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The lipopolysaccharide (LPS) of enterobacteria frequently contains various numbers of charged non-stoichiometric substituents such as phosphate (P) and ethanolamine (EtN) groups and a third residue of 3-deoxy-D-manno-2-octulosonic acid (KDO) on the R-core polysaccharide backbone. These substituents can modify the biological activities of LPS including varying the stability of the outer membrane, tolerance to cationic antibiotics, pathogenicity, and sensitivity to enterobacteria bacteriophages. These diverse substituents can be clearly detected in degraded samples of LPS from E. coli C using electrospray ionization single quadrupole mass spectrometry (ESI-Q-MS) from a 0.1 mg/mL solution in a 50:50 mixture of methanol and 10 mM ammonium acetate (pH 6.8). The O-deacylated derivative showed multiple peaks of [M-3H](3-) ions which corresponded to species having up to eight phosphates, two ethanolamines, and an additional KDO on the backbone of Hex(5) Hep(3) KDO(2) GlcN(2) C14:0(3-OH)(2). The major components of the O,N-deacylated derivative were the species associated with four and five phosphates on Hex(5) Hep(3) KDO(2) GlcN(2). The polysaccharide portion of LPS also revealed species which corresponded to Hex(5) Hep(3) KDO associated with two to four phosphates and an ethanolamine. The present method was proved to be useful to investigate the structural diversity of enterobacterial LPS.

The O,N-deacylated derivative (deON) and polysaccharide part (PS) from the lipopolysaccharide (LPS) of Escherichia coli C strain were separated by strongly basic anion-exchange chromatography (SAX) based on the differences in the number of charged phosphate and ethanolamine substituents. They were also successfully separated and characterized by capillary zone electrophoresis and subsequent ESI-ion trap-MS (CZE/ESI-IT-MS). The O-deacylated LPS (deO) presented as a broad peak in CZE/ESI-IT-MS. However, more than twelve species could be discriminated by an extracted ion electropherogram (EIE) and monitoring the species which have different numbers of phosphate and ethanolamine substituents on polysaccharide backbone.

The lipopolysaccharide (LPS) of enterobacteria frequently contains various numbers of charged non-stoichiometric substituents such as phosphate (P) and ethanolamine (EtN) groups and a third residue of 3-deoxy-D-manno-2-octulosonic acid (KDO) on the R-core polysaccharide backbone. These substituents can modify the biological activities of LPS including varying the stability of the outer membrane, tolerance to cationic antibiotics, pathogenicity, and sensitivity to enterobacteria bacteriophages. These diverse substituents can be clearly detected in degraded samples of LPS from E. coli C using electrospray ionization single quadrupole mass spectrometry (ESI-Q-MS) from a 0.1 mg/mL solution in a 50:50 mixture of methanol and 10 mM ammonium acetate (pH 6.8). The O-deacylated derivative showed multiple peaks of [M-3H](3-) ions which corresponded to species having up to eight phosphates, two ethanolamines, and an additional KDO on the backbone of Hex(5) Hep(3) KDO(2) GlcN(2) C14:0(3-OH)(2). The major components of the O,N-deacylated derivative were the species associated with four and five phosphates on Hex(5) Hep(3) KDO(2) GlcN(2). The polysaccharide portion of LPS also revealed species which corresponded to Hex(5) Hep(3) KDO associated with two to four phosphates and an ethanolamine. The present method was proved to be useful to investigate the structural diversity of enterobacterial LPS.

A new approach for the separation and inline characterization of lipopolysaccharide (LPS) related compounds has been developed. The separation was based on the difference in the number of charged phosphate and ethanolamine groups, as non-stoichiometric substituents, on the polysaccharide backbone, and was achieved with reverse phase ion-pairing chromatography (RPIP-HPLC). Tributylamine was used as an ion-pair reagent. In the conditions used in this study, tributylammonium then binds to the LPS related compounds through the negatively charged phosphate groups. This changes the hydrophobicity of the analytes at different positions and allows for separation based on both the number and position of the substituents on the analyte. The RPIP-HPLC was found to be effective for the separation of the O,N-deacylated derivative (deON) and polysaccharide portion (PS) from the LPS of Escherichia coli C strain. Post-column fluorescence derivatization (FLD), using sodium periodate and taurine, was used to detect the separated LPS related species. On the other hand, the separated species were also detected by direct infusion into the ESI-Q-MS using a volatile ammonium acetate buffer rather than the more traditional potassium phosphate buffer. The signal to noise ratio (S/N ratio) was low for the total ion chromatogram, however, high S/N ratios as well as good resolution were attained by selected ion monitoring (SIM) using m/z numbers corresponding to species with different numbers of non-stoichiometric substituents. