Engineering of the active site of human lysozyme: conversion of aspartic acid 53 to glutamic acid and tyrosine 63 to tryptophan or phenylalanine

Three human lysozymes containing a mutation either at Asp-53 to Glu or at Tyr-63 to Trp or Phe were synthesized and examined for their immunological and enzymatical activities in comparison with the native one. All mutants were immunologically indistinguishable from native human lysozyme. The [Trp63] and [Phe63] mutants catalysed the hydrolysis of Micrococcus lysodeikticus cell wall and glycol chitin effectively, while the [Glu53] mutant displayed very low activity toward M. lysodeikticus cells and no detectable activity toward glycol chitin.     

Developmental changes in the effects of carbachol and morphine on cGMP contents of plasma, heart, and lung of mice

It is considered that carbachol increases plasma cGMP levels by acting on muscarinic receptors and morphine increases these levels by acting on opioid receptors, followed by stimulation of muscarinic receptors. We investigated the ability of carbachol and morphine to increase cGMP contents of plasma, heart, and lung and the guanylate cyclase activity of heart and lung homogenate in 1-, 2-, 3-, and 7-week-old mice. The increase in plasma cGMP levels induced by carbachol showed a peak at 2 and 3 weeks of age. The basal cGMP contents in heart and lung and their rise induced by carbachol, as well as the guanylate cyclase activity of these organs, were decreased in 7-week-old mice. The effects of morphine on the cGMP contents showed a similar developmental change, except for no effect in 1-week-old mice. These changes in the effects of carbachol and morphine may be the result of developmental changes of the muscarinic receptor--guanylate cyclase system and opioid receptors.     

X-ray structure of Glu 53 human lysozyme.

The three-dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 A resolution by X-ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild-type HL. The root mean square difference for the superposition of equivalent C alpha atoms is 0.141 A. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild-type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 A between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild-type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction.     

Xenopsin-related peptide(s) are formed from xenopsin precursor by leukocyte protease(s) and cathepsin D

Lysates of isolated rat polymorphonuclear leukocytes and macrophages were found to generate xenopsin-related peptides when incubated with a liver extract used as a source of precursor. The lysosomal enzyme, cathepsin D, was also shown to display this property and to share with the lysate a similar pH dependence (optimum, approximately pH 3.5) and sensitivity to the acid protease inhibitor, pepstatin A (ID50: lysate, 10 nM; cathepsin D, 30 nM). When subjected to HPLC on mu-Bondapak C-18, the xenopsin-related peptides generated by the lysate eluted near to those formed by cathepsin D and when tested in a radioreceptor assay for neurotensin, they displayed similar cross-reactivities (peak 2, approximately 50%; peak 1, approximately 100%). These results indicate that cathepsin D from lysed granulocytes can process precursor protein(s) to form radioreceptor-active xenopsin-related peptides.     

Antibody responses to early antigens of varicella-zoster virus (VZV) during varicella and zoster

The antibody responses to membrane and early antigens and thymidine kinase of varicella-zoster virus (VZV) were studied in sera during both varicella and zoster by a test with fluorescent antibody to membrane antigen (FAMA), staining the biochemically transformed cells by the immunofluorescent technique and neutralization of virus-specific thymidine kinase activity, respectively. Similar increases in FAMA antibody titers were demonstrated in sera from patients with varicella and zoster. IgM was detected in both groups, but appeared earlier during varicella than during zoster. Furthermore, the antibody titers to early antigens and virus-specific thymidine kinase were higher in patients with zoster than in those with varicella. These data suggest that different types of antibody responses occur during varicella and zoster.     

Histochemical staining of exogenous chromium and iron with the catalytic oxidation of phenylamines

Summary A highly sensitive and specific method for staining exogenous chromium and iron in tissues is described. This method is superior to conventional complex-forming methods with regard to its sensitivity and specificity for these metals. The staining reaction is based on the metalcatalysed oxidation of phenylamines. Tissue sections were incubated in a medium containing hydrogen peroxide and phenylamines, p-phenylenediamine or phenylhydrazine. Results obtained from test-tube experiments concerning the catalytic activities of metals indicated that the staining reactions depends on the activities of metals in tissues.     

Expression of synthetic human-lysozyme gene in Saccharomyces cerevisiae: use of a synthetic chicken-lysozyme signal sequence for secretion and processing

A multicopy plasmid was constructed to direct the synthesis and secretion of human lysozyme (HLY) in Saccharomyces cerevisiae. This plasmid contains a synthetic chicken-lysozyme signal sequence (SIG) and a synthetic HLY structural gene, both inserted between the yeast GAL10 promoter and 2 mu plasmid FLP (flip-flop recombination gene) terminator. The resulting plasmid directed the expression of the hybrid pre-lysozyme, with most of the HLY activity secreted into the culture medium and extracellular periplasmic space. The HLY activity in the culture medium increased with cell growth. The yeast accurately processed the hybrid precursor at the junction between the chicken SIG and the coding sequence downstream, yielding mature HLY. HLY purified from the culture medium was homogeneous and displayed specific activity identical to that of authentic HLY.     

Effect of maximal arm exercise on skin blood flux in the paralyzed lower limbs in persons with spinal cord injury

The purpose of the present study was to examine the effect of maximal arm exercise on the skin blood circulation of the paralyzed lower limbs in persons with spinal cord injury (PSCI). Eight male PSCI with complete lesions located between T3 and L1 performed graded maximal arm-cranking exercise (MACE) to exhaustion. The skin blood flux at the thigh (SBFT) and that at the calf (SBFC) were monitored using laser-Doppler flowmeter at rest and for 15 s immediately after the MACE. The subject's mean peak oxygen uptake and peak heart rate was 1.41 ± 0.22 1·min⁻¹ and 171.6 ± 19.2 beats·min⁻¹, respectively. No PSCI showed any increase in either SBFT or SBFC after the MACE, when compared with the values at rest. These results suggest that the blood circulation of the skin in the paralyzed lower limbs in PSCI is unaffected by the MACE.     

Development of secondary pine forests after pine wilt disease in western Japan

Abstract. The development of secondary Pinus densiflora (Japanese red pine) forests after pine wilt disease was studied through phytosociological analysis, estimation of forest structure before disease and size-structure, tree ring and stem analyses. Following the end of the disease, the growth of previously suppressed small oak trees was accelerated. This is quite different from the development of forests following fire, which starts with the establishment of pine seedlings. Pine wilt disease shifted the dominance of secondary forests from Pinus densiflora to Quercus serrata oak forest. In pine forests, disturbance by fire is important for forest maintenance. In contrast, disturbance by pine wilt disease leads to an acceleration of succession from pine forest to oak forest.     

Identification of Influenza C Virus Phosphoproteins

The HMV-II cells infected with influenza C virus were labeled with inorganic [³²P]phosphate to identify phosphorylated proteins. Analysis by radioimmunoprecipitation with antiviral serum or monoclonal antibodies revealed that three major structural proteins of the virus, hemagglutinin-esterase (HE), nucleoprotein (NP), and matrix protein (M1) are all phosphorylated in both infected cells and virions. It was also observed that, in the presence of trypsin (10 μg/ml), the unphosphorylated form of the HE glycoprotein was cleaved efficiently whereas the phosphorylated form was not, raising the possibility that phosphorylation of HE may influence its susceptibility to degradation by proteolytic enzymes.