Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin.

We have previously shown that replacing the P1-site residue (Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lys results in the acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively. However, the inhibitory activities thus induced are not strong. In the present study, we introduced additional amino acid replacements around the reactive site to try to make the P1-site mutants more effective inhibitors of chymotrypsin or trypsin. The amino acid replacement Asp-->Tyr at the P2' site of OMCHI3(P1Met) resulted in conversion to a 35000-fold more effective inhibitor of chymotrypsin with an inhibitor constant (K(i)) of 1. 17x10(-11) M. The K(i) value of OMCHI3(P1Met, P2'Ala) indicated that the effect on the interaction with chymotrypsin of removing a negative charge from the P2' site was greater than that of introducing an aromatic ring. Similarly, enhanced inhibition of trypsin was observed when the Asp-->Tyr replacement was introduced into the P2' site of OMCHI3(P1Lys). Two additional replacements, Asp-->Ala at the P4 site and Arg-->Ala at the P3' site, made the mutant a more effective inhibitor of trypsin with a K(i) value of 1. 44x10(-9) M. By contrast, Arg-->Ala replacement at the P3' site of OMCHI3(P1Met, P2'Tyr) resulted in a greatly reduced inhibition of chymotrypsin, and Asp-->Ala replacement at the P4 site produced only a small change when compared with a natural variant of OMCHI3. These results clearly indicate that not only the P1-site residue but also the characteristics, particularly the electrostatic properties, of the amino acid residues around the reactive site of the protease inhibitor determine the strength of its interactions with proteases. Furthermore, amino acids with different characteristics are required around the reactive site for strong inhibition of chymotrypsin and trypsin.     

Mechanism of inhibition caused by long-chain fatty acids in anaerobic digestion process

The inhibitory effect of long-chain fatty acids on the anaerobic digestion process was examined in batch experiments using synthetic substrates. The addition of long-chain fatty acids caused the appearance of the appearance of the lag period in the methane production from acetate and in the degradation of both long-chain fatty acids and n-butyrate. Methane production from hydrogen proceeded without lag period although its rate was lowered. Fermentation of glucose was not inhibited. Neutral fat in the whole milk was easily hydrolyzed to long-chain fatty acids, which brought about the inhibition. The addition of calcium chloride reduced the inhibitory effect of long-chain fatty but it did not do so after the culture had been exposed to long-chain fatty acids for more than several hours. The addition of calcium carbonate could not reduce the inhibition because of its insolubility.     

Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis

Abstract Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus . This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis .     

Characterization of the bacteriophage T3 DNA packaging reaction in vitro in a defined system

The bacteriophage T3 DNA packaging system in vitro defined here is composed of purified proheads and two non-capsid proteins, the products of genes 18 and 19 (gp18 and gp19). In this system, a precursor complex (50 S complex) accumulates in the presence of adenosine 5'-O-(3'-thiotriphosphate) (ATP-gamma-S), a non-hydrolyzable analog of ATP. The 50 S complex is converted to a filled head in the presence of ATP. The conversion of the 50 S complex, formed by preincubation with ATP-gamma-S, to the mature head proceeds in a synchronous manner after the addition of ATP. The lag time for formation of mature heads from the 50 S complex is 1.8, 4.5 and 6.8 minutes at 30, 25 and 20 degrees C, respectively. DNA is translocated into the capsid at a constant rate of 5.7 x 10(3) base-pairs per minute at 20 degrees C. The conversion of the 50 S complex to the mature head exhibits a sigmoidal relationship with respect to the concentration of ATP, the concentration for half-maximal activity being about 20 microM. The transition of the prohead to the expanded capsid occurs at 20 degrees C at one minute 40 seconds after the initiation of DNA translocation, when one-fourth of the genome has been packaged into a prohead. At the same time, the capsid-DNA complex becomes stable to high concentrations of salt. When DNA translocation is interrupted by the addition of ATP-gamma-S, packaged DNA exists at 0 degrees C as well as at 20 degrees C but the exit of DNA stops after one-third of the genome is inside the capsid. After exit, DNA is retranslocated into the expanded capsid by the addition of ATP at a rate of about 5.7 x 10(3) base-pairs per minute at 20 degrees C. The decrease in concentration of ATP interrupts DNA translocation into the capsid but does not induce DNA exit. Interrupted DNA translocation may be reinitiated by the addition of ATP. DNA exit is not induced by the addition of ATP-gamma-S to mature heads or partially filled heads pretreated with DNase.     

