The extra-embryonic space and the local contour are crucial geometric constraints regulating cell arrangement.

In multicellular systems, cells communicate with adjacent cells to determine their positions and fates, an arrangement important for cellular development. Orientation of cell division, cell-cell interactions (i.e. attraction and repulsion) and geometric constraints are three major factors that define cell arrangement. In particular, geometric constraints are difficult to reveal in experiments, and the contribution of the local contour of the boundary has remained elusive. In this study, we developed a multicellular morphology model based on the phase-field method so that precise geometric constraints can be incorporated. Our application of the model to nematode embryos predicted that the amount of extra-embryonic space, the empty space within the eggshell that is not occupied by embryonic cells, affects cell arrangement in a manner dependent on the local contour and other factors. The prediction was validated experimentally by increasing the extra-embryonic space in the Caenorhabditis elegans embryo. Overall, our analyses characterized the roles of geometrical contributors, specifically the amount of extra-embryonic space and the local contour, on cell arrangements. These factors should be considered for multicellular systems.

An asymmetric attraction model for the diversity and robustness of cell arrangement in nematodes.

During early embryogenesis in animals, cells are arranged into a species-specific pattern in a robust manner. Diverse cell arrangement patterns are observed, even among close relatives. In the present study, we evaluated the mechanisms by which the diversity and robustness of cell arrangements are achieved in developing embryos. We successfully reproduced various patterns of cell arrangements observed in various nematode species in Caenorhabditis elegans embryos by altering the eggshell shapes. The findings suggest that the observed diversity of cell arrangements can be explained by differences in the eggshell shape. Additionally, we found that the cell arrangement was robust against eggshell deformation. Computational modeling revealed that, in addition to repulsive forces, attractive forces are sufficient to achieve such robustness. The present model is also capable of simulating the effect of changing cell division orientation. Genetic perturbation experiments demonstrated that attractive forces derived from cell adhesion are necessary for the robustness. The proposed model accounts for both diversity and robustness of cell arrangements, and contributes to our understanding of how the diversity and robustness of cell arrangements are achieved in developing embryos.

Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis.

Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.

Efficient production of lumichrome by Microbacterium sp. strain TPU 3598.

Lumichrome is a photodegradation product of riboflavin and is available as a photosensitizer and fluorescent dye. To develop new efficient methods of lumichrome production, we isolated bacterial strains with high lumichrome productivity from soil. The strain with highest productivity was identified as Microbacterium sp. strain TPU 3598. Since this strain inductively produced lumichrome when cultivated with riboflavin, we developed two different methods, a cultivation method and a resting cell method, for the production of large amounts of lumichrome using the strain. In the cultivation method, 2.4 g (9.9 mmol) of lumichrome was produced from 3.8 g (10.1 mmol) of riboflavin at the 500-ml scale (98% yield). The strain also produced 4.7 g (19.4 mmol) of lumichrome from 7.6 g (20.2 mmol) of riboflavin (96% yield) by addition of riboflavin during cultivation at the 500-ml scale. In the resting cell method, 20 g of cells (wet weight) in 100 ml of potassium phosphate buffer, pH 7.0, produced 2.4 g of lumichrome from 3.8 g of riboflavin (98% yield). Since the lumichrome production by these methods was carried out in suspension, the resulting lumichrome was easily purified from the cultivation medium or reaction mixture by centrifugation and crystallization. Thus, the biochemical methods we describe here are a significant improvement in terms of simplicity and yield over the existing chemical, photolytic, and other biochemical methods of lumichrome production.

Identification and characterization of CYP79D16 and CYP71AN24 catalyzing the first and second steps in L-phenylalanine-derived cyanogenic glycoside biosynthesis in the Japanese apricot, Prunus mume Sieb. et Zucc.

