Phosphorylation of a heme-regulated eukaryotic initiation factor 2α kinase enhances the interaction with heat-shock protein 90 and substantially upregulates kinase activity

Heme-regulated eukaryotic initiation factor 2α kinase (HRI) functions under conditions of heme shortage caused by blood diseases such as erythropoietic protoporphyria and β-thalassemia, and retains the heme:globin ratio at 1:1 by sensing the heme concentration in reticulocytes. This HRI function is regulated by various factors including autophosphorylation and protein-protein interactions. A heat-shock protein controls HRI function, however, the molecular mechanism of catalytic regulation of HRI by the heat-shock protein is unclear. In the present study, we examined the interactions of HRI with a heat-shock protein, Hsp90, under various conditions, using a pull-down assay and measuring catalytic activity. It was found that [1] an interaction between Hsp90 and phosphorylated HRI was evident, whereas no interaction was observed between Hsp90 and HRI dephosphorylated by treatment with λ protein phosphatase; [2] Hsp90 enhanced the kinase activity of phosphorylated HRI but not dephosphorylated HRI, but this enhancement was not observed in the presence of heme; and, [3] autophosphorylation of HRI was not influenced by Hsp90. Therefore, we propose that autophosphorylation of HRI is critical for catalytic regulation by Hsp90 under heme-shortage conditions.     

Long‐term dynamics of a cladoceran community from an early stage of lake formation in Lake Fukami‐ike,Japan

An increase in nutrient levels due to eutrophication has considerable effects on lake ecosystems. Cladocerans are intermediate consumers in lake ecosystems; thus, they are influenced by both the bottom‐up and top‐down effects that occur as eutrophication progresses. The long‐term community succession of cladocerans and the effects cladocerans experience through the various eutrophication stages have rarely been investigated from the perspective of the early‐stage cladoceran community assemblage during lake formation. In our research, long‐term cladoceran community succession was examined via paleolimnological analysis in the currently eutrophic Lake Fukami‐ike, Japan. We measured the concentration of total phosphorus and phytoplankton pigments and counted cladoceran and other invertebrate subfossils in all layers of collected sediment cores, and then assessed changes in the factors controlling the cladoceran community over a 354‐year period from lake formation to the present. The cladoceran community consisted only of benthic taxa at the time of lake formation. When rapid eutrophication occurred and phytoplankton increased, the benthic community was replaced by a pelagic community. After further eutrophication, large Daphnia and high‐order consumers became established. The statistical analysis suggested that bottom‐up effects mainly controlled the cladoceran community in the lake''s early stages, and the importance of top‐down effects increased after eutrophication occurred. Total phosphorus and phytoplankton pigments had positive effects on pelagic Bosmina, leading to the replacement of the benthic cladoceran community by the pelagic one. In contrast, the taxa established posteutrophication were affected more by predators than by nutrient levels. A decrease in planktivorous fish possibly allowed large Daphnia to establish, and the subsequent increase in planktivorous fish reduced the body size of the cladoceran community.     

Novel Antibodies Reactive with Sialyl Lewis X in Both Humans and Mice Define Its Critical Role in Leukocyte Trafficking and Contact Hypersensitivity Responses

Sialyl Lewis X (sLe^x) antigen functions as a common carbohydrate determinant recognized by all three members of the selectin family. However, its expression and function in mice remain undefined due to the poor reactivity of conventional anti-sLe^x monoclonal antibodies (mAbs) with mouse tissues. Here, we developed novel anti-sLe^x mAbs, termed F1 and F2, which react well with both human and mouse sLe^x, by immunizing fucosyltransferase (FucT)-IV and FucT-VII doubly deficient mice with 6-sulfo-sLe^x-expressing cells transiently transfected with an expression vector encoding CMP-N-acetylneuraminic acid hydroxylase. F1 and F2 specifically bound both the N-acetyl and the N-glycolyl forms of sLe^x as well as 6-sulfo-sLe^x, a major ligand for L-selectin expressed in high endothelial venules, and efficiently blocked physiological lymphocyte homing to lymph nodes in mice. Importantly, both of the mAbs inhibited contact hypersensitivity responses not only when administered in the L-selectin-dependent sensitization phase but also when administered in the elicitation phase in mice. When administered in the latter phase, F1 and F2 efficiently blocked rolling of mouse leukocytes along blood vessels expressing P- and E-selectin in the auricular skin in vivo. Consistent with these findings, the mAbs blocked P- and E-selectin-dependent leukocyte rolling in a flow chamber assay. Taken together, these results indicate that novel anti-sLe^x mAbs reactive with both human and mouse tissues, with the blocking ability against leukocyte trafficking mediated by all three selectins, have been established. These mAbs should be useful in determining the role of sLe^x antigen under physiological and pathological conditions.     

