Identification of intracellular target proteins of the calcium-signaling protein S100A12.

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+-dependent, whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone-like function which is Ca2+-dependent.     

Rice plant development: from zygote to spikelet

Rice is becoming a model plant in monocotyledons and a model cereal crop. For better understanding of the rice plant, it is essential to elucidate the developmental programs of the life cycle. To date, several attempts have been made in rice to categorize the developmental processes of some organs into substages. These studies are based exclusively on the morphological and anatomical viewpoints. Recent advancement in genetics and molecular biology has given us new aspects of developmental processes. In this review, we first describe the phasic development of the rice plant, and then describe in detail the developmental courses of major organs, leaf, root and spikelet, and specific organs/tissues. Also, for the facility of future studies, we propose a staging system for each organ.     

Biochemical properties and immunohistochemical localization of carbonic anhydrase in the sacculus of the inner ear in the salmon Oncorhynchus masou

Carbonic anhydrase (CA) in the inner ear sacculus was examined by activity assay, Western blotting and immunohistochemistry to determine its role in otolith calcification. An immunoreactive protein with a molecular mass of approximately 28 kDa was detected by Western blotting. The CO2 hydration activity in the cytosol fraction of the sacculus was 5.4 units/mg protein, while little or no activity was detected in the nuclear and mitochondrial fractions. The enzyme activity was highly inhibited by acetazolamide. The concentration of 50% inhibition was 8.16 nM and the inhibition constant of the activity was 8.25 nM. Transitional and squamous epithelial cells of the sacculus were immunopositive with an anti-CA II antibody, but sensory epithelial cells and mitochondria-rich cells in the transitional epithelium were not. These results suggest that transitional epithelial cells other than mitochondria-rich cells and squamous epithelial cells play an important role in otolith calcification by supplying bicarbonate to otoliths and/or by eliminating protons from endolymph.     

Rice DECUSSATE controls phyllotaxy by affecting the cytokinin signaling pathway

Phyllotaxy is defined as the spatial arrangement of leaves on the stem. The mechanism responsible for this extremely regular pattern is one of the most fascinating enigmas in plant biology. In this study, we identified a gene regulating the phyllotactic pattern in rice. Loss‐of‐function mutants of the DECUSSATE (DEC) gene displayed a phyllotactic conversion from normal distichous pattern to decussate. The dec mutants had an enlarged shoot apical meristem with enhanced cell division activity. In contrast to the shoot apical meristem, the size of the root apical meristem in the dec mutants was reduced, and cell division activity was suppressed. These phenotypes indicate that DEC has opposite functions in the shoot apical meristem and root apical meristem. Map‐based cloning revealed that DEC encodes a plant‐specific protein containing a glutamine‐rich region and a conserved motif. Although its molecular function is unclear, the conserved domain is shared with fungi and animals. Expression analysis showed that several type A response regulator genes that act in the cytokinin signaling pathway were down‐regulated in the dec mutant. In addition, dec seedlings showed a reduced responsiveness to exogenous cytokinin. Our results suggest that DEC controls the phyllotactic pattern by affecting cytokinin signaling in rice.     

Histochemical detection of l-gulonolactone: phenazine methosulfate oxidoreductase activity in several mammals with special reference to synthesis of vitamin C in primates

Summary An improved detection of activity of l-gulonolactone oxidase, which is responsible for the final oxidative step in the synthetic process of l-ascorbate from glucose in animals, was achieved using phenazine methosulfate and cyanide. Cold acetone fixation eliminated non-specific deposition of formazan on lipid droplets. The specificity of the method was tested and proven by a biological control, histochemical controls, inhibitors and activators. By application of the method, strong reactivity was found in the cytoplasm of centrilobular parenchymal cells of livers of the opossum, rat, ground squirrel and flying squirrel. Staining of dog liver was moderate and centrilobular. Prosimians were strongly positive: The centrilobular localization was found in the tree shrew and galago; slow lorises and some pottos showed strong reactivity in centrilobular cells and some peripheral cells as well. These prosimians seem to be able to synthesize l-ascorbate as many lower mammals are. On the contrary, true simians (i.e. the squirrel monkey, spider monkey, rhesus monkey and chimpanzee) were negative as guinea pigs were, suggesting their probable inability for l-ascorbate synthesis.Visiting scientist from the Department of Anatomy, Tokyo Medical and Dental University, Tokyo, Japan. T. R. Shanthaveerappa in previous publications, also fellow, Department of Anesthesiology, Emory University.     

