Oviducal Contribution to Alteration of the Vitelline Coat in the Frog, Rana japonica. An Electron Microscopic Study

The vitelline coat (VC) surrounding coelomic eggs of the frog, Rana japonica , comprises bundles of filaments running both parallel and perpendicular to the egg surface. The coat gives little or no staining reaction with PA-CrA-Silver methenamine. In contrast, in the VC of uterine eggs the filament bundles are less conspicuous. and the interstices between the filament bundles stain strongly for carbohydrate. This alteration occurs during passage of the eggs down the first 1/20 th of the oviduct, the pars recta. The epithelium of the p. recta contains secretory cells, which contain electron-dense granules distinct from those in the jelly-secreting cells in more caudal portions of the oviduct. Treatment of coelomic eggs with an extract of p. recta followed by exposure to a sperm suspension resulted in marked swelling and softening of the VC. These results indicate that the contents of the granules secreted from the epithelial cells in the p. recta are deposited in the VC to increase its susceptibility to a fertilizing sperm.     

Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.     

Properties of the Coat Protein of a New Tobacco Mosaic Virus Coat Protein ts-Mutant

Amino acid substitutions in a majority of tobacco mosaic virus (TMV) coat protein (CP) ts-mutants have previously been mapped to the same region of the CP molecule tertiary structure, located at a distance of about 70 Å from TMV virion axis. In the present work some properties of a new TMV CP ts-mutant ts21-66 (two substitutions I21 T and D66 G, both in the 70-Å region) were studied. Thermal inactivation characteristics, sedimentation properties, circular dichroism spectra, and modification by a lysine-specific reagent, trinitrobenzensulfonic acid, of ts21–66 CP were compared with those of wild-type (U1) TMV CP. It is concluded that the 70-Å region represents the most labile portion of the TMV CP molecule. Partial disordering of this region in the mutant CP at permissive temperatures leads to loss of the capacity to form two-layer aggregates of the cylindrical type, while further disordering induced by mild heating results also in the loss of the ability to form ordered helical aggregates.     

Abscisic Acid-Triggered Persulfidation of the Cys Protease ATG4 Mediates Regulation of Autophagy by Sulfide

Hydrogen sulfide is a signaling molecule that regulates essential processes in plants, such as autophagy. In Arabidopsis (Arabidopsis thaliana), hydrogen sulfide negatively regulates autophagy independently of reactive oxygen species via an unknown mechanism. Comparative and quantitative proteomic analysis was used to detect abscisic acid-triggered persulfidation that reveals a main role in the control of autophagy mediated by the autophagy-related (ATG) Cys protease AtATG4a. This protease undergoes specific persulfidation of Cys170 that is a part of the characteristic catalytic Cys-His-Asp triad of Cys proteases. Regulation of the ATG4 activity by persulfidation was tested in a heterologous assay using the Chlamydomonas reinhardtii CrATG8 protein as a substrate. Sulfide significantly and reversibly inactivates AtATG4a. The biological significance of the reversible inhibition of the ATG4 by sulfide is supported by the results obtained in Arabidopsis leaves under basal and autophagy-activating conditions. A significant increase in the overall ATG4 proteolytic activity in Arabidopsis was detected under nitrogen starvation and osmotic stress and can be inhibited by sulfide. Therefore, the data strongly suggest that the negative regulation of autophagy by sulfide is mediated by specific persulfidation of the ATG4 protease.     

The Bipartite Geminivirus Coat Protein Aids BR1 Function in Viral Movement by Affecting the Accumulation of Viral Single-Stranded DNA

The movement of bipartite geminiviruses such as squash leaf curl virus (SqLCV) requires the cooperative interaction of two essential virus-encoded movement proteins, BR1 and BL1. While the viral coat protein AR1 is not essential for systemic infection, genetic studies demonstrate that its presence masks the defective phenotype of certain BR1 missense mutants, thus suggesting that coat protein does interact with the viral movement pathway. To further examine the mechanism of this interaction, we have constructed alanine-scanning mutants of AR1 and studied them for the ability to mask the infectivity defects of appropriate BR1 mutants, for the ability to target to the nucleus and to bind viral single-stranded DNA (ssDNA) and multimerize, and for effects on the accumulation of replicated viral ssDNA. We identified a specific region of AR1 required for masking of appropriate BR1 mutants and showed that this same region of AR1 was also important for ssDNA binding and the accumulation of viral replicated ssDNA. This region of AR1 also overlapped that involved in multimerization of the coat protein. We also found that the accumulation in protoplasts of single-stranded forms of a recombinant plasmid that included the SqLCV replication origin but was too large to be encapsidated was dependent on the presence of AR1 but did not appear to require encapsidation. These findings extend our model for SqLCV movement, demonstrating that coat protein affects viral movement through its ability to induce the accumulation of replicated viral ssDNA genomes. They further suggested that encapsidation was not required for the AR1-dependent accumulation of viral ssDNA.     