Japanese apricot, Prunus mume Sieb. et Zucc., belonging to the Rosaceae family, produces as defensive agents the cyanogenic glycosides prunasin and amygdalin, which are presumably derived from L-phenylalanine. In this study, we identified and characterized cytochrome P450s catalyzing the conversion of L-phenylalanine into mandelonitrile via phenylacetaldoxime. Full-length cDNAs encoding CYP79D16, CYP79A68, CYP71AN24, CYP71AP13, CYP71AU50, and CYP736A117 were cloned from P. mume 'Nanko' using publicly available P. mume RNA-sequencing data, followed by 5'- and 3'-RACE. CYP79D16 was expressed in seedlings, whereas CYP71AN24 was expressed in seedlings and leaves. Enzyme activity of these cytochrome P450s expressed in Saccharomyces cerevisiae was evaluated by liquid and gas chromatography­mass spectrometry. CYP79D16, but not CYP79A68, catalyzed the conversion of L-phenylalanine into phenylacetaldoxime. CYP79D16 showed no activity toward other amino acids. CYP71AN24, but not CYP71AP13, CYP71AU50, and CYP736A117, catalyzed the conversion of phenylacetaldoxime into mandelonitrile. CYP71AN24 also showed lower conversions of various aromatic aldoximes and nitriles. The K m value and turnover rate of CYP71AN24 for phenylacetaldoxime were 3.9 µM and 46.3 min(−1), respectively. The K m value and turnover of CYP71AN24 may cause the efficient metabolism of phenylacetaldoxime, avoiding the release of the toxic intermediate to the cytosol. These results suggest that cyanogenic glycoside biosynthesis in P. mume is regulated in concert with catalysis by CYP79D16 in the parental and sequential reaction of CYP71AN24 in the seedling.

Switching from weak to strong cortical attachment of microtubules accounts for the transition from nuclear centration to spindle elongation in metazoans.

The centrosome is a major microtubule organizing center in animal cells. The position of the centrosomes inside the cell is important for cell functions such as cell cycle, and thus should be tightly regulated. Theoretical models based on the forces generated along the microtubules have been proposed to account for the dynamic movements of the centrosomes during the cell cycle. These models, however, often adopted inconsistent assumptions to explain distinct but successive movements, thus preventing a unified model for centrosome positioning. For the centration of the centrosomes, weak attachment of the astral microtubules to the cell cortex was assumed. In contrast, for the separation of the centrosomes during spindle elongation, strong attachment was assumed. Here, we mathematically analyzed these processes at steady state and found that the different assumptions are proper for each process. We experimentally validated our conclusion using nematode and sea urchin embryos by manipulating their shapes. Our results suggest the existence of a molecular mechanism that converts the cortical attachment from weak to strong during the transition from centrosome centration to spindle elongation.

Development of a method for long-term preservation of Bombyx mori silkworm strains using frozen ovaries.

Development of long-term preservation is essential for conservation of stocks of silkworm genetic resources. Thus far, a few methods have been reported, but more improvement is required for practical use. We have developed two effective modifications of a method for long-term preservation using frozen ovaries. One was slow cooling (1 °C per min) until -80 °C of the donor ovaries made possible by use of a BICELL freezing vessel. Using donor ovaries of 4th instar larvae, the average number of eggs laid per moth increased significantly from 110.7 ± 53.4 eggs per moth by slow cooling with the BICELL vessel vs 12.3 ± 10.3 eggs per moth by direct cooling in liquid nitrogen. A second improvement was connecting the thread bodies of the donor ovaries with those of the host in the transplantation step. Females operated on with the new method yielded a significantly higher percentage of moths that laid fertilized eggs than those transplanted with the standard procedure (70.4 ± 21.6% vs 22.9 ± 9.3%).

Cell polarity: Adapting the PAR cascade to diverse cellular contexts.

Two new studies shed light on the intricacies of Caenorhabditis elegans embryo patterning, revealing how the conserved interaction and crosstalk of PAR proteins are adapted to perceive distinct cues, ultimately shaping unique asymmetries in form and function.

Caffeine intoxication as a result of excessive consumption of bottled coffee products: a case report.