Spatial and seasonal changes in species diversity of epilithic fungi along environmental gradients of a river

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Detection of Core2 β-1,6-N-Acetylglucosaminyltransferase in Post-Digital Rectal Examination Urine Is a Reliable Indicator for Extracapsular Extension of Prostate Cancer

To identify appropriate candidates for aggressive treatment such as radical prostatectomy or radiation therapy of localized prostate cancer (PCa), novel predictive biomarkers of PCa aggressiveness are essential. Core2 β-1,6-N-acetylglucosaminyltransferase-1 (GCNT1) is a key enzyme that forms core 2-branched O-glycans. Its expression is associated with the progression of several cancers. We established a mouse IgG monoclonal antibody (mAb) against GCNT1 and examined the relationship of GCNT1 expression to the clinicopathological status of PCa. Paraffin-embedded PCa specimens were analyzed by immunohistochemistry for GCNT1 expression using a newly established mouse anti-GCNT1 mAb by ourselves. GCNT1-positive tumor showed significantly higher Gleason score and larger tumor volume. The number of GCNT1-positive cases was significantly lower in cases of organ-confined disease than in cases of extracapsular extension. GCNT1-negative tumors were associated with significantly better prostate-specific antigen (PSA)-free survival compared with GCNT1-positive tumors. Multivariate analysis revealed that detection of GCNT1 expression was an independent risk factor for PSA recurrence. We established new methods for GCNT1 detection from PCa specimens. Immunoblotting was used to examine post-digital rectal examination (DRE) urine from PCa patients. Over 90% of GCNT1-positive PCa patients with high concentrations of PSA showed extracapsular extension. In conclusion, GCNT1 expression closely associates with the aggressive potential of PCa. Further research aims to develop GCNT1 detection in post-DRE urine as a marker for PCa aggressiveness.     

Contrasting effects of a cladoceran (Daphnia galeata) and a calanoid copepod (Eodiaptomus japonicus) on algal and microbial plankton in a Japanese lake, Lake Biwa

Macrozooplankton may affect algal and microbial plankton directly through grazing or predation and indirectly through nutrient regeneration. They may also affect potential prey positively by removing alternative predators. Here, we examined the effects of a cladoceran (Daphnia) and a calanoid copepod (Eodiaptomus) on algal and microbial plankton in a Japanese lake using in situ experiments in which we manipulated the nutrient supply and biomass of these macrozooplankton. The response of algal and microbial plankton to macrozooplankton was diverse and varied depending on the level of nutrient supply. Eodiaptomus seemed to feed mainly on large algae (>20 µm) and microzooplankton, while direct grazing by Daphnia on algae, bacteria, heterotrophic nanoflagellates (HNF), and microzooplankton (ciliates, heliozoa, and rotifers) was pronounced. Trophic linkages within these microbial plankton was also suggested; bacteria were grazed by HNF and these in turn were grazed by microzooplankton. When the nutrient supply was high, both HNF and microzooplankton were exposed to higher amounts of algae and lower bacterial abundance. Moreover, nutrient regeneration by daphnids and Eodiaptomus copepods seemed to differentially stimulate the growth of algae and bacteria. The results suggest that the relationship between macrozooplankton and microbial plankton cannot be fully understood without taking into consideration not only the feeding characteristics of the macrozooplankton, but also the food web structure, the subsidized algal resource, and nutrient regeneration from the macrozooplankton.     

The taxonomic position of Asian Holopedium (Crustacea: Cladocera) confirmed by morphological and genetic analyses

Limnology - The cladoceran Holopedium gibberum Zaddach, 1855 (Ctenopoda: Holopediidae) was once thought to occur broadly in the northern hemisphere, but its cryptic sister species was recently...     

Phosphorus acquisition and competitive abilities of two herbivorous zooplankton, <Emphasis Type="Italic">Daphnia pulex</Emphasis> and <Emphasis Type="Italic">Ceriodaphnia quadrangula</Emphasis>

Contrary to an expectation from the size-efficiency hypothesis, small herbivore zooplankton such as Ceriodaphnia often competitively predominate against large species such as Daphnia. However, little is known about critical feeding conditions favoring Ceriodaphnia over Daphnia. To elucidate these conditions, a series of growth experiments was performed with various types of foods in terms of phosphorus (P) contents and composition (algae and bacteria). An experiment with P-rich algae showed that the threshold food level, at which an individual’s growth rate equals zero, was not significantly different between the two species. However, the food P:C ratio, at which the growth rate becomes zero, was lower for Daphnia than for Ceriodaphnia, suggesting that the latter species is rather disfavored by P-poor algae. Ceriodaphnia showed a higher growth rate than Daphnia only when a substantial amount of bacteria was supplied together with a low amount of P-poor algae as food. These results suggest that an abundance of bacteria relative to algae plays a crucial role in favoring Ceriodaphnia over Daphnia because these are an important food resource for the former species but not for the latter.     

Structural studies of constitutive nitric oxide synthases with diatomic ligands bound

Crystal structures are reported for the endothelial nitric oxide synthase (eNOS)–arginine–CO ternary complex as well as the neuronal nitric oxide synthase (nNOS) heme domain complexed with l-arginine and diatomic ligands, CO or NO, in the presence of the native cofactor, tetrahydrobiopterin, or its oxidized analogs, dihydrobiopterin and 4-aminobiopterin. The nature of the biopterin has no influence on the diatomic ligand binding. The binding geometries of diatomic ligands to nitric oxide synthase (NOS) follow the {MXY} ⁿ formalism developed from the inorganic diatomic–metal complexes. The structures reveal some subtle structural differences between eNOS and nNOS when CO is bound to the heme which correlate well with the differences in CO stretching frequencies observed by resonance Raman techniques. The detailed hydrogen-bonding geometries depicted in the active site of nNOS structures indicate that it is the ordered active-site water molecule rather than the substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (CO, NO, as well as O₂) bound to the heme. This has important implications for the oxygen activation mechanism critical to NOS catalysis.