Alteration of Bacteriolytic Enzyme Profile of Staphylococcus aureus during Growth

Profiles of cell-associated bacteriolytic activities and those in the culture supernatant of Staphylococcus aureus FDA209P at various stages of growth were analyzed using sodium dodecyl sulfate-polyacrylamide gels containing Micrococcus luteus or S. aureus. In the logarithmic growth phase, the cell-associated bacteriolytic activities extracted with Triton X-100 contained a number of bacteriolytic proteins, the profiles of which were similar to those we reported elsewhere (Sugai, M., Akiyama, T., Komatsuzawa, H., Miyake, Y., and Suginaka, H.(1990) J. Bacteriol., 172, 6494-6498). The proteins include P1, P2, P7, P9, PX, P13, P18 and other minor components. At the stationary growth phase, the bacteriolytic band-profile of the Triton X-100 extract changed dramatically. P1, P7 and P9 disappeared, and the other minor bands had markedly decreased band intensities. On the other hand, P2, PX, P13, and P18 retained their band intensities during the stationary growth phase. The band intensities of P7, P13, PX, and P18 increased in the supernatant during the logarithmic growth phase. These results indicated that the bacteriolytic band-profile changes during growth.     

Determination of the solution structure of the SH3 domain of human p56 Lck tyrosine kinase

Summary The solution structure of the SH3 domain of human p56 Lck tyrosine kinase (Lck-SH3) has been determined by multidimensional heteronuclear NMR spectroscopy. The structure was calculated from a total of 935 experimental restraints comprising 785 distance restraints derived from 1017 assigned NOE cross peaks and 150 dihedral angle restraints derived from 160 vicinal coupling constants. A novel combination of the constant-time ¹H–¹³C NMR correlation experiment recorded with various delays of the constant-time refocusing delays and a fractionally ¹³C-labelled sample was exploited for the stereo-specific assignment of prochiral methyl groups. Additionally, 28 restraints of 14 identified hydrogen bonds were included. A family of 25 conformers was selected to characterize the solution structure. The average root-mean-square deviations of the backbone atoms (N, C, C, O) among the 25 conformers is 0.42 Å for residues 7 to 63. The N- and C-terminal residues, 1 to 6 and 64 to 81, are disordered, while the well-converged residues 7 to 63 correspond to the conserved sequences of other SH3 domains. The topology of the SH3 structure comprises five anti-parallel -strands arranged to form two perpendicular -sheets, which are concave and twisted in the middle part. The overall secondary structure and the backbone conformation of the core -strands are almost identical to the X-ray structure of the fragment containing the SH2-SH3 domains of p56 Lck [Eck et al. (1994) Nature, 368, 764–769]. The X-ray structure of the SH3 domain in the tandem SH2-SH3 fragment is spatially included within the ensemble of the 25 NMR conformers, except for the segment of residues 14 to 18, which makes intermolecular contacts with an adjacent SH2 molecule and the phosphopeptide ligand in the crystal lattice. Local structural differences from other known SH3 domains are also observed, the most prominent of which is the absence in Lck-SH3 of the two additional short -strands in the regions Ser¹⁵ to Glu¹⁷ and Gly²⁵ to Glu²⁷ flanking the so-called RT-Src loop. This loop (residues Glu¹⁷ to Leu²⁴), together with the n-Src loop (residues Gln³⁷ to Ser⁴⁶) confines the ligand interaction site which is formed by a shallow patch of hydrophobic amino acids (His¹⁴, Tyr¹⁶, Trp⁴¹, Phe⁵⁴ and Phe⁵⁹). Both loops are flexible and belong to the most mobile regions of the protein, which is assessed by the heteronuclear ¹⁵N,¹H-NOE values characterizing the degree of internal backbone motions. The aromatic residues of the ligand binding site are arranged such that they form three pockets for interactions with the polyproline ligand.Abbreviations CT constant time - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - SH2 Src homology domain 2 - SH3 Src homology domain 3     

Establishment of a set of combined immunodeficient DA/Slc-Foxn1(rnu) Lyst(bg) congenic rat strains.

The congenitally athymic nude rat is used for studying cancer and transplantation owing to its hairlessness and T-cell defective function caused by the Foxn1(rnu) gene. However, NK cell activity of the nude rat is markedly increased. It is known that NK cells play a major role in rejection of xenografts and in cytotoxicity against tumor cells. Thus, the athymic nude rat with impaired NK cell activity should be a useful model for extensive studies. The DA-Lyst(bg)/Lyst(bg) rat, a model for human Chediak-Higashi syndrome (CHS) is characterized by diluted-coat color and impairment of NK cell activity. We planned to establish a combined immunodeficient double mutant rat introgressed with the Foxn1(rnu) and Lyst(bg) genes and a set of congenic strains having an identical genetic backgrounds simultaneously. Based on the phenotypic and genetic characteristics of the parental rat strains, the new strains were produced using continuous backcross and diagnosis with molecular genetic techniques. Each disease gene was diagnosed with PCR-RFLP or the long-nested PCR method. Furthermore, we used a marker-assisted congenic strategy based on scanning the genetic backgrounds of the parental rats with 461 rat microsatellite markers. We think that the newly established DA/Slc-Foxn1(rnu)/Foxn1(rnu) Lyst(bg)/Lyst(bg) double mutant will be useful as a severe disease model for human CHS, and the set of DA/Slc-Foxn1(rnu) Lyst(bg) congenic strains which have impaired NK cell activity and/or defective T cell function should be useful for studying in cancer research, xenotransplantation, immune function and other wide-ranging studies.     