Objectives: Most cases of caffeine intoxication result from the excessive intake of over-the-counter drugs and energy drinks. However, few cases of caffeine intoxication due to the excessive consumption of bottled coffee products have been reported. Herein, we present a case report of caffeine intoxication. Patient: A 39-year-old man experienced numbness and weakness in the extremities for three nights over five days. Results: Blood tests revealed hypophosphatemia and low 25-OH vitamin D concentration. The symptoms disappeared the next day without any additional treatment. A lifestyle interview revealed that he regularly consumed bottled coffee like it was water and had approximately 1 L of it from evening to night. He was diagnosed with weakness in the extremities due to hypophosphatemia caused by caffeine intoxication. Upon investigating some bottled coffee products, we found that only a few of them had labels disclosing caffeine content and warnings of the risks of excessive caffeine intake. Conclusion: We encountered a case of caffeine intoxication via coffee. Although rare in the past, caffeine intoxication might increase owing to the widespread use of bottled coffee products. The caffeine content of coffee products should be indicated on labels to warn consumers.

Photo-products of retinal pigment epithelial bisretinoids react with cellular thiols.

PURPOSE: Bisretinoids such as A2E that accumulate as components of the lipofuscin of retinal pigment epithelial cells are implicated in some retinal disease processes. These compounds undergo light-induced oxidation and cleavage with the latter releasing of a mixture of aldehyde-bearing fragments, including dicarbonyl methylglyoxal. We tested for the reactivity of photooxidation and photodegradation products of A2E with thiol-containing glutathione (GSH). METHODS: In cell-free assays, we measured the ability of photooxo-A2E to competitively inhibit the GSH-mediated reduction of the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid). Cellular GSH was assayed colorimetrically. Products of GSH reduction and GSH-adducts were detected by electrospray ionization mass spectrometry (ESI-MS) and GSH and oxidized GSH (glutathione disulfide [GSSG]) were quantified from chromatographic peak areas. RESULTS: We found that GSH can donate hydrogen atoms to, and form conjugates with, photooxidized forms of the bisretinoid A2E and with its photocleavage products. Reaction with non-photooxidized A2E was not observed. Chemical reduction by GSH involved the donation of a hydrogen atom from each of two GSHs. The ratio of GSH consumed to GSSG formed was consistent with GSH being used for both reduction and adduct formation. With the aid of synthesized standards, methylglyoxal-GSH adducts were identified within mixtures of GSH and photooxidized A2E; the adducts formed noncatalytically and by glutathione-S-transferase mediation. CONCLUSIONS: Reduction and adduct formation by GSH likely limits the reactivity of bisretinoid photoproducts and may aid their elimination from the cells. These findings are significant to forms of macular degeneration associated with bisretinoid formation and maculopathy stemming from GSH synthase deficiency.

Syntheses of cellotriose and cellotetraose analogues as transition state mimics for mechanistic studies of cellulases.

Cellotriose and cellotetraose analogues carrying cyclohexene rings were developed as molecular probes which are expected to mimic the transition state conformation of hydrolysis by cellulases. The cyclohexene ring was placed at the pyranose ring being expected to locate the -1 subsite of the enzyme. In order to evaluate these probes, sulfur derivatives of cellotriose and cellotetraose were also synthesized as the enzyme tolerant analogues which mimic the stable conformations of the natural cellulose. The binding assays using differential scanning calorimetry revealed that introduction of the cyclohexene ring is effective to the complexation with an endoglucanase, NCE5 from Humicola insolens.

Fundus autofluorescence and the bisretinoids of retina.

Imaging of the human fundus of the eye with excitation wavelengths in the visible spectrum reveals a natural autofluorescence, that in a healthy retina originates primarily from the bisretinoids that constitute the lipofuscin of retinal pigment epithelial (RPE) cells. Since the intensity and distribution of fundus autofluorescence is altered in the presence of retinal disease, we have examined the fluorescence properties of the retinal bisretinoids with a view to aiding clinical interpretations. As is also observed for fundus autofluorescence, fluorescence emission from RPE lipofuscin was generated with a wide range of exciting wavelengths; with increasing excitation wavelength, the emission maximum shifted towards longer wavelengths and spectral width was decreased. These features are consistent with fluorescence generation from a mixture of compounds. While the bisretinoids that constitute RPE lipofuscin all fluoresced with maxima that were centered around 600 nm, fluorescence intensities varied when excited at 488 nm, the excitation wavelength utilized for fundus autofuorescence imaging. For instance the fluorescence efficiency of the bisretinoid A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE) was greater than A2E and relative to both of the latter, all-trans-retinal dimer-phosphatidylethanolamine was weakly fluorescent. On the other hand, certain photooxidized forms of the bisretinoids present in both RPE and photoreceptor cells were more strongly fluorescent than the parent compound. We also sought to evaluate whether diffuse puncta of autofluorescence observed in some retinal disorders of monogenic origin are attributable to retinoid accumulation. However, two retinoids of the visual cycle, all-trans-retinyl ester and all-trans-retinal, did not exhibit fluorescence at 488 nm excitation.

