Tandem repeat of a seven-bladed beta-propeller domain in oligoxyloglucan reducing-end-specific cellobiohydrolase

Oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH; EC 3.2.1.150) is an exoglucanase that recognizes the reducing end of oligoxyloglucan and releases two glucosyl residue segments from the main chain. The X-ray crystal structure of OXG-RCBH determined at 2.2 A resolution reveals a unique feature of this enzyme; OXG-RCBH consists of a tandem repeat of two similar domains, which are both folded into seven-bladed beta-propeller structures. The sequence alignment of the propeller blades, based on the structure, indicates that a weak repeat of the amino acid sequence occurred seven times to construct each domain. There is a cleft that can accommodate the substrate oligosaccharide between the two domains, which is a putative substrate binding subsite. Mutation of either Asp35 or Asp465, located in the putative catalytic center, to Asn resulted in a protein with no detectable catalytic activity, indicating the critical role of these amino acids in catalysis.     

Content and chemical compositions of cerebrosides in lactose-assimilating yeasts

Cerebrosides were found in ten lactose-assimilating yeasts. Representative component ceramide moieties of cerebrosides from nine of these yeasts contained 9-methyl-4-trans, 8-trans-sphingadienine, and 2-hydroxy fatty acid with carbon chain lengths of 16 or 18. The major ceramide moieties in Brettanomyces anomalus, however, differed from those in other yeasts, and were predominately moieties containing 2-hydroxymyristic acid. Thus we found that various cerebroside molecular species are present in yeasts.     

Pre- and postsynaptic inhibition mediated by GABA(B) receptors in rat ventrolateral periaqueductal gray neurons

The present study examined the actions of a GABA(B)-receptor agonist, baclofen, on synaptic transmission in rat ventrolateral periaqueductal gray (PAG) neurons of brainstem slices by using whole-cell voltage-clamp recordings. Baclofen (10 microM) induced a slow outward current (peak amplitude: 30.1+/-3.1pA, n=13) at -70mV, which persisted in the presence of tetrodotoxin (0.5 microM) and was diminished in the presence of postsynaptic intracellular K(+)-channel blockers (Cs(+) and TEA) and GDP-beta-S, indicating a direct postsynaptic depression mediated by K(+) channels and G proteins. Baclofen (10 microM) also decreased the frequency of both glutamatergic spontaneous EPSC (by 36+/-7%, n=11) and GABAergic spontaneous IPSC (by 37+/-12%, n=6) without changes in their amplitudes, indicating its presynaptic inhibitions. Taken together, the activation of postsynaptic GABA(B) receptors inhibits ventrolateral PAG neurons directly. At the same time, activating presynaptic GABA(B) receptors on glutamatergic and GABAergic nerve terminals inhibits glutamate and GABA release, respectively. The overall effects might influence an output of ventrolateral PAG neurons that build up the descending pain control system to the spinal dorsal horn.     

Correlation between development of female flower buds and expression of the CS-ACS2 gene in cucumber plants

Ethylene plays a key role in sex determination of cucumber flowers. Gynoecious cucumber shoots produce more ethylene than monoecious shoots. Because monoecious cucumbers produce both male and female flower buds in the shoot apex and because the relative proportions of male and female flowers vary due to growing conditions, the question arises as to whether the regulation of ethylene biosynthesis in each flower bud determines the sex of the flower. Therefore, the expression of a 1-aminocyclopropane-1-carboxylic acid synthase gene, CS-ACS2, was examined in cucumber flower buds at different stages of development. The results revealed that CS-ACS2 mRNA began to accumulate just beneath the pistil primordia of flower buds at the bisexual stage, but was not detected prior to the formation of the pistil primordia. In buds determined to develop as female flowers, CS-ACS2 mRNA continued to accumulate in the central region of the developing ovary where ovules and placenta form. In gynoecious cucumber plants that produce only female flowers, accumulation of CS-ACS2 mRNA was detected in all flower buds at the bisexual stage and at later developmental stages. In monoecious cucumber, flower buds situated on some nodes accumulated CS-ACS2 mRNA, but others did not. The proportion of male and female flowers in monoecious cucumbers varied depending on the growth conditions, but was correlated with changes in accumulation of CS-ACS2 mRNA in flower buds. These results demonstrate that CS-ACS2-mediated biosynthesis of ethylene in individual flower buds is associated with the differentiation and development of female flowers.     