Glutathione S-transferase 4 is a putative DIF-binding protein that regulates the size of fruiting bodies in Dictyostelium discoideum

In the development of the cellular slime mold Dictyostelium discoideum, two chlorinated compounds, the differentiation-inducing factors DIF-1 and DIF-2, play important roles in the regulation of both cell differentiation and chemotactic cell movement. However, the receptors of DIFs and the components of DIF signaling systems have not previously been elucidated. To identify the receptors for DIF-1 and DIF-2, we here performed DIF-conjugated affinity gel chromatography and liquid chromatography–tandem mass spectrometry and identified the glutathione S-transferase GST4 as a major DIF-binding protein. Knockout and overexpression mutants of gst4 (gst4^– and gst4^OE, respectively) formed fruiting bodies, but the fruiting bodies of gst4^– cells were smaller than those of wild-type Ax2 cells, and those of gst4^OE cells were larger than those of Ax2 cells. Both chemotaxis regulation and in vitro stalk cell formation by DIFs in the gst4 mutants were similar to those of Ax2 cells. These results suggest that GST4 is a DIF-binding protein that regulates the sizes of cell aggregates and fruiting bodies in D. discoideum.     

Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas

Two hybridoma systems, mouse·human-human (m·h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m·h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semicontinuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.     

Purification and Properties of Poly(3-Hydroxybutyrate) Depolymerase from the Fungus Paecilomyces lilacinus D218

Poly(3-hydroxybutyrate) depolymerase was purified to homogeneity from the culture filtrate of Paecilomyces lilacinus D218 by column chromatography on CM-Toyopearl 650M and hydroxylapatite. The molecular weight of the enzyme was estimated to be 48,000 by SDS-PAGE. Maximal activity was observed near pH 7.0 and 45°C. The K _m and V _max values for PHB were 0.13 (mg/ml) and 3750 (U/mg protein), respectively. The enzyme hydrolyzed PHB and p-nitrophenyl fatty acids but not polycaprolactone and triglycerides. Received: 29 August 1996 / Accepted: 30 September 1996     

Development of 99mTc radiolabeled A85380 derivatives targeting cerebral nicotinic acetylcholine receptor: Novel radiopharmaceutical ligand 99mTc-A-YN-IDA-C4

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that have been implicated in higher brain functions. To elucidate the functional mechanisms underlying nAChRs and contribute significantly to development of drugs targeting neurological and neuropsychiatric diseases, non-invasive nuclear medical imaging can be used for evaluation. In addition, technetium-99m (^99mTc) is a versatile radionuclide used clinically as a tracer in single-photon emission computed tomography. Because A85380 is known as a potent α4β2-nAChR agonist, we prepared A85380 derivatives labeled with ^99mTc using a bifunctional chelate system. A computational scientific approach was used to design the probe efficiently. We used non-radioactive rhenium (Re) for a ^99mTc analog and found that one of the derivatives, Re-A-YN-IDA-C4, exhibited high binding affinity at α4β2-nAChR in both the docking simulation (?19.3 kcal/mol) and binding assay (Ki = 0.4 ± 0.04 nM). Further, ^99mTc-A-YN-IDA-C4 was synthesized using microwaves, and its properties were examined. Consequently, we found that ^99mTc-A-YN-IDA-C4, with a structure optimized by using computational chemistry techniques, maintained affinity and selectivity for nAChR in vitro and possessed efficient characteristics as a nuclear medicine molecular imaging probe, demonstrated usefulness of computational scientific approach for molecular improvement strategy.     

Facile preparation of the N‐acetyl‐glucosaminylated asparagine derivative with TFA‐sensitive protecting groups useful for solid‐phase glycopeptide synthesis

In this study, a novel N‐acetyl‐glucosaminylated asparagine derivative was developed. This derivative carried TFA‐sensitive protecting groups and was derived from commercially available compounds only in three steps. It was applicable to the ordinary 9‐fluorenylmethoxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (SPPS) method, and the protecting groups on the carbohydrate moiety could be removed by a single step of TFA cocktail treatment generally used for the final deprotection step in Fmoc‐SPPS. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.