Rapid viability assessment of spores of several fungi by an ionic intensified fluorescein diacetate method

Fluorescein diacetate (FDA) was applied to the viability assessment of spores of Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, and Penicillium citrinum. The fluorescence of individual cells was quantitated with a charge coupled device (CCD) detector. When staining was carried out in a phosphate buffer solution (10 mM, pH 7.0), weak or no fluorescence was emitted from viable spores of A. niger and R. stolonifer, which made it difficult to distinguish between viable (nontreated) and nonviable (heat treated at 90°C for 30 min) spores. The addition of NaCl, KCl, or MgCl₂ to the staining solution caused an increase in the fluorescence intensity of A. niger viable spores, from which nonviable spores could be distinguished. The same effect of NaCl was observed in staining the spores of other species.     

A VPE family supporting various vacuolar functions in plants

Vacuolar processing enzyme (VPE) is a cysteine protease that has substrate specificity toward Asn and Asp residues, and found in various eukaryotic organisms including higher plants and mammals. Plant VPEs are separated into three subfamilies: seed-type, vegetative-type and uncharacterized-type. VPE was originally identified as a protease responsible for the maturation of seed storage proteins, and recent research has shown that it is a key protease responsible for the maturation of various vacuolar proteins not only in maturating cotyledons, but also in vegetative tissues. Thus, the VPE-mediated processing system is important for various vacuolar functions in the plant. Vegetative-type VPEs are expressed during senescence or pathogen-induced hypersensitive response. A VPE-deficiency abolished programmed cell death during hypersensitive response in tobacco leaves after TMV infection. This suggests that vegetative-type VPEs are involved in vacuolar-organized programmed cell death.     

The Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1

Adaptor protein (AP) complexes play critical roles in protein sorting among different post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP-1–AP-5) in plants, AP-4 is one of the most poorly understood; the AP-4 components, AP-4 cargo motifs, and AP-4 functional mechanism are not known. Here, we identify the AP-4 components and show that the AP-4 complex regulates receptor-mediated vacuolar protein sorting by recognizing VACUOLAR SORTING RECEPTOR1 (VSR1), which was originally identified as a sorting receptor for seed storage proteins to target protein storage vacuoles in Arabidopsis (Arabidopsis thaliana). From the vacuolar sorting mutant library GREEN FLUORESCENT SEED (GFS), we isolated three gfs mutants that accumulate abnormally high levels of VSR1 in seeds and designated them as gfs4, gfs5, and gfs6. Their responsible genes encode three (AP4B, AP4M, and AP4S) of the four subunits of the AP-4 complex, respectively, and an Arabidopsis mutant (ap4e) lacking the fourth subunit, AP4E, also had the same phenotype. Mass spectrometry demonstrated that these four proteins form a complex in vivo. The four mutants showed defects in the vacuolar sorting of the major storage protein 12S globulins, indicating a role for the AP-4 complex in vacuolar protein transport. AP4M bound to the tyrosine-based motif of VSR1. AP4M localized at the trans-Golgi network (TGN) subdomain that is distinct from the AP-1-localized TGN subdomain. This study provides a novel function for the AP-4 complex in VSR1-mediated vacuolar protein sorting at the specialized domain of the TGN.Membrane trafficking in plants shares many fundamental features with those in yeast and animals (Bassham et al., 2008). In general, vacuolar proteins are synthesized on the rough endoplasmic reticulum and then transported to vacuoles via the Golgi apparatus (Xiang et al., 2013; Robinson and Pimpl, 2014). The vacuolar trafficking in plants has been studied by monitoring the transport of reporter proteins to lytic vacuoles in vegetative cells and tissues (Jin et al., 2001; Pimpl et al., 2003; Miao et al., 2008; Niemes et al., 2010). Recently, seed storage proteins became a model cargo for monitoring the transport of endogenous vacuolar proteins in plants (Shimada et al., 2003a; Sanmartín et al., 2007; Isono et al., 2010; Pourcher et al., 2010; Uemura et al., 2012; Shirakawa et al., 2014). During seed maturation, a large amount of storage proteins are synthesized and sorted to specialized vacuoles, the protein storage vacuoles (PSVs). To properly deliver vacuolar proteins, sorting receptors play a critical role in recognizing the vacuole-targeting signal of the proteins. VACUOLAR PROTEIN SORTING10 and Man-6-P receptor function as sorting receptors for vacuolar/lysosomal proteins in the trans-Golgi network (TGN) of yeast and mammals, respectively. The best-characterized sorting receptors in plants are VACUOLAR SORTING RECEPTOR (VSR) family proteins (De Marcos Lousa et al., 2012). VSRs have been shown to function in sorting both storage proteins to PSVs (Shimada et al., 2003a; Fuji et al., 2007) and lytic cargos to lytic vacuoles (Zouhar et al., 2010).To sort the receptors in the TGN into vacuoles/lysosomes, the adaptor protein (AP) complex binds the cytosolic domain of the receptors. The AP complexes form evolutionarily conserved machinery that mediates the post-Golgi trafficking in eukaryotic cells (Robinson, 2004). There are five types of AP complexes, AP-1 to AP-5. The functions of AP-1, AP-2, and AP-3 have been established. AP-1 appears to be involved in trafficking between the TGN and endosomes (Hirst et al., 2012), AP-2 is involved in clathrin-mediated endocytosis (McMahon and Boucrot, 2011), and AP-3 is involved in protein trafficking from the TGN/endosomes to the vacuole/lysosomes (Dell’Angelica, 2009). However, little is known about AP-4 and AP-5. Mammalian AP-4 may be involved in basolateral sorting in polarized cells and in the transport of specific cargo proteins, such as the amyloid precursor protein APP, from the TGN to endosomes (Burgos et al., 2010). The fifth AP complex, AP-5, was recently identified, and its orthologs are widely conserved in the eukaryotic genomes (Hirst et al., 2011). The AP complexes exist as heterotetrameric proteins that consist of two large subunits (β1-5 and one each of ɣ/α/δ/ε/ζ), one medium subunit (µ1-5), and one small subunit (σ1-5). The sorting mechanism is best characterized for the medium (µ) subunit, which is known to recognize the Tyr-based YXXФ motif (where Ф represents Leu, Ile, Phe, Met, or Val) that is present in the cytosolic domains of cargo proteins (Ohno et al., 1995). Mutations of the YXXФ motif abolish the interaction with µ and alter the subcellular localization of the cargo proteins.The genome of Arabidopsis (Arabidopsis thaliana) contains all five sets of putative AP genes (Bassham et al., 2008; Hirst et al., 2011). The function of AP-4 in membrane trafficking and its physiological roles in plants are largely unknown. In this study, we identified and characterized the AP-4 complex in Arabidopsis. Mutants lacking the AP-4 subunits exhibited defects in VSR1-mediated vacuolar sorting of storage proteins in seeds. Our results provide new insights into the receptor-mediated vacuolar trafficking in post-Golgi pathways.     

