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.     

Salicylate-promoted permeation of cefoxitin, insulin and phenylalanine across red cell membrane. Possible mechanism

Uptake of sodium cefoxitin, D-phenylalanine and insulin into human red blood cells was significantly enhanced by the presence of salicylate and 5-methoxysalicylate in the medium. The mechanism of adjuvant action appeared to depend on an affinity between the adjuvant and the protein fraction in the erythrocyte membrane. The inhibitory effect of DIDS and phlorizin on the salicylate-enhanced uptake of these compounds strongly suggests that the ability of salicylate to permeate the membrane may be essential for it to act as an adjuvant.     

Asexual reproductive organ-specific expression of the glyceraldehyde-3-phosphate dehydrogenase 2 gene of Pilobolus crystallinus

Pilobolus crystallinus has three putative glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes (pcgapdh1, pcgapdh2 and pcgapdh3). The results of this study demonstrate that expression of pcgapdh2 was increased by irradiation and that this increased expression was correlated with the formation of asexual reproductive organs (trophocysts). Interestingly, expression of pcgapdh2 was restricted to trophocysts. The formation of trophocysts was likely promoted by light, and the expression of pcgapdh2 was increased as a result of trophocyst formation. This is the first report that shows the regulation of a gapdh gene in an organ-specific manner in fungi.     

Evidence of increased synthesis of δ-aminolevulinic acid dehydratase in experimental lead-poisoned rats

δ-Aminolevulinic acid dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) was purified from rat and rabbit erythrocytes to a homogeneous state. Specific activities were 26.0 and 26.6 units/mg protein for the rat and rabbit enzymes, respectively, and their estimated molecular weight was 280 000, each consisting of 8 subunits of M_r 35 000. In order to quantitate rat δ-aminolevulinic acid dehydratase at several stages of lead-poisoning, a radioimmunoassay technique using goat antiserum against the rat enzyme was developed for the first time. This technique was specific, reproducible and high sensitive allowing determination of 1 ng enzyme. When drinking water containing 25 mM lead acetate was given daily to rats ad lib. the δ-aminolevulinic acid dehydratase activity in the blood, assayed without any pretreatment, decreased to 8% of the control level on the next day. On the contrary, the restored enzyme activity, assayed in the presence of Zn²⁺ and dithiothreitol, was greater than normal by the fourth day of lead administration in bone-marrow cells and by the ninth day in the peripheral blood. The increased activity level stayed the same from the ninth day onward. The enzyme content as determined directly by the radioimmunoassay technique at this stage was about 2-fold above that the control. There was no significant difference in the number of reticulocytes and the distribution profile of different types of reticulocytes between the lead-exposed and non-exposed rats. Therefore, the increase in the amount of δ-aminolevulinic acid dehydratase in erythrocytes of lead-poisoned rats was suggested to be due to an increased rate of synthesis in the bone-marrow cells.     

Geographical Distribution of Adolescent Body Height with Respect to Effective Day Length in Japan: An Ecological Analysis

The height of Japanese youth raised in the northern region tends to be greater than that of youth raised in the southern region; therefore, a geographical gradient in youth body height exists. Although this gradient has existed for about 100 years, the reasons for it remain unclear. Consideration of the nutritional improvement, economic growth, and intense migration that has occurred in this period indicates that it is probably the result of environmental rather than nutritional or genetic factors. To identify possible environmental factors, ecological analysis of prefecture-level data on the body size of 8- to 17-year-old youth averaged over a 13-year period (1996 to 2008) and Japanese mesh climatic data on the climatic variables of temperature, solar radiation, and effective day length (duration of photoperiod exceeding the threshold of light intensity) was performed. The geographical distribution of the standardized height of Japanese adolescents was found to be inversely correlated to a great extent with the distribution of effective day length at a light intensity greater than 4000 lx. The results of multiple regression analysis of effective day length, temperature, and weight (as an index of food intake) indicated that a combination of effective day length and weight was statistically significant as predictors of height in early adolescence; however, only effective day length was statistically significant as a predictor of height in late adolescence. Day length may affect height by affecting the secretion of melatonin, a hormone that inhibits sexual and skeletal maturation, which in turn induces increases in height. By affecting melatonin production, regional differences in the duration of the photoperiod may lead to regional differences in height. Exposure to light intensity greater than 4000 lx appears to be the threshold at which light intensity begins to affect the melatonin secretion of humans who spend much of their time indoors.     

Human T-cell hybridomas producing migration inhibitory factor and macrophage activating factors

Studies were carried out to determine the heterogeneity of factors that affect macrophage functions using human hybridomas constructed by fusing PHA-activated human peripheral blood lymphocytes with emetine-actinomycin D-pretreated cloned human acute lymphatic leukemia cells (CEM). Three assay systems were used to investigate the activity of the macrophage migration inhibitory factor and of the macrophage activation factors for glucose consumption (MAF-G) and for O2- formation. In the culture supernatant of hybridomas and other cells, various combinations of these activities were detected. The results indicate that at least three molecules are concerned in each of these activities.     