Three multi-allelic gene pairs are responsible for self-sterility in the ascidian Ciona intestinalis.

Many hermaphroditic organisms possess a self-incompatibility system to avoid inbreeding. Although the mechanisms of self-incompatibility in flowering plants are well known, little is known about the mechanisms of self-sterility in hermaphroditic marine invertebrates. Ascidians are hermaphroditic sessile marine invertebrates that release sperm and eggs into the surrounding seawater. Several species, including Ciona intestinalis type A (Ciona robusta), exhibit strict self-sterility. In a previous study, we found that the candidate genes responsible for self-sterility in Ciona reside in chromosome 2q (locus A) and chromosome 7q (locus B). Two pairs of multi-allelic genes, named s(sperm)-Themis-A and v(vitelline-coat)-Themis-A in locus A and s-Themis-B and v-Themis-B in locus B, are responsible for self-sterility. In this study, we identified a third multi-allelic gene pair, s-Themis-B2 and v-Themis-B2, within locus B that is also involved in this system. Genetic analysis revealed that the haplotypes of s/v-Themis-A, s/v-Themis-B and s/v-Themis-B2 play essential roles in self-sterility. When three haplotypes were matched between s-Themis and v-Themis, fertilization never occurred even in nonself crossing. Interestingly, gene targeting of either s/v-Themis-B/B2 or s/v-Themis-A by genome editing enabled self-fertilization. These results indicate that s/v-Themis-A, -B and -B2 are S-determinant genes responsible for self-sterility in the ascidian C. intestinalis type A.

A high optical-gain organic crystal comprising thiophene/phenylene co-oligomer nanomolecules.

We have estimated the optical gain for a crystal of a thiophene/phenylene co-oligomer, 1,4-bis(5-phenylthiophen-2-yl)benzene. We prepared the crystal by a vapor phase growth method and measured emission spectra by exciting it with a pulse laser and changing the pumped stripe lengths. With increasing the stripe lengths, the emission intensity was increased and the emission spectra were gain narrowed around 516 nm with its full width at half maximum down to approximately 6 nm. The optical gain spectrum was determined from the emission spectra that varied as a function of the pumped stripe lengths. The gain spectrum was peaked at 516 nm with the maximum net optical gain coefficient being 75 cm(-1). This peak location is in excellent agreement with the peak position of the gain narrowed emission spectra. The results indicate that the determination of the gain spectrum is important for laser applications of the material. The oligomer crystal in the present studies is a good candidate for them.

Does Gd@C82 have an anomalous endohedral structure? Synthesis and single crystal X-ray structure of the carbene adduct.

We report here the results on single crystal X-ray crystallographic analysis of the Gd@C82 carbene adduct (Gd@C82(Ad), Ad = adamantylidene). The Gd atom in Gd@C82(Ad) is located at an off-centered position near a hexagonal ring in the C2v-C82 cage, as found for M@C82 (M = Sc and La) and La@C82(Ad). Theoretical calculation also confirms the position of the Gd atom in the X-ray crystal structure.

Strongly pH-buffered ringer's solution expands the receptive field size of horizontal cells in the carp retina.