Reducing Short-Wavelength Blue Light in Dry Eye Patients with Unstable Tear Film Improves Performance on Tests of Visual Acuity

PurposeTo investigate whether suppression of blue light can improve visual function in patients with short tear break up time (BUT) dry eye (DE).MethodsTwenty-two patients with short BUT DE (10 men, 12 women; mean age, 32.4 ± 6.4 years; age range, 23–43 years) and 18 healthy controls (10 men, 8 women; mean age, 30.1 ± 7.4 years; age range, 20–49 years) underwent functional visual acuity (VA) examinations with and without wearing eyeglasses with 50% blue light blocked lenses. The functional VA parameters were starting VA, functional VA, and visual maintenance ratio.ResultsThe baseline mean values (logarithm of the minimum angle of resolution, logMAR) of functional VA and the visual maintenance ratio were significantly worse in the DE patients than in the controls (P < 0.05), while no significant difference was observed in the baseline starting VA (P > 0.05). The DE patients had significant improvement in mean functional VA and visual maintenance ratio while wearing the glasses (P < 0.05), while there were no significant changes with and without the glasses in the control group (P > 0.05),ConclusionsProtecting the eyes from short-wavelength blue light may help to ameliorate visual impairment associated with tear instability in patients with DE. This finding represents a new concept, which is that the blue light exposure might be harmful to visual function in patients with short BUT DE.     

2,4-D-Sustained Photomixotrophic Growth of a Chlorophyllous Cell Suspension Culture of Nicotiana tabacum

