A Zn2+-dependent and histone H1-specific protease in sea urchin sperm

Protease activity was extracted from sea urchin sperm with 1% Triton X-100 and partially purified by DEAE-cellulose and Sephadex G-100 chromatography. The enzyme preferentially degraded histone H1, while showing only a weak activity toward other histones. Heat-denatured casein and bovine serum albumin were not digested by this enzyme under the present experimental conditions. This protease hydrolyzed only Boc-Val-Leu-Lys-MCA among various peptidyl-MCAs. The optimal pH ranged from 7 to 11. Its molecular weight was about 41,000. Among various known inhibitors of proteases, only omicron-phenanthroline effectively inhibited the activity. The enzyme was stimulated by Zn2+ or Co2+. It was inactivated by omicron-phenanthroline but could be reactivated by the addition of Zn2+ or Co2+. Therefore, this protease seems to be a metalloprotease dependent on Zn2+ or Co2+. The insensitivity of this enzyme to phosphoramidon and its very restricted substrate specificity suggest that this enzyme is very different from other metalloproteases described hitherto.     

Replication timing: histone genes replicate during early S phase in cleavage-stage embryos of sea urchin.

Newly synthesized DNA was separated from the bulk of the DNA by pulse-labeling with BUdR and centrifugation in an alkaline CsCl buoyant density gradient. The content of histone gene in the newly synthesized DNA was determined by DNA dot hybridization. The gene contents in DNA replicated during the early half of S phase and during the whole S phase were compared. Results showed that histone genes were replicated during the first half of the S phase in embryos in the early cleavage stage.     

Nucleosomelike structures associated with chromosomes of the archaebacterium Halobacterium salinarium.

Chromosomes of the halophilic archaebacterium Halobacterium salinarium were examined by electron microscopy after being spread onto water. The major part of the chromosomal DNA was associated with protein particles with diameter of 9.4 nm, arranged tandemly along the DNA fibers. Thus, the primary structure of the chromosome resembles that of eucaryote chromosomes.     

An ATP-driven Cl- pump in the brain

EDTA-treated microsomes prepared from rat brain mainly consisted of sealed membrane vesicles 200-500 nm in diameter and were rich in both Cl- -ATPase and Na+,K+-ATPase activities. Such Cl- -ATPase-rich membrane vesicles accumulated Cl- in an ATP-dependent and osmotically reactive manner in the presence of 1 nM ouabain. The Cl- uptake was maximally stimulated by ATP with a Km value of 1.5 mM; GTP, ITP, and UTP partially stimulated Cl- uptake, but CTP, beta, gamma-methylene ATP, ADP, and AMP did not. The ATP-dependent Cl- uptake was accelerated by an increase in the medium Cl- concentration with a Km value of 7.4 mM. Such stimulation of Cl- uptake by ATP was dependent on the pH of the medium, with an optimal pH of 7.4, and also on the temperature of the medium, with an optimal range of 37-42 degrees C. Ethacrynic acid dose dependently inhibited the ATP-dependent Cl- uptake with a concentration for half-maximal inhibition at 57 microM. N-ethylmaleimide (0.1 mM) completely inhibited and sodium vanadate (1 mM) partially inhibited the ATP-dependent Cl- uptake. The membrane vesicles did not accumulate H+ in the Cl- uptake assay medium. The ATP-dependent Cl- uptake profile agreed with that of Cl- -ATPase activity reported previously (Inagaki, C., Tanaka, T., Hara, M., and Ishiko, J. (1985) Biochem. Pharmacol. 34, 1705-1712), and this strongly supports the idea that Cl- -ATPase in the brain actively transports Cl-.     

Translocation of organic compounds in sunflower III. Effect of oligomycin, ouabain and phlorizin on translocation of sugars

The apical portions of intact sunflower leaves were infiltratedwith ¹⁴C-glucose, ¹⁴C-fructose or 3-O-methyl-¹⁴C-glucose andthe basal portions were treated with inhibitors. The effects of oligomycin, ouabain and phlorizin on translocationwere studied. Inhibition of translocation from the basal portionof the leaf to the stem was determined by experiments usingoligomycin. In other experiments, each leaf was divided intothree parts. The apical portion was fed with ¹⁴C-glucose andthe basal part treated with oligomycin. The effects of oligomycinon the distribution of ¹⁴C-glucose, ¹⁴C-sucrose, ¹⁴C-fructoseand ¹⁴C-sugar phosphate along the three parts of the leaf wereinvestigated. Inhibition of sucrose synthesis in the leavestreated with oligomycin was observed. Oligomycin inhibited ¹⁴Ctranslocation from the leaf. ¹Present address: Department of Biology, Faculty of Science,Science University of Tokyo, Kagurazaka, Shinjuku-ku, Tokyo,Japan. (Received August 17, 1978; )     