Arabidopsis RAN 1 Mediates Seed Development through Its Parental .Ratio by Affecting the Onset of Endosperm Cellularization

Although previous studies have demonstrated that endosperm development is influenced by its parental genome constitution, the genetic basis and molecular mechanisms that control parent-of-origin effects require further elucidation. Here we show that the Ras-related nuclear protein 1 （RAN1） regulates endosperm development in Arabidopsis thaliana. Reciprocal crosses between wild-type （WT） and transgenic lines misexpressing RAN1 （msRAN1） gave rise to small F1 seeds when RAN1 down-regulated/up-regulated individuals were used as a male/female parent; in contrast, F1 seeds were aborted when RAN1 down-regulated/up-regulated plants were used as a female/male parent, suggesting that seed development is affected by the parental genome ratio of RAN1. Whereas RAN1 expression in wild-type plants is reduced before the onset of endosperm cellularization, F1 seeds from reciprocal crosses between WT and msRAN1 showed abnormal endosperm cellularization and ectopic expression of RAN1. The expression of MINISEED3 （MINI3）-a gene that also controls endosperm cellularization-was also affected in these reciprocal crosses, and the misregulation of MINI3 activity rescued F1 seeds when msRAN1 plants were used in reciprocal crosses. Taken together, our results suggest that the parental ratio of RAN1 regulates the onset of endosperm cellularization through its genetic interaction with MINI3.     

Egg jelly triggers a calcium influx which inactivates and is inhibited by calmodulin antagonists in the sea urchin sperm

Sea urchin sperm must undergo the acrosome reaction to fertilize eggs. The natural inducer of this reaction is the most external coat of the egg, named 'jelly'. The ionic composition of the extracellular and intracellular media and the permeability properties of the sperm plasma membrane are fundamental in this reaction. As Ca2+ is required for the acrosome reaction to occur, its intracellular concentration ([Ca2+]i) was measured with fura-2. In 10 mM Ca2+, egg jelly induced the acrosome reaction and an increase in [Ca2+]i that lasted for several minutes. However, at 0.5 or 2 mM Ca2+, it became evident that the Ca2+-influx pathway activated by jelly opened only for a few seconds; this prevented both the full increase in [Ca2+]i and the acrosome reaction even after the concentration of Ca2+ was raised to 10 mM. In the presence of jelly, the time this permeability pathway remained open was inversely related to the extracellular concentration of Ca2+ ([ Ca2+]e). Using Bisoxonol (a permeant fluorescent membrane potential probe), it was found that the jelly-induced depolarization depended on [Ca2+]e and was proportional to the increase in [Ca2+]i. Since [Ca2+]i could affect the jelly-induced Ca2+ influx through calmodulin, two of its antagonists, trifluoperazine and W-7, were tested. Both compounds blocked the acrosome reaction by inhibiting the jelly-induced increase in [Ca2+]i. W-5 at the same concentration had no effect. The results suggest that one of the jelly-activated Ca2+-influx pathways, probably a channel, is the target of the calmodulin antagonists.     

Electron Microscopic Observations on the Cortical Reaction of the Brittle-Star Amphipholis kochii Lütken, with Special Reference to its Vitelline Coat Modification as Revealed by the Surface Replica Method

This paper describes the fine structural changes of the egg of the brittle-star Amphipholis kochii Lütken during the cortical reaction. The vitelline coat is 20 nm thick, when Ruthenium Red stain is used, and consists of a dense network of fibers. The cortical granules are large, 1.5–2.0 μm in diameter, and exist in several layers in the egg cortex, unlike the monolayer arrangement found in many other animals. The contents of the cortical granules are clearly distinguished into two components: peripheral fibrous (PF) material and central fibrous (CF) material that consists of two components differing in electron density. The PF material is densely stained by periodic acid-chromic acid-silver methenamine stain, while the CF material is stained little if at all by this technique. The vitelline coat and some PF materials form the fertilization membrane, which is about 40 nm thick and consists of three layers; the outer and the inner layer of the fertilization membrane each have a trilaminated structure. The vitelline coat substances are probably located in the upper part of the fertilization membrane. The hyaline layer, 7–8 μm thick, consists mainly of CF materials. These observations on the morphology of the ophiuroid egg are discussed in comparison with those on other echinoderms, especially echinoids and asteroids.     

Protease production by a thermophilic Bacillus species (P-001A) which degrades various kinds of fibrous proteins

An aerobic Gram-positive sporeforming bacterium was isolated from an alkaline hot spring at Wondo Genet, Ethiopia. In an optimized culture medium it produced maximum activity of protease at 55°C and pH 9.5. The protease activity against casein was 65 units/ml. Enzyme activity was detected between 30–70°C and pH of 4.5–11.5. The enzyme had a half-life of 55 and 30 min at 60° and 70°C, respectively. The isolate hydrolysed 90, 60 and 50% of the skin, feather and horn used in the optimized medium within 120 h.     