By intracellular recordings, we studied the effects of pH buffering on the size of the receptive field and the extent of dye coupling of horizontal cells (HCs) in the light-adapted carp retina. These parameters were compared between data obtained in fortified Ringer's solution and those obtained in control bicarbonate Ringer's of the same pH (7.60). In Ringer's fortified with 10 mM HEPES or 15 mM Tris, the dye-coupling ratio of HCs increased by 71% and 70%, respectively. These fortified Ringer's solutions also depolarized the dark membrane potential and increased the light-evoked response. The HC receptive field profile could be described by the exponential decline in peak response amplitude to a slit of light moved tangentially from the recording electrode. Thus, the receptive field size was determined as a space constant proportional to (gj/gm)(1/2), where gj and gm denote gap and non-gap-junctional conductances. The HEPES- or Tris-fortified Ringer's significantly increased the space constant by 43% and 41%, respectively. Since dye coupling was increased in the fortified Ringer's, it is likely that gj increased more than gm as a result of alkalinization of the cytosol. Since HEPES has an aminosulfonate moiety, it has been assumed to close the hemi-channels of connexin 26, but the pH-buffering effects were essentially the same as those of Tris that has no aminosulfonate moiety. Therefore, it is unlikely that the closure of connexin 26 hemichannels is responsible for the change in the receptive field size of HCs.

Synthesis of D-trigalacturonic acid methylglycoside and conformational comparison with its sulfur analogue.

D-Trigalacturonic acid methylglycoside (3) was synthesized to evaluate the previously synthesized sulfur analogue 1 by comparison. The NOE experiments revealed that both 3 and 1 took on a similar conformation around their glycosyl linkage.

Translucent larval integument and flaccid paralysis caused by genome editing in a gene governing molybdenum cofactor biosynthesis in Bombyx mori.

Translucency of the larval integument in Bombyx mori is caused by a lack of uric acid in the epidermis. Hime'nichi translucent (ohi) is a unique mutation causing intermediate translucency of the larval integument and male-specific flaccid paralysis. To determine the gene associated with the ohi mutation, the ohi locus was mapped to a 400-kb region containing 29 predicted genes. Among the genes in this region, we focused on Bombyx homolog of mammalian Gephyrin (BmGphn), which regulates molybdenum cofactor (MoCo) biosynthesis, because MoCo is indispensable for the activity of xanthine dehydrogenase (XDH), a key enzyme in uric acid biosynthesis. The translucent integument of ohi larvae turned opaque after injection of bovine xanthine oxidase, which is a mammalian equivalent to XDH, indicating that XDH activity is defective in ohi larvae. RT-PCR and sequencing analysis showed that (i) in ohi larvae, expression of the BmGphn gene was repressed in the fat body where uric acid is synthesized, and (ii) there was no amino acid substitution in the ohi mutant allele. Finally, we obtained BmGphn knockout alleles (hereafter denoted as BmGphnΔ) by using CRISPR/Cas9. The resulting ohi/BmGphnΔ larvae had translucent integuments, demonstrating that BmGphn is the gene responsible for the ohi phenotype. Our results show that repressed expression of BmGphn is a causative factor for the defective MoCo biosynthesis and XDH activity observed in ohi larvae. Interestingly, all male BmGphnΔ homozygotes died before pupation and showed a flaccid paralysis phenotype. The genetic and physiological mechanisms underlying this flaccid paralysis phenotype are also discussed.

Acidification decouples gap junctions but enlarges the receptive field size of horizontal cells in carp retina.

The receptive field size of retinal horizontal cells is much larger than their dendritic field size due to gap junctional coupling between the same sub-types of cell. Thus, horizontal cells form syncytia by electrical coupling. The basic receptive field profile of horizontal cells can be described by an exponential function based on measurement of responses to a slit of light moved tangentially from a recording electrode. The space constant of this exponential function is proportional to (g(s)/g(m))(1/2), where g(s) and g(m) represent gap junctional conductance and non-gap junctional conductance, respectively. Acidifying the superfusing solution by lowering the pH from 7.60 to 7.30 decreased the dye-coupling, hyperpolarised the resting membrane potential and reduced the photoresponses of H1 type horizontal cells. Surprisingly, however, the receptive field size expanded significantly. Raising the pH from 7.30 to 7.60 or 7.90 produced opposite effects. These results were consistent with alkaline extracellular pH producing a greater increase in g(m) than in g(s) and enhancing release of transmitter from cones acting upon horizontal cells.