lines showed respective typical gene expression profiles and classified into clear four groups, PEL, TCL, BL, and normal PBMCs. Two B lymphocyte-originated tumor cell lines, PEL and BL cell lines, clearly exhibited distinct gene expression profiles, respectively. Even though there was only one line that was co-infected with both Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), KSHV seemed to govern the gene expression profile of the co-infected line. These data suggested not only that established typical tumor cell lines show a distinct gene expression profile but also that this profile may be governed by certain viruses.     

Essential role of NMDA receptor channel ε4 subunit (GluN2D) in the effects of phencyclidine, but not methamphetamine

Phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, increases locomotor activity in rodents and causes schizophrenia-like symptoms in humans. Although activation of the dopamine (DA) pathway is hypothesized to mediate these effects of PCP, the precise mechanisms by which PCP induces its effects remain to be elucidated. The present study investigated the effect of PCP on extracellular levels of DA (DA(ex)) in the striatum and prefrontal cortex (PFC) using in vivo microdialysis in mice lacking the NMDA receptor channel ε1 or ε4 subunit (GluRε1 [GluN2A] or GluRε4 [GluN2D]) and locomotor activity. PCP significantly increased DA(ex) in wildtype and GluRε1 knockout mice, but not in GluRε4 knockout mice, in the striatum and PFC. Acute and repeated administration of PCP did not increase locomotor activity in GluRε4 knockout mice. The present results suggest that PCP enhances dopaminergic transmission and increases locomotor activity by acting at GluRε4.     

Analysis of stream water temperature changes during rainfall events in forested watersheds

Despite continued interest in stream water temperature (Tw) analysis, there are few studies of Tw response to rainfall events at forested watersheds. We examined 61 sets of data on Tw for 21 rainfall events at 16 watersheds with various slope gradients (from 0.08 to 0.56) in four regions of Japan from June 2004 to December 2005. The investigation focused on the changes of specific discharge (ΔQs) and ΔTw at medium-sized watersheds (0.5–100 ha). The results clearly demonstrated different flow patterns expressed by Qs vs. Tw hysteretic loops. Those were clockwise in Period I (April–September) and counterclockwise in Period II (October–March), except for lower slope gradient at Aichi, where counterclockwise loops were observed in both periods. These differences in hysteretic loops could be explained by the differences in Tw and in response times to rainfall between surface/subsurface and groundwater flows. The response times were probably determined by the slope gradient and the vertical level of groundwater. We also found that the changes in air temperature (ΔTa) influenced ΔTw to a lesser degree than Qs. The average rainfall intensities in Period I and Period II (9.3 ± 1.7 and 5.4 ± 0.2 mm/h, respectively) affected the average values of ΔQs and ΔTw (6.62 ± 4.08 mm/h and 1.7 ± 0.4°C; 0.85 ± 0.68 mm/h and 0.9 ± 0.3°C, respectively). This indicates that slope gradient and Qs influenced ΔTw by changing the relative proportions of flow paths. In addition, the water table changes influenced the percentage of groundwater flow to the stream.