Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203

Abstract Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M⁺ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M⁺ and M⁻ bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M⁺ bacteria after treatment with N -acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M⁻ bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.     

Immune response induced by live attenuated varicella vaccine and short term follow-up on immunity of the vaccinated children

Live attenuated varicella vaccine (Oka strain, 500 to 750 PFU) was inoculated into 234 children with various underlying diseases. Varicella-zoster virus (VZV) specific immune adherence hemagglutination antibody developed in 95% of initially seronegative subjects. VZV specific cellular immune responses were detected in 91% of subjects within 6 weeks after vaccination. A preliminary survey of 46 vaccinated normal-risk subjects during the first and the third year revealed excellent protection with the exception of one case who had not shown any antibody response at the fourth week.     

Effects of polyamines on the activities of Escherichia coli ribonuclease I and II.

The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by E. coli ribonuclease I [ribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.23] and ribonuclease II [EC 3.1.4.1] have been studied. The degradation of poly(C) by RNase II was stimulated by spermine and spermidine, while that of poly(A) by RNase II was not affected by polyamines. Under our standard experimental conditions, the breakdown of poly(U) by RNase II was inhibited slightly by polyamines. The stimulatory effect of spermine and spermidine on the breakdown of poly(C) occurred in the absence of monovalent cations but not in the absence of divalent cations. When polyamines were used as a stimulant of RNase II, the ratio of poly(C) degradation to poly(U) degradation was greater in the presence of inhibitors such as poly(G) than in their absence. Although the breakdown of all synthetic polynucleotides by RNase I was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C)greater than poly(A)(see text)poly(U). However, the difference in degree of stimulation among polynucleotides decreased as monovalent cation concentration was increased.     

Studies on the restoration of the activities of Ribonucleases by polyamines in the presence of various ribonuclease inhibitors.

The effect of polyamines on ribonucleases in the presence of various inhibitors (poly(G), heparin, and rat liver RNase inhibitor) has been studied. Bovine pancreatic RNas A and a ribonuclease from horse submaxillary gland (RNase HS) were inhibited by the inhibitors, but RNase T1 and RNase M were not inhibited. Polyamines were found to restore the activites of RNase A and RNase HS inhibited by poly(G) or heparin but not those activities inhibited by rat liver RNase inhibitor. When poly(U) and poly(C) were used as substrates, the inhibitory effects of poly(G) and heparin were greater with poly(U) than poly(C) as a substrate. However, when poly(C) was used as a substrate in the presence of either of the above inhibitors, the restoration of RNase activity by sperimine was more efficient. In fact, a stimulatory effect was observed. From the double-reciprocal plots, it was concluded that polyamines restored the activiities of RNases by increasing the availability of the substrate and enzyme to each other. The restoration of enzyme activity by polyamines occurred through the binding of the polyamines to the inhibitor and the subsequent release of enzyme from the inhibitor.     

Properties of membrane-bound α-glucosidases: Possible sugar receptor protein of the blowfly, Phormia regina

Previously reported PII-type α-glucosidase located in the precipitate of the labellar homogenate of the blowfly Phormia regina was solubilized by sodium deoxycholate (DOC) and further separated into three isozymes with different molecular weight: PII-M (mol. wt 9 × 10⁴). PII-D (mol. wt 2 × 10⁵) and PII-T (mol. wt 8 × 10⁵) by molecular sieve chromatography on Biogel P-300 or Ultragel AcA-34. These three isozymes had almost the same K_m's and relative values of V_m's for several substrates, suggesting that they had the same common active site.PII-D and PII-T are more strongly embedded in the membrane than PII-M, because the proportion of PII-D and PII-T was much increased when the remaining glucosidase in the precipitate after the first solubilization was reextracted by DOC. A large peak of α-glucosidase isozyme P-IV which preferentially hydrolyze sucrose eluted just after P-II (soluble P-II) when the supernatant fraction of the labellar homogenate was chromatographed on DEAE-Sephadex A-50. P-IV was scarcely present in the precipitate fraction.Soluble P-II had the same mol. wt as PII-M and had similar properties to PII-M except for the ratio of V_m's.A large proportion of PII-D was contained in the well washed labellar integuments, a preparation rich in labellar chemosensilla. It suggests that most of the insoluble α-glucosidase contained in the dendrite in labellar chemosensilla is PII-D. PII-D (and PII-T) are possible sites of the pyranose receptor molecule because their properties and localization agree well with those of the receptor.     