Primary Infection of Barley by Erysiphe graminis f. sp. hordei in Relation to Leaf-Age Dependent Resistance and the Roles of the Epidermis and Mesophyll in this Resistance

Abstract The infection frequency of both compatible and incompatible races of Erysiphe graminis f. sp. hordei decreased gradually with increasing leaf age on undetached primary barley leaves. The length of secondary hyphae of the compatible race was approximately the same regardless of age, but secondary hyphae were slightly longer on younger seedlings than on older seedlings in the case of the incompatible race. Both the infection frequency and length of secondary hyphae of the two races weredistinctly different. On composite sections produced by exchanging the epidermal layers of young and relatively mature primary leaves, the infection frequency of the compatible race was higher on the epidermis of young leaves than on the epidermis of older, leaves, regardless of which mesophyll was under the epidermis. The epidermis appears to play a major role in age-dependent resistance, while the mesophyll may act disparately by providing a factor promotive to mildew infection in addition to supporting the resistance function of the epidermis.     

Inhibition of ATPase Activity in Pea Plasma Membranes In Situ by a Suppressor from a Pea Pathogen, Mycosphaerella pinodes

A pathogenic fungus of pea, Mycosphaerella pinodes, secretesa so-called "suppressor" in its pycnospore germination fluid.The suppressor blocks the defense responses and induces localsusceptibility (accessibility) in pea plants to agents thatare not pathogenic in pea. The suppressor nonspecifically inhibitsthe ATPase activity in plasma membranes prepared from pea, soybean,kidney bean, cowpea and barley plants. However, cytochemicalstudies by electron microscopy indicate that the suppressorspecifically inhibits the ATPase in pea cell membranes, butnot in those of four other plant species tested. That is, thespecificity of the suppressor appears at the cell and/or tissuelevel, but is not evident in vitro. Furthermore, the inhibitoryeffect of the suppressor is temporary because the ATPase activityrecovers 9 h after the treatment. A similar effect was observedafter inoculation with M. pinodes but not with a nonpathogenof pea, M. ligulicola. The role of the suppressor in host-parasitespecificity is discussed. (Received April 9, 1991; Accepted August 6, 1991)     

Conformational dynamics of DNA affected by intercalation and minor groove binding: direct observation of large DNA.

Using fluorescence microscopy, we have observed moving DNA molecules in solution and analyzed the "higher-order" structure in a quantitative manner. It was found that EB (ethidium bromide), an intercalator, has the effect to increase the persistent length. In other words, EB expands DNA. Whereas, DAPI (4',6-diamidino-2-phenylindole), a minor groove binding drug, decreases the persistent length. It is demonstrated that the direct observation of DNA molecules with fluorescence microscopy is quite useful to study the interaction of various chemical compounds with DNA molecules.     

Carboxyl Terminal Segment of T4 Gene 32 Protein is Dispensable in Vivo

We obtained a mutant of bacteriophage T4 which overcame thedeficiency in gene 49 endonuclease. The new mutation occurredin gene 32 and the mutant, which was viable, produced an amberfragment under non-suppressed conditions, lacking about 30 aminoacid residues at the carboxyl terminus. Its growth, recombination,and resistance to UV irradiation were affected to various degreesby the particular suppressor tRNA present. Growth was increasedby Su2⁺ to nearly that of the wild type, but growth of all otherswas reduced in the presence and absence of suppressors, suggestingthat the terminal domain of gene 32 protein is not indispensablefor the function but modulates it. We discuss the mechanismby which the mutation overcomes the defect in gene 49 endonuclease. ¹ This paper is dedicated to the memory of the late Dr. JojiAshida. (Received November 22, 1982; Accepted February 21, 1983)     

Fine structure and lectin histochemistry of the apical surface of the free neuromast of Lampetra japonica

The surface coat, ciliary process, and microvilli of the lamprey neuromast were examined with electron microscopy after tannic acid prefixation and lectin histochemistry. The neuromast was found to exist in the form of a dermal mound with a furrow in the middle. On the bottom of the furrow, the hair cell was characterized by a kinocilium and 15–20 stereocilia, arranged along the longitudinal axis of the furrow. Spanning structures were demonstrated between the kinocilium and stereocilia as well as between stereocilia. The surface coat, enhanced by tannic acid prefixation, was particularly rich over the surface of the supporting cell; by contrast, it was thin over the hair cell. Some lectins (PNA, GS-I, SBA, WGA) showed affinity to the surface coat of the supporting cell as well as the hair cell, and the others (RCA-I, MPA, ConA) showed affinity only to the supporting cell. These differences in the structure and affinities of the surface coat suggest an extracellular milieu highly specialized for the hair cell in this particular form of the mechanoreceptor.