A novel beta-lactamase activity from a penicillin-binding protein of Treponema pallidum and why syphilis is still treatable with penicillin

Treponema pallidum, the causative agent of syphilis, is sensitive to penicillins. Yet, an abundant membrane-bound protein of this organism, Tp47, turns over penicillins. It is shown herein that the turnover process is a hydrolytic reaction that results in the corresponding penicilloates, products that have their beta-lactam bonds hydrolyzed. This is the reaction of beta-lactamases, bona fide resistance enzymes to beta-lactam antibiotics. Remarkably, the x-ray structure of Tp47 bears no resemblance to any other beta-lactamases or the related penicillin-binding proteins. Furthermore, evidence is presented that the reaction of Tp47 takes place in the absence of the zinc ion and does not involve intermediary acyl enzyme species. Hence, the beta-lactamase activity of Tp47 is the fifth known mechanism for turnover of beta-lactam antibiotics. Tp47 also exhibits a penicillin binding reaction, in the process of which the enzyme is covalently modified in the active site. The two reactions take place in two different active sites, and the events of the beta-lactamase activity are over 2,000-fold more rapid than the penicillin binding reaction. The level of beta-lactamase activity is high and is held back only by a strong product-inhibition component to the catalytic process. If natural selection would result in a mutant variant of Tp47 that overcomes product inhibition for the beta-lactamase activity, a novel bona fide resistance to penicillins will emerge in Treponema, which will be a disconcerting clinical development. The physiological functions of Tp47 are not known, but it is likely that this is at least a bifunctional enzyme involved in the processing of the Treponema peptidoglycan as a substrate.     

Biofilm formation by a fimbriae-deficient mutant of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans strain 310-TR produces fimbriae and forms a tight biofilm in broth cultures, without turbid growth. The fimbriae-deficient mutant 310-DF, constructed in this study, was grown as a relatively fragile biofilm at the bottom of a culture vessel. Scanning electron microscopy revealed that on glass coverslips, 310-TR formed tight and spherical microcolonies, while 310-DF produced looser ones. These findings suggest that fimbriae are not essential for the surface-adherent growth but are required for enhancing cell-to-surface and cell-to-cell interactions to stabilize the biofilm. Treatment of the 310-DF biofilm with either sodium metaperiodate or DNase resulted in significant desorption of cells from glass surfaces, indicating that both carbohydrate residues and DNA molecules present on the cell surface are also involved in the biofilm formation.     

Establishment and characterization of a human ovarian granulosa tumor cell line (HSOGT)

We successfully established a novel ovarian granulosa tumor cell line (HSOGT). The tumor tissue of the ovary was derived from a 25 year-old Japanese woman under her consent. The cell line was maintained for over 14 months, subcultured more than 73 times, and had a population doubling time of 18.9 hours. Phase contrast microscopy displayed a pavement-like arrangement without contact inhibition. The chromosome number showed a wide distribution of aneuploidy and the mode was 83; many marker chromosomes were observed. The HSOGT was also successfully xenotransplanted into nude mice. The cell line produced estradiol and has preserved some characters of granulosa cells with stable growth in vitro. We firmly believe that this cell line will be a most useful tool for endocrinological investigation of human granulosa cells.     

New approach for the establishment of an hepatocyte cell line derived from rat early embryonic stem cells

A cell line with the characteristics of hepatocytes was established from rat early embryonic stem cells (REES). This cell line was established using a new novel method of Ishiwata et al. from two cell embryos taken from the spontaneous dwarf rat (SDR). The hepatocyte cell line (REES-hep) was instituted from dark red colored tissue in embryos during embryogenesis using REES cell line cultured in the presence of embryotrophic factors. These cell lines were cultured with DMEM/F12 medium supplemented 10% FBS and 1 ng/ml of LIF. They were found to maintain their diploid state, were characterized with 42 normal chromosomes and proliferated to confluence; contact inhibition was also present. These cells produced albumin when cultured using a collagen sponge gel system and reconstructed in a funicular form resembling the cell cords of liver. The cells also produced albumin and bilirubin when transplanted into the spleen of SDR Reconstruction of a REES-hep cell line from early embryonic stem cells should help in treating hepatic insufficient patients. It will be valuable for further research, as an introduction to cell transplantation and application for use in a bio-hybrid typed liver apparatus.     