A chlorophyllous, photomixotrophic cell suspension culture oftobacco (Nicotiana tabacum L.) was established using mediumcontaining 30 g/liter of sucrose and 1.5 µM 2,4-D. The2,4-D-sustained photomixotrophic line was able to show rapidregreening in the light after bleaching in the dark and characterizedwith a much slower and longer growth cycle than a heterotrophicline derived from the same original callus (cell doubling timeof 100 h vs. 40 h and duration of logarithmic phase of 17 daysvs. 7 days). The photomixotrophic line took up sucrose morerapidly than the heterotrophic line and accumulated starch duringthe early logarithmic phase when it showed a maximum photosyntheticcapacity on a chlorophyll basis (6.3µmol O₂/min/mg Chl).Chlorophyll content and photosynthetic capacity on a per cellbasis and on a cell fresh weight basis, on the other hand, decreasedduring this phase and reincreased later to reach maximum levels(310 µg Chl/g fr wt; 1.4 µmol O₂/min/g fr wt) whenthe line exhibited the highest activities of dark respiration(1.0 µmol; O₂/min/g fr wt) and cell division (mitoticindex of 3.0%). These characteristics of the photomixotrophicline were lost if it was grown in the dark to become non-chlorophyllous.Although net O₂ evolution could not be detected in the photomixotrophicline throughout the growth cycle when assayed under suboptimumlight intensity, reaccumulation of starch and a marked increasein cell fresh weight upon addition of minerals, vitamins and2,4-D without sucrose at the late logarithmic phase indicatedthe development of photosynthetic activity under the cultureconditions. ¹The investigations reported were included in the thesis submittedto the Graduate School, Faculty of Agriculture, Kobe University,in partial fulfillment of the requirement for M. Agr. degree. (Received May 30, 1988; Accepted October 5, 1988)     

Tryptophan 5-hydroxylase in rat intestine

Tryptophan 5-hydroxylase was partially purified from rat small intestine and characterized. The enzyme activity was mainly localized in the distal one-fourth of the small intestine. The enzyme required Fe(2+), 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine and oxygen for full activity. The pH optimum of the reaction was 8.0. The hydroxylation rate of d-tryptophan by the enzyme was one-third that of l-tryptophan. l-Phenylalanine and l-tyrosine could not serve as substrates. The physiological significance of the enzyme is discussed.     

Revisiting the mystery of fibronectin multimers: The fibronectin matrix is composed of fibronectin dimers cross-linked by non-covalent bonds

Fibronectin (FN) matrix fibrils have long been thought to be formed by disulfide-bonded FN multimers, although there is no direct evidence that they are covalently linked with each other. To understand the biochemical properties of these fibrils, we extracted a crude FN matrix from FN-YPet transfected 3T3 cell culture using 0.2% deoxycholate and DNase. The insoluble extracted matrix preserved fibrillar structures and a major portion of the extracted proteins migrated as FN monomers on an SDS gel under reducing conditions. Under non-reducing conditions, some FN molecules appeared to be trapped at the top of the stacking gel. We tested this by mixing fluorescently labeled FN dimers with the extracted matrix just before loading on an SDS gel, and found that most of them were trapped with the extracted proteins at the top of the stacking gel. These results suggested that some components of the extracted matrix plugged the stacking gel and FN dimers were trapped with them. Rotary shadowing electron microscopy showed that the extracted matrix had some fibers that resembled fibrillin microfibrils. Peptide mass fingerprinting confirmed the presence of fibrillin in the extracted matrix. Fibrillin is known to form disulfide-bonded multimers and it is likely to be one of the components that plug the stacking gel and trap FN molecules in this system. The phenomenon by which FN molecules appear to migrate as multimers on SDS gels is thus an artifact rising from the presence of other large components in the extract. We conclude that FN matrix fibrils are made of FN dimers that are further cross-linked by non-covalent protein–protein bonds.     

Seasonal changes in spermatogenic cell degeneration in the seminiferous epithelium of adult Japanese macaques (Macaca fuscata fuscata)

The degenerating pattern of spermatogenic cells in the seminiferous tubule of Japanese macaques was studied to clarify a relationship between seasonal changes of reproductive performances and cytological findings in the Japanese macaque. For light microscopy, testis samples were obtained from five adult animals by biopsy in April (nonmating season) and October (mating season). For electron microscopy, specimens from four additional macaques were used. Degenerating cells were found in all steps of spermatogenesis. In stages I to V of the cycle of the seminiferous epithelium, morphologically atypical pachytene spermatocytes were observed in 14.7 and 10.0% of the cells in the nonmating and mating seasons, respectively, although the difference in percentage was not significant. Mature spermatids with atypical features in those stages occupied 59.6% of the cells in the nonmating season, which significantly decreased to 34.1% in the mating season. These results imply that the seasonal change of sperm production is related, at least in part, to the process of degeneration of the spermatogenic cells in this species.     

Vacuolar Processing Enzyme of Soybean That Converts Proproteins to the Corresponding Mature Forms

A vacuolar processing enzyme was detected in soybean proteinbodies. A 39-kDa immunore-active polypeptide obtained by chromatographyon a hydroxyapatite column processed both a decapeptide substrateand proproteins. A cDNA was isolated for a 55-kDa protein with71% identity to the castor bean vacuolar processing enzyme. (Received March 18, 1994; Accepted April 18, 1994)