Purification and Properties of an Extracellular Protease Produced by the Entomopathogenic Fungus Beauveria bassiana

Beauveria bassiana GK2016 grown in a medium with gelatin as the sole carbon and nitrogen source produced an extracellular protease. The protease production was highest when the fungus was grown on a semiliquid medium and was purified about 18-fold, with a recovery of 21%. The protease molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 35,000. It had an optimum activity at pH 8.5 and 37 degrees C and was rapidly inactivated at 50 degrees C. Its enzymatic activity was that of an endopeptidase which hydrolyzed elastin, casein, and gelatin but was much less active on bovine serum albumin and collagen. No trypsinlike activity was detected on N-alpha-benzoyl-dl-arginine-p-nitroanilide. It was, however, inhibited by phenylmethylsulfonyl fluoride, indicating that a serine residue is present in the active site. The protease was unaffected by metal-chelating agents, sulfhydryl reagents, trypsin inhibitor, and chymotrypsin inhibitor.     

The Role of Potassium in Active Transport of Sodium by the Toad Bladder

Studies were carried out on the isolated urinary bladder of the toad, Bufo marinus, in order to explain the dependence of active sodium transport on the presence of potassium, in the serosal medium. Attempts to obtain evidence for coupled sodium-potassium transport by the serosal pump were unsuccessful; no relation between sodium transport and uptake of K⁴² from the serosal medium was demonstrable. Rather, the predominant effect of serosal potassium appeared to be operative at the mucosal permeability barrier, influencing the permeability of this surface to sodium. The mucosal effects of serosal potassium were correlated with effects on cellular cation content. When sodium Ringer's solution was used as serosal medium, removal of potassium resulted in significant decrease in tissue potassium content, commensurate increase in tissue sodium content, and marked depression of mucosal permeability and sodium transport. When choline replaced sodium in the serosal medium, removal of potassium resulted in only slight alterations of tissue electrolyte content, and effects on mucosal permeability and sodium transport were minimal.     

Purification and Properties of a Protease Produced by Bacillus Subtilis CN2 Isolated from a Vietnamese Fish Sauce

Bacillus subtilis CN2 isolated from a Vietnamese fish sauce produced a large quantity of an alkaline protease, when grown on a soy peptone medium. The protease was purified to an electrophoretically homogeneous state and crystallized in its pure condensed solution. The molecular weight was determined to be 27,636 Da, and the N-terminal amino acid sequence was AQSVPYGISQIKAPAL. The optimum pH and temperature were pH 10.0 and 50 °C, respectively. The protease was active over a wide pH range of pH 7.0–11.0, and also active over a broad temperature range of 30–60 °C. The enzyme was potently inhibited by 1 mM phenylmethanesulphonyl fluoride, but resistant to 1 mM sodium dodecyl sulphate (SDS). This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and Some Properties of a Protease from the Sarcocarp of Musk Melon Fruit

A protease has been purified from sarcocarp of musk melon, Cucumis melo ssp. melo var. reticulatus Naud. Earl’s Favourite. The protease was mostly present in the placenta part of the fruit and next in the inside mesocarp. The molecular mass of the enzyme was estimated to be about 62kDa on SDS-PAGE. The enzyme had a carbohydrate moiety. The optimum pH of the enzyme was 11 at 35°C using casein as a substrate. The enzyme was stable between pH 6 and 11. The enzyme was strongly inhibited by diisopropyl fluorophosphate, but was not inhibited by EDTA or cysteine protease inhibitors. From the digestion of Ala-Ala-Pro-X-pNA (X = Phe, Leu, Val, Ala, Gly, Lys, Glu, Pro, and diaminopropionic acid (Dap) substrates the specificity of the protease was found to be approximately broad, but the preferential cleavage sites were C-terminal sites of hydrophobic or acidic amino acid residues at P, position. It was proved that the enzymatic properties of musk melon protease are similar to those of cucumisin [EC 3.4.21.25]. The enzyme was not inhibited by typical proteinous inhibitors such as STI or ovomucoid. Therefore, this enzyme seems to be a useful protease for the food industries.     

Properties of a filamentous phage which adsorbs to pili coded by plasmids of the IncI complex

A filamentous phage, PR64FS, which adsorbs to tips of I pili was isolated. PR64FS is shorter than the I pilus-adsorbing phage If1 and differs from it in plaque morphology. Phages PR64FS and Ifl are serologically related and, like the latter, PR64FS adsorbs to pili coded by all Inc groups of the I plasmid complex.