Erysiphe kenjiana (Erysiphales), a new invasive fungus in Europe

An Asian powdery mildew fungus Erysiphe (Uncinula) kenjiana (Erysiphales, Ascomycota) has been found in Ukraine. This is the first record of this fungus in Europe. In 2007, E. kenjiana was collected on four Ulmus species in Kiev. All locations adjoined railways or an airport. Development of E. kenjiana was epiphytotic. This species was not found on elms surveyed at towns situated north-east, east or south of Kiev. The fungus may have been brought directly to Ukraine by rail or air transport. In 2008, the fungus was also collected in Chernihiv situated north-east of Kiev. It is likely that E. kenjiana will spread over all Ukraine and into countries of central and western Europe in 2009 or later. Molecular phylogenetic analyses using D1/D2 domains of the 28S rDNA and ITS sequences revealed that the Ulmaceae-Cannabaceae-parasitic powdery mildews, including E. kenjiana, form a clade with strong supports, suggesting that these species diverged from a single ancestor and expanded their host ranges within the Ulmaceae and allied Cannabaceae. This hypothesis is supported by these species sharing the unique morphology of enlarged apices on their chasmothecial appendages. These fungi formed part of a larger grouping with species on Fagaceae, Nothofagaceae, Rosaceae, and Sapindaceae with strong statistical supports. These results suggest that Uncinula-like powdery mildew fungi on these plant families exhibit close evolutionary relationships with their hosts.     

Coiled Filamentous Structure in the Sperm Acrosome of Gastropoda, Sulculus aquatilis and Turbo cornutus

A helically coiled filamentous structure, termed the truncated cone originally identified in Haliotis discus , was demonstrated to exist in the apex of the acrosome subjacent to the outer acrosomal membrane of the sperm in two species of Gastropoda, Sulculus aquatilis and Turbo cornutus . Thin section and whole mount preparations revealed that in intact sperm this filamentous structure consisted of about 12 helically arranged filaments with a diameter of 10–12 nm which were tightly packed in a truncated shape. This truncated cone elongated anteriorly from the acrosomal opening and transformed into a cylinder which closely surrounds the acrosomal process during the acrosome reaction. In S. aquatilis and T. cornutus , the truncated cone elongated more than 3 and 1.5 times as long as the original height, respectively. The elongated truncated cone was characterized by striations with increase in its periodic spacing and inclinations in thin sections. The truncated cone in both the species was fundamentally analogous to that of Haliotis discus , further suggesting that the truncated cone plays a role in fertilization as a common cytoskeletal structure among the species of Gastropoda.     

Translocation of organic substances in sunflower II. Role of sugar phosphate in translocation of sugars

A single sunflower leaf was fed ¹⁴C-glucose for one min. Aftervarions periods, two sections of the plant stem above and belowthe fed leaf were cut into several sections and examined forradioactivity. Sucrose phophate was identified in the extracts after neutronirradiation of the chromatograms. A correlation was noted between the proportion of sucrose phosphatein the fed leaf and the rate of translocation from it. (Received May 22, 1978; )     

Chemical modifications of histidyl and tyrosyl residues of inorganic pyrophosphatase from Escherichia coli

Chemical modifications by photooxidation in the presence of rose bengal (RB) and with tetranitromethane (TNM) were carried out to elucidate the amino acid residues involved in the active site of inorganic pyrophosphatase (pyrophosphate phosphohydrolase) [EC 3.6.1.1] from Escherichia coli Q13. The photooxidation caused almost complete inactivation, which followed pseudo-first-order kinetics depending on pH and concentration of RB. The presence of Mg2+ or complex between Mg2+ and substrate or substrate analogues, imidodiphosphate and sodium methylenediphosphate, gave partial protection against the photoinactivation, whereas the substrate alone showed no protective effect. The enzyme was almost completely inactivated by chemical modification with TNM, depending upon the concentration of TNM. The amino acid analyses and enzyme activity measurements revealed that 2 histidyl residues among 5 photooxidized residues and 2 tyrosyl residues per subunit were essential for the enzyme activity. The circular dichroism (CD) spectra in the far ultraviolet region showed no significant alteration during these two modifications, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modifications altered considerably the CD bands in the near ultraviolet region and the fluorescence spectra, indicating that subtle change in conformation had occurred in the vicinity of the active site in the enzyme molecule. These results strongly suggest that histidyl and tyrosyl residues may be involved in the active site or be located in the vicinity of the active site and seem to participate in the mechanism of stability against heat inactivation.