Action spectra for the light inhibition of flowering and its reversal inLemna paucicostata T-101

Lemna paucicostata Hegelm. T-101, a short-day plant, flowers when plants preirradiated with red light (R) for 24 h are subjected to inductive darkness for 72 h followed by two short-day cycles (6 h R+ 18 h dark). However, flowering is inhibited by blue-or far-red-light pulses applied at the beginning of the inductive dark period. These inhibitory light effects are fully reversible by a R pulse. The action spectra for the inhibitory light effect and for its reversal show that the light pulses act exclusively through phytochrome. It is concluded that a low level of Pfr at the beginning of the inductive dark period prevents flowering.Abbreviations R red (light) - B blue (light) - FR far-red (light)     

Human pituitary corticotrophs: their amphophilic stainability and immunohistochemical characterization

Immunohistochemical characterization of the human pituitary beta(R) cells was investigated through the findings of the immunoreactivities with anti-porcine ACTH, -rat TSH, -rat FSH sera. Immunostained corticotrophs are oval or round in shape and localized in the anteromedial wedge. It is shown on the adjacent sections that they correspond to the beta(R) cells with amphophilic stainability with PAS-iron hematoxylin. In this wedge, amphophilic cells are preponderant, but PAS-positive thyrotrophs and gonadotrophs are not numerous. Amphophilic stainability varies in degree from cell to cell: One cell contains numerous medium-size of secretory granules weakly stained with iron hematoxylin and strongly with PAS in the PAS-positive cytoplasm, and the other cell is filled with big secretory granules intensively stained with iron hematoxylin and weakly with PAS. The immunostained TSH, LH and FSH cells are different from the beta(R) corticotrophs, because anti-ACTH serum never reacts to the TSH, LH and FSH cells in the two adjacent sections. LH and FSH reactivities are observed in the single cells. It is concluded that human corticotrophs are amphophilic beta(R) cells filled with secretory granules, and that they have quite a different appearance from the rat chromophobic stellate corticotrophs with a row arrangement of secretory granules along the plasma membrane.     

An improved method for estimating sequence divergence between related DNAs from changes in restriction endonuclease cleavage sites

Summary We have developed a theory to estimate the degree of sequence divergence between related DNAs from the comparison of restriction endonuclease recognition sites. Two major improvements have been made upon a similar method reported by Upholt (1977). First, the most probable value is calculated by the collective use of all available data. This reduces intrinsic statistical error and extends the analyzable range of sequence divergence. Second, all variables are redefined so that they have strict mathematical implications. This corrects a serious error arising from the misinterpretation of the meaning of the fraction of conserved cleavage sites. With this refined method, sequence divergence between rat and mouse mitochondrial DNAs (mtDNAs) was calculated to be about 25% substitutions/nucleotide, which is in good agreement with the DNA-DNA hybridization data obtained by Jakovcic et al. (1975). It was also estimated that the three types of rat mtDNAs differ from one another by 0.3 ~1% of total base pairs. These values are 2 ~5 times smaller than those obtained with the conventional method.     

Effects of 2 water-soluble contrast media, meglumine iothalamate (Conray) and meglumine iocarmate (Dimer-X), on the electric activity of identifiable giant neurons of the African giant snail (Achatina fulica Ferussac)]

The influences of two water soluble contrast media, meglumine iothalamate and meglumine iocarmate, on the neuronal excitability and on the neuronal sensitivity to putative transmitters were examined in comparison with those of sucrose using two identifiable giant neurones of Achatina fulica Férussac (the TAN and the PON). A relatively low increase of osmotic pressure of the extracellular fluid, produced by the application of contrast media, reversed the Cl- dependent inhibition caused by a putative transmitter. The same increase of this osmotic pressure, however, did not influence the Cl- independent inhibition and the excitation of the neurone examined. The hyperpolarization of neuromembrane was caused by an increase of osmotic pressure of the extracellular fluid. Its relatively high increase was necessary to make spontaneous spike discharges disappear totally. All effects of the two contrast media, observed in this study, were due to the increase of osmotic pressure of the extracellular fluid ; no specific effect of the contrast media containing the iodine on the indicators used was observed.     

Giant oil cells in the cotyledons ofPharbitis nil and other convolvulacean plants

Suspension of protoplasts (ca. 13–25 μm in diameter) that were isolated from the mesophyll of the cotyledons ofPharbitis nil, strain Violet, contained many large spherical or spheroidal bodies (ca. 100 μm in diameter). Microscopic observation of these bodies and some anatomic studies of the cotyledons during embryogenesis and after germination showed that these bodies are giant cells containing many oil drops stainable with Sudan dyes. Such giant cells were found in four otherPharbitis nil strains, Nepal, Tendan, Africa and Tokyo-kokei, and in six other Convolvulacean plants,Ipomoea batatas, cv. Koukei-14,Calystegia japonica, Calystegia hederacea, Calonyction aculeatum, Quamoclit pennata andCuscuta japonica.