Relationship between the size of the bottleneck 15 A from iron in the main channel and the reactivity of catalase corresponding to the molecular size of substrates

A catalase that exhibits a high level of activity and a rapid reaction with organic peroxides has been purified from Exiguobacterium oxidotolerans T-2-2T (EKTA catalase). The amino acid sequence of EKTA catalase revealed that it is a novel clade 1 catalase. Amino acid residues in the active site around the protoheme are conserved in the primary structure of EKTA catalase. Although the general interactions of molecules larger than hydrogen peroxide with catalases are strongly inhibited because of the selection role of long and narrow channels in the substrate reaching the active site, the formation rate of reactive intermediates (compound I) in the reaction of EKTA catalase with peracetic acid is 77 times higher than that of bovine liver catalase (BLC) and 1200 times higher than that of Micrococcus luteus catalase (MLC). The crystal structure of EKTA catalase has been determined and refined to 2.4 A resolution. The main channel structure of EKTA catalase is different from those of BLC and MLC. The rate constant of compound I formation in catalases decreased with an increase in the molecular size of the substrate. For EKTA catalase with a larger bottleneck 15 A from the iron (entrance of narrow channel) in the main channel, a lower rate of reduction in compound I formation rate with an increase in the molecular size of substrates was found. The increase in the rate constant of compound I formation in these catalases was directly proportional to the increase in the size of the bottleneck in the main channel when molecules of substrates larger than H2O2, such as organic peroxides, are used in the reaction. The results indicate that the size of the bottleneck in the main channel in catalase is an important factor in defining the rate of compound I formation corresponding to the molecular size of the substrates, and this was demonstrated. The Leu149-Ile180 and Asp109-Met167 combinations at the entrance of the narrow channel in EKTA catalase determine the size of the bottleneck, and each atom-to-atom distance for the combination of residues was larger than those of corresponding combinations of amino acid residues in BLC and MLC. The combination of these four amino acids is quite specific in EKTA catalase as compared with the combinations in other catalases in the gene database (compared with more than 432 catalase genes in the database).     

Functional Visual Acuity of Early Presbyopia

Purpose

To evaluate visual function in patients with early presbyopia using the functional visual acuity (FVA) test.

Methods

This study included 27 eyes of 27 healthy older volunteers (mean age, 44.1 ± 2.6 years) and 14 eyes of 14 healthy young volunteers (mean age, 28.4±4.8 years). The distance-corrected visual acuity (DCVA), distance-corrected near VA (DCNVA), subjective amplitude of accommodation (AA), and distance and near pupillary diameters were measured. The distance FVA and distance-corrected near FVA (DCNFVA) were measured using the FVA Measurement System. The standard Schirmer test and standard tear break-up time measurement also were performed.

Results

The logarithm of the minimum angle of resolution (logMAR) DCVA was better than 0 in all subjects. The percentages of subjects with logMAR DCNVA below 0 was significantly lower in the presbyopia group than in the young group. The DCNFVA in the presbyopia group was significantly (P < 0.001) poorer than the DCNVA in that group. Significant linear negative correlations were seen between the DCNVA and AA (r = -0.507, P < 0.001) and the DCNFVA and AA (r = -0.681, P < 0.001) in the older subjects. Stepwise regression analysis showed that only the AA was a significant factor predictive of the DCNFVA in the presbyopia group. Tear function parameters were not adopted in the regression model.

Conclusions

Measurement of the DCNFVA can detect decreased AA in early presbyopia better than measurement of the conventional near VA. The DCNFVA is a good index for early presbyopia.     

Production of a New Acidic Polysaccharide,Succinoglucan by Alcaligenes faecalis var. myxogenes

A slimy non-spore-forming bacterium strain 10C3 isolated from soil was motile with peritrichous flagella and named Alcaligenes faecalis var. myxogenes. Studies were made on the conditions necessary for maximal production of a new acidic succinoglucan polysaccharide by this strain in shaken cultures. Much production was observed with sucrose, glucose, xylose, galactose, cellobiose, maltose, fructose, mannose and rhamnose. The yield was greatest with sucrose and decreased in order with the above sugars from about 36 to 23 per cent. The most suitable medium contained 4 per cent sugar, 0.5 per cent yeast extract and one per cent calcium carbonate in tap water. The optimum temperature was 28°C.     

Three-Dimensional Distribution of Sensory Stimulation-Evoked Neuronal Activity of Spinal Dorsal Horn Neurons Analyzed by In Vivo Calcium Imaging

The spinal dorsal horn comprises heterogeneous populations of interneurons and projection neurons, which form neuronal circuits crucial for processing of primary sensory information. Although electrophysiological analyses have uncovered sensory stimulation-evoked neuronal activity of various spinal dorsal horn neurons, monitoring these activities from large ensembles of neurons is needed to obtain a comprehensive view of the spinal dorsal horn circuitry. In the present study, we established in vivo calcium imaging of multiple spinal dorsal horn neurons by using a two-photon microscope and extracted three-dimensional neuronal activity maps of these neurons in response to cutaneous sensory stimulation. For calcium imaging, a fluorescence resonance energy transfer (FRET)-based calcium indicator protein, Yellow Cameleon, which is insensitive to motion artifacts of living animals was introduced into spinal dorsal horn neurons by in utero electroporation. In vivo calcium imaging following pinch, brush, and heat stimulation suggests that laminar distribution of sensory stimulation-evoked neuronal activity in the spinal dorsal horn largely corresponds to that of primary afferent inputs. In addition, cutaneous pinch stimulation elicited activities of neurons in the spinal cord at least until 2 spinal segments away from the central projection field of primary sensory neurons responsible for the stimulated skin point. These results provide a clue to understand neuronal processing of sensory information in the spinal dorsal horn.