Changes in predominant energy substrate after hepatectomy

The changes in the energy substrate utilized by the remnant liver were studied in relation to the changes in the cellular energy status of 25 and 70% hepatectomized rabbits. In 25% hepatectomized rabbits, the energy charge $\text{(}\text{(}\text{ATP}\text{+0.5}\text{ADP}\text{)}\text{(}\text{ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}\text{)}$ level of the remnant liver remained unchanged, the energy substrate of which was predominantly glucose, rather than fatty acid. In contrast, in 70% hepatectomized rabbits, the energy production by the mitochondria was mainly dependent upon fatty acid oxidation at the early period after hepatectomy when the energy charge level decreased remarkably, and then upon glucose oxidation, concomitant with the restoration of the energy charge. It is suggested that the changes in the energy substrate utilized are closely related to those in the energy charge level and the mitochondrial phosphorylative activity of the remnant liver following hepatectomy.     

Detection of polyglutamine protein oligomers in cells by fluorescence correlation spectroscopy

Abnormal aggregation of misfolded proteins and their deposition as inclusion bodies in the brain have been implicated as a common molecular pathogenesis of neurodegenerative diseases including Alzheimer, Parkinson, and the polyglutamine (poly(Q)) diseases, which are collectively called the conformational diseases. The poly(Q) diseases, including Huntington disease and various types of spinocerebellar ataxia, are caused by abnormal expansions of the poly(Q) stretch within disease-causing proteins, which triggers the disease-causing proteins to aggregate into insoluble beta-sheet-rich amyloid fibrils. Although oligomeric structures formed in vitro are believed to be more toxic than mature amyloid fibrils in these diseases, the existence of oligomers in vivo has remained controversial. To explore oligomer formation in cells, we employed fluorescence correlation spectroscopy (FCS), which is a highly sensitive technique for investigating the dynamics of fluorescent molecules in solution. Here we demonstrate direct evidence for oligomer formation of poly(Q)-green fluorescent protein (GFP) fusion proteins expressed in cultured cells, by showing a time-dependent increase in their diffusion time and particle size by FCS. We show that the poly(Q)-binding peptide QBP1 inhibits poly(Q)-GFP oligomer formation, whereas Congo red only inhibits the growth of oligomers, but not the initial formation of the poly(Q)-GFP oligomers, suggesting that FCS is capable of identifying poly(Q) oligomer inhibitors. We therefore conclude that FCS is a useful technique to monitor the oligomerization of disease-causing proteins in cells as well as its inhibition in the conformational diseases.     

Hindgut microbes, fermentation and their seasonal variations in Hokkaido native horses compared to light horses

Fecal bacteria and protozoa of Hokkaido native horses and light horses were enumerated to compare seasonal variation in hindgut microbes and fermentation between the two breeds. Fecal samples were collected in winter and summer from eight horses (four for each breed) that had been reared together under the same conditions after birth (on woodland pasture in winter and on grassland pasture for the rest of the year). Total fecal bacteria counts for both breeds showed temporal variation, with the highest levels occurring in summer (P<0.05). For both breeds, Gram-negative rods were the major constituents (58–69%) and showed higher counts in winter (P<0.05) than in summer. Total protozoa counts in both breeds were lower in winter than in summer (P<0.05). The proportion of large cellulolytic protozoa such as Cochliatoxum periachtum was increased (P<0.05) in winter, and this tended to be more pronounced in native horses. Although total volatile fatty acids (VFA) in feces were lower in winter (P<0.05), the reduction was smaller in native horses (P<0.05). Fecal VFA pattern showed a shift toward more acetate and less propionate production in winter regardless of the horse breed. Evaluation of digestive tract organs in 12 animals showed that the relative weight of the colon in body weight or total digestive tract weight is larger in native horses than in light horses (P<0.05). The present results suggest that hindgut microbial adaptation to winter diets occurs to a greater extent in native horses, as partly characterized by advantages in anatomy.     

Occurrence of an alpha-galacturonosyl-ceramide in the dioxin-degrading bacterium Sphingomonas wittichii

The chemical structure of two glycosphingolipids (GSLs) found in the dioxin-degrading bacterium Sphingomonas wittichii strain RW1 was investigated by means of mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. One of the GSLs was alpha-D-glucuronosyl-ceramide, commonly present in Sphingomonas spp., and the other was proved to be alpha-D-galacturonosyl-ceramide, whose sugar configuration has not been reported before. In both GSLs the ceramide portion was composed of myristic acid or 2-hydroxy-myristic acid as the fatty acid, and 2-amino-1,3-octadecanediol or 2-amino-cis-13,14-methylene-1,3-eicosanediol as the dihydrosphingosine.