Establishment and characterization of a pluripotent stem cell line derived from human amniotic membranes and initiation of germ layers in vitro

OBJECTIVES: Pluripotent stem cells are proposed to be used in regenerative therapy and may exist in the human amniotic membrane. The present article is aimed at establishing a pluripotent stem cell line from human placenta. METHODS: HAM-1 (stem cell line derived from human amniotic membranes) was established by the colonial cloning technique using aMEM culture medium containing 10 ng/ml of EGF, 10 ng/ml of hLIF and 10% fetal bovine serum. RESULTS: HAM-1 cells appeared to maintain a normal karyotype indefinitely in vitro and expressed markers characteristic of stem cells from mice and human, namely alkaline phosphatase. Also, these cells contributed to the formation of chimeric mouse embryoid bodies and gave rise to cells of all germ layers in vitro. CONCLUSIONS: This study demonstrates that human amniotic membranes derived stem cells have a wide developmental capability and might be utilized to regenerate different types of cells or tissues for transplantation therapy.     

Identification of a novel selenium metabolite, Se-methyl-N-acetylselenohexosamine, in rat urine by high-performance liquid chromatography--inductively coupled plasma mass spectrometry and--electrospray ionization tandem mass spectrometry

The major urinary metabolite of selenium (Se) in rats was identified by HPLC-inductively coupled argon plasma mass spectrometry (ICP-MS) and--electrospray tandem mass spectrometry (ESI-MS/MS). As the urine sample was rich in matrices such as sodium chloride and urea, it was partially purified to meet the requirements for ESI-MS. The group of signals corresponding to the Se isotope ratio was detected in both the positive and negative ion modes at m/z 300 ([M+H]+) and 358 ([M+CH3COO]-) for 80Se, respectively. These results suggested that the molecular mass of the Se metabolite was 299 Da for 80Se. The Se metabolite was deduced to contain one methylselenyl group, one acetyl group and at least two hydroxyl groups from the mass spectra of the fragment ions. The spectrum of the Se metabolite was completely identical to that of the synthetic selenosugar, 2-acetamide-1,2-dideoxy-beta-D-glucopyranosyl methylselenide. However, the chromatographic behavior of the Se metabolite was slightly different from that of the synthetic selenosugar. Thus, the major urinary Se metabolite was assigned as a diastereomer of a selenosugar, Se-methyl-N-acetyl-selenohexosamine.     

Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself.     

DIEL PATTERNS IN LIGHT ABSORPTION AND ABSORPTION EFFICIENCY FACTORS OF ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE)1

The chl a specific absorption coefficients [a* (λ), m²·mg chl a ^{? 1}] were examined in chemostat culture of the Prymnesiophyceae Isochrysis galbana (Parke) under a 12:12‐h light:dark cycle at low light (75 μmol photons·m ^{? 2}·s ^{? 1}) and high light (500 μmol photons· m ^{? 2}·s ^{? 1}) conditions. Other associated measurements such as pigment composition, cell density, and diameter as the measure of cell size were also made at the two light regimes every 2 h for 2 days to confirm the periodicity. A distinct diel variability was observed for the a* (λ) with maxima near dawn and minima near dusk. The magnitude of diel variation in a* (440) was 15% at low light and 22% at high light. Pronounced diel patterns were observed for cell size with minima near dawn and maxima near dusk. The magnitude of diel variation in cell size was 9.3% at low light and 21% at high light. The absorption efficiency factors [Q _a (440)] were determined by reconstruction using intracellular concentrations of pigments and cell size. The Q _a (440) also showed a distinct diel variability, with minima near dawn and maxima near dusk. The diel variation in a* (λ) and Q _a (λ) was primarily caused by changes in cell size due to growth, although there was some influence from diel variations in the intracellular pigment concentrations. The results presented here indicated that diel variation in a* (λ) was an important component of the optical characterization of phytoplankton.     

A human/mouse chimeric monoclonal antibody against CA125 for radioimmunoimaging of ovarian cancer

Murine monoclonal antibody 196-14 recognizes the ovarian-cancer-associated antigen CA 125, but the epitope it recognizes is different from that of monoclonal antibody OC125. We developed a human/mouse chimeric 196-14 using the variable regions of the murine 196-14 and human heavy-chain (l) and light-chain () constant regions. Cell binding and competitive inhibition assays using chimeric 196-14 labeled with¹²⁵I,¹¹¹In or^99mTc demonstrated that the in vitro immunoreactivity of the chimeric antibody was identical to that of the parental murine monoclonal antibody. However, in mice bearing human ovarian cancer xenografts, the clearance from blood was faster and absolute levels of accumulation in the tumor were lower for the¹²⁵I-labeled or^99mTc-labeled chimeric antibody than for the murine antibody labeled with the corresponding radionuclides. The tumor-to-blood radioactivity ratio was not significantly different between the chimeric antibody and the murine antibody, regardless of the radionuclide used for labeling. Chimeric antibody 196-14 labeled with¹³¹I,¹¹¹In or^99mTc is promising for the radioimmunoimaging of ovarian cancer.