Protein kinase A inhibitors enhance radiation-induced apoptosis

In addition to a role for de novo protein synthesis in apoptosis we have previously shown that activation of a protein phosphatase or loss of activity of a kinase is also important in radiation-induced apoptosis in human cells [Baxter, and Lavin (1992): J Immunol 148:149–1954]. We show here that some inhibitors of protein kinases exacerbate radiation-induced apoptosis in the human cell line BM13674. The specific protein kinase A inhibitor isoquinoline sulfonamide (20 μM) gave rise to significantly increased levels of apoptosis at 2–6 h postirradiation compared to values after radiation exposure only. The same concentration of isoquinolinesulfonamide, which was effective in increasing apoptosis, reduced activity markedly. A 66% inhibition of cyclic AMP-dependent protein kinase A activity occurred in unirradiated cells at this concentration of H89 and activity was reduced to 58% in irradiated cells. Calphostin C, a specific inhibitor of protein kinase C, at a concentration of 0.1 μM, which caused 68% inhibition of enzyme activity in irradiated cells, failed to enhance the level of radiation-induced apoptosis. Other kinase inhibitors did not lead to an additional increase in apoptosis over and above that observed after irradiation. The results obtained here provide further support for an important role for modification of existing proteins during radiation-induced apoptosis.     

A Novel Enhanced Green FluorescentProtein-Expressing NOG Mouse for Analyzingthe Microenvironment of Xenograft Tissues

The interaction between transplanted cells and host tissues is important for the growth and maintenance of transplanted cells. To analyze the mechanisms of these interactions, a systemic fluorescent protein-expressing mouse is a useful recipient. In this study, we generated a novel NOG strain, which strongly expresses enhanced green fluorescent protein (EGFP; PgkEGFP-NOG), especially in the liver, kidney, gastrointestinal tract, and testis. Because the host tissues expressed EGFP, xenotransplanted human cancer cells were clearly identified as EGFP-negative colonies in PgkEGFP-NOG mice. Immunohistochemical analysis revealed that EGFP-expressing stromal tissues formed a complicated tumor microenvironment within xenograft tissues. Moreover, a similar microenvironment was observed in human iPS cell-derived teratomas. Collectively, these results indicated that a suitable microenvironment is essential for the growth and maintenance of xenotransplanted cells and that PgkEGFP-NOG mice represent a useful animal model for analyzing the mechanisms of microenvironment formation.     

Interactions of poly-N6-methyladenylic Acid and N6-substituted-9-methyladenines with poly-5-bromouridylic Acid: A Novel Base-pairing

Abstract

The interaction of poly-N⁶-methyladenylic acid (poly(m⁶A)) with poly-5-bromouridylic acid (poly(BU)) was studied by the mixing curve method. A 1 m⁶A: 2 BU stoichiometry was clearly indicated over a wide range of ionic strengths at neutral pH, while the binding of poly(m⁶A) to poly(U) is known to occur with 1 m⁶A:1 U. Digestion by nuclease S1 confirmed this stoichiometry, indicating the absence of single strands in a 1:2 mixture. Heating profile analysis and hydroxyapatite column chromatography provided further confirmation of this finding. To determine whether 1:2 stoichiometry holds in a monomer-polymer system, the interaction of N⁶-methyl-9-methyladenine (m⁶m⁹A), a corresponding monomer of poly(m⁶A), with poly(BU) was investigated.

Equilibrium dialysis experiments showed the stoichiometry of the interaction to be 1 m⁶A: 2 BU. Thus, we would describe some structural studies of the above complexes using c.d. and i. r. spectroscopy. Poly (m⁶A)·2poly(BU) and m⁶m⁹A·2poly(BU) are helical and analogous to each other in structure, and the bases in the complexes are all bound by hydrogen-bonding. N⁶-(Δ²-isopentenyl)- and N⁶-allyl-9-methyladenine were also found to form complexes with poly(BU), giving similar c.d. spectra with that of m⁶m⁹A·2poly(BU). The melting experiments indicated the T_ms to be substantially decreased, compared to the parent unmodified complexes, even though the T_m dependence of the polymer complex on salt concentration conforms to the typical triple strand. In the following, the biological significance of this novel pairing will be discussed.     

Mouse and human CD14 (myeloid cell-specific leucine-rich glycoprotein) primary structure deduced from cDNA clones

cDNA clones complementary to MS7-4 (Setoguchi et al. (1988) Somat. Cell Mol. Genet. 14, 427-438) from a mouse macrophage cDNA library were separated. Sequence analysis of these clones demonstrated that the longest cDNA clone, MS7X, had a 1366 bp insert and high homology with that of the human CD14 gene (Ferrero and Goyert (1988) Nucleic Acids Res. 16, 4173). Using the MS7X cDNA probe, cDNA clones were separated from cDNA libraries constructed from a human macrophage cell line and macrophages. The total cDNA sequence was 1364 bp in length, with an open reading frame of 1125 nucleotides matching that of the human CD14 gene except for one nucleotide difference. The amino-acid sequence (mouse CD14), deduced from the nucleotide sequence of the MS7X insert consisted of 351 amino-acid residues with a high leucine content (17.66%) and five putative N-glycosylation sites, and in vitro translation predicted a protein of molecular mass of 37.5 kDa. Human CD14 had 356 amino-acid residues, with high leucine content (15.5%), and contained four putative N-glycosylation sites. Mouse CD14 showed 13 building blocks, of which internal nine blocks have a conserved leucine motif and significant homology with human leucine-rich alpha 2-glycoprotein.