Hypermethylation of the <Emphasis Type="Italic">CaSR</Emphasis> and <Emphasis Type="Italic">VDR</Emphasis> genes in the parathyroid glands in chronic kidney disease rats with high-phosphate diet

Chronic kidney disease (CKD) disrupts mineral homeostasis and its representative pathosis is defined as secondary hyperparathyroidism (SHPT). SHPT occurs during the early course of progressive renal insufficiency, and is associated with mortality and cardiovascular events. SHPT results in reduction of calcium-sensing receptor (CaSR) and vitamin D receptor (VDR) in the parathyroid glands during CKD. However, the precise mechanism of CaSR and VDR reduction is largely unknown. CKD was induced through two-step 5/6 nephrectomy, and then CKD rats and sham-operated rats were maintained for 8 weeks on diets containing 0.7 % phosphorus (normal phosphate) or 1.2 % phosphorus (high phosphate). In gene expression analysis, TaqMan probes were used for quantitative real-time polymerase chain reaction. Finally, CaSR and VDR protein expressions were analyzed using immunohistochemistry. DNA methylation analysis was performed using a restriction digestion and quantitative PCR. CaSR and VDR mRNA were reduced only in CKD rats fed the high-phosphorus diets (CKD HP), then CaSR and VDR immunohistochemical expressions were compatible with gene expression assay. SHPT was then confirmed only in CKD HP rats. Furthermore, sole CKD HP rats showed the hypermethylation in CaSR and VDR genes; however, the percentage methylation of both genes was low. Although CaSR and VDR hypermethylation was demonstrated in PTGs of CKD HP rats, the extent of hypermethylation was insufficient to support the relevance between hypermethylation and down-regulation of gene expression because of the low percentage of methylation. Consequently, our data suggest that mechanisms, other than DNA hypermethylation, were responsible for the reduction in mRNA and protein levels of CaSR and VDR in PTGs of CKD HP rats.     

Analysis of intranuclear binding process of glucocorticoid receptor using fluorescence correlation spectroscopy

The diffusion properties of EGFP-hGRalpha and mutants C421G, A458T and I566 in living cells were analyzed. The wild type and mutants C421G and A458T translocated from the cytoplasm to the nucleus after addition of Dex; however, the Brownian motions of the proteins were different. The diffusion constant of wild-type GRalpha after addition of Dex slowed to 15.6% of that in the absence of Dex, whereas those of A458T and C421G slowed to 34.8% and 61.7%, respectively. This is the first report that dimer formation is less important than the binding activity of GRalpha to GRE in the living cell.     

A COMPARATIVE STUDY OF MITOSIS IN THE ONAGRACEAE

Kurabayashi , Masataka , Harlan Lewis , and Peter H. Raven . (U. California, Los Angeles.) A comparative study of mitosis in the Onagraceae. Amer. Jour. Bot. 49(9): 1003–1026. Illus. 1962. —Mitotic cycles of 46 species of Onagraceae, representing 15 genera and all of the tribes, have been examined and were found to fall into 3 modally distinct groups: (1) the tribes Fuchsieae, Lopezieae, and Circaeae; (2) the Epilobieae and Jussiaeae; (3) the Onagreae and Hauyeae. All species characterized by translocation systems, whether complex heterozygotes or variable frequencies of individual translocations, belong to group 3. Group 1 has no definite chromocenters during interphase and the chromosomes contract more or less evenly along a gradient during prophase. Groups 2 and 3 have definite chromocenters during interphase and the chromosomes become differentiated at early prophase into dense proximal and diffuse distal regions; contraction of the chromosomes does not seem to follow a gradient. In group 2, the chromosomes within a genome are much more heterogeneous in size and in the relative proportions of the differentiated segments than in group 3. Only in species belonging to group 3 are all or most of the chromosomes metacentric, subequal in size, and uniformly differentiated. The differences between the groups and within group 3 are discussed in relation to the development of translocation systems. Chromosome numbers of 9 taxa are reported for the first time.     

