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.     

Tail-associated structural protein gp61 of Staphylococcus aureus phage φMR11 has bifunctional lytic activity

A tailed bacteriophage, φMR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1–150] and the lysozyme (aa 401–624) domains but not the linker domain (aa 151–400) caused efficient lysis of S . aureus . Immunoelectron microscopy localized gp61 to the tail tip of the φMR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of φMR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with φMR11 was also lysed by both proteins. Staphylococcus aureus strains on which φMR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.     

Use of paired abdominal flaps for release of adduction contractures of the thumb.

A method for correction of an adduction contracture of the thumb is presented. Paired flaps provide good cover to the palmar and dorsal sides of the web space. This method produces better cosmetic and functional results than the traditional methods.     

Automated high-performance liquid chromatographic method for determination of furosemide in dog plasma

An automated high-performance liquid chromatographic method for the determination of the diuretic drug furosemide has been established. Dog plasma was injected directly into a two-column system with a BSA—ODS (ODS column coated with bovine serum albumin) precolumn and a C₁₈ analytical column for the separation of furosemide. The two columns were automatically switched. Furosemide remained trapped on the precolumn while proteins were eluted to waste. After column switching, furosemide was washed onto the analytical column and analysed without interference. The greatest advantage of the method is its easy performance without manual sample preparation; it requires no extraction or deproteinization. The method allows determination of 0.1–10 μg/ml of furosemide with accuracy and precision comparable with previously reported values. The coefficients of variation obtained from replicate measurements of 1 μg/ml and 5 μg/ml samples were 1.65% and 2.40%, respectively. This method was used to measure the plasma levels of furosemide in beagle dogs to whom the drugs was administered, as a reference, in a toxicological study.     

Identification and characteristics of a novel mitochondrial ATPase in rat liver

A novel ATPase is postulated for isolated mitochondria and mitoblasts of rat liver. The enzyme is active in the presence of oligomycin and carboxyatractyloside. It can be distinguished from other well-known mitochondrial and non-mitochondrial ATPases by its insensitivity to common ATPase inhibitors and effectors and by digitonin treatment. The ATPase is localized on the outer side of the inner mitochondrial membrane. It is activated by Mg2+ in the alkaline pH range and exhibits a biphasic kinetics. The novel external ATPase of rat liver mitochondria possesses similar properties with respect to ATP-dependent protease.     

Effects of Exogenous Amino Acids on Iron Uptake in Relation to their Effects on Photoperiodic Flowering in Lemna paucicostata 6746

The flowering of Lemna paucicostata 6746 grown on 14-h photoperiodwas enhanced by the addition of high concentrations of ironto the medium, which also increased the endogenous iron concentration.The addition of asparagine, aspartate, glutamate, -alanine,glycine or serine to the medium also increased the endogenousiron level, resulting in the promotion of flowering. In contrast,the addition of cysteine, cystine, glutamine, arginine, threonineor phenylalanine lowered the endogenous iron level, resultingin the inhibition of flowering. Glycine and asparagine added to the medium during an inductive96-h dark period did not promote iron uptake and had no effecton flowering, but when added during the subsequent 120-h lightperiod, they promoted both iron uptake and flowering response.The increase in the endogenous iron level seems to favor floraldevelopment rather than induction of photoperiodic floweringof Lemna paucicostata 6746. (Received September 8, 1986; Accepted March 31, 1987)     

Colchicine activates cell cycle-dependent genes in growth-arrested rat 3Y1 cells

When growth-arrested 3Y1 cells (Fischer rat fibroblasts) were exposed to 3 X 10(-5) M colchicine, they entered S phase after a 12-h lag period which is the same as that in serum-stimulated cells. The expression of genes such as c-fos, c-myc, JE, KC, ornithine decarboxylase, and histone H3, analyzed by Northern blotting, increased in a cell-cycle dependent manner after colchicine treatment. The increased level of mRNAs was much smaller in colchicine-stimulated cells than in serum-stimulated cells, corresponding to the lower frequency of the former cells entering S phase. The course of the prereplicative phase seems to be similar in terms of the expression of cell cycle-dependent genes in cells stimulated with colchicine and in those stimulated with serum.     

Molecular cloning and sequencing of cDNA encoding goat pre alpha-lactalbumin

In poly(A)+RNA extracted from a lactating goat mammary gland, mRNA of about 750 nucleotides was shown to encode pre alpha-lactalbumin by using in vitro translation and immunoprecipitation. From the total poly(A)+RNA, the cDNA library was constructed using the Escherichia coli plasmid pUC18; it was screened with the oligodeoxyribonucleotide probe corresponding to the amino acid sequence of Trp60-Gln65 of goat alpha-lactalbumin. A plasmid containing almost full-length cDNA of goat pre alpha-lactalbumin, pGLA-1, was identified. The cDNA insert of pGLA-1 comprises 727 base pairs and contains the signal peptide and mature protein sequence.