Cellular expression of C<Subscript>3</Subscript> and C<Subscript>4</Subscript> photosynthetic enzymes in the amphibious sedge<Emphasis Type="Italic"> Eleocharis retroflexa</Emphasis> ssp.<Emphasis Type="Italic"> chaetaria</Emphasis>

The amphibious leafless sedge Eleocharis retroflexa ssp. chaetaria expresses C₄-like biochemical characteristics in both the terrestrial and submerged forms. Culms of the terrestrial form have Kranz anatomy, whereas those of the submerged form have Kranz-like anatomy combined with anatomical features of aquatic plant leaves. We examined the immunolocalization of C₃ and C₄ enzymes in culms of the two forms. In both forms, phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase; and NAD-malic enzyme were compartmentalized between the mesophyll (M) and Kranz cells, but their levels were somewhat reduced in the submerged form. In the terrestrial form, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) occurred mainly in the Kranz cells, and weakly in the M chloroplasts. In the submerged form, the rubisco occurred at higher levels in the M cells than in the terrestrial form. In both forms, the C₄ pattern of enzyme expression was clearer in the M cells adjacent to Kranz cells than in distant M cells. During the transition from terrestrial to submerged conditions, the enzyme expression pattern changed in submerged mature culms that had been formed in air before submergence, and matched that in culms newly developed underwater. It seems that effects of both environmental and developmental factors overlap in the C₄ pattern expression in this plant.     

Role of Vanilloid Receptors in the Capsaicin-Mediated Induction of iNOS in PC12 Cells

The vanilloid receptor 1(VR1) is a nonselective cation channel that is activated by pungent vanilloid compound, extracellular protons, or noxious heat. mRNA of VR1 and vanilloid receptor 1-like receptor (VRL1) were expressed in PC12 cells, and only VRI mRNA was detected in glioma and A10 cell lines. VRI protein was demonstrated in PC12 cells by immunocytochemistry and Western blotting. Capsaicin (CPS), the VRI receptor agonist, led to an increase in intracellular calcium ion, and this effect was blocked by pretreatment with VR1 receptor antagonist capsazepin (CPZ). Treatment of PC12 cells with low concentration of CPS (5-50 microM) increased reactive oxygen species (ROS) production, and inducible nitric oxide synthase (iNOS) was expressed after CPS treatment for 24 h. These CPS-induced changes are inhibited by pretreatment of CPZ. These findings suggest that CPS-induced iNOS expression through the VR1 and/or VRL1-mediated pathway, and this may explain the CPS-mediated physiological and pathological effects in neuron system.     

Strategies for bioremediation of polychlorinated biphenyls

Polychlorinated biphenyls (PCBs) are serious environmental pollutants that threaten both the natural ecosystem and human health. For remediation of environments contaminated with PCBs, several approaches that exploit the potential of microbes to degrade PCBs have been developed. These approaches include improvement of PCB solubilization and entry into the cell, pathway and enzyme engineering, and control of enzyme expression. In this mini-review, we briefly summarize these strategies and provide potentially useful knowledge for the further improvement of the bacterial breakdown of PCBs.     

Migration from plant to plant: an important factor controlling densities of the epiphytic cladoceran <Emphasis Type="Italic">Alona</Emphasis> (Chydoridae,Anomopoda) on lake vegetation

We investigated seasonal changes in the density of epiphytic cladocerans Alona spp. (Chydoridae, Anomopoda) in two habitats, emergent and submerged aquatic plants, in Lake Suwa, Japan, from April to August 1998 and from April to November 2000. Alona had a density peak in early June on reeds (emergent) and in late June on Potamogeton malaianus (submerged). In summer, Alona density remained low in both habitats. Although density was positively correlated with the abundance of epiphytic algae, the birth rate was constant and no correlation between algal abundance and clutch size was detected. In a field experiment using ropes as an artificial substrate covered with high and low densities of epiphytic algae as food, more Alona attached to the ropes with the high density of algae. These results suggest that Alona may select food-rich habitats and migrate seasonally, and that migration is an important factor in the population dynamics of epiphytic chydorid cladocerans such as Alona. In Lake Suwa, Alona may migrate from the reed zone to the submerged macrophyte zone in June.     

Protein folding by the effects of macromolecular crowding

Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence-labeled PEG as a test macromolecule. The size of Fluorescence-labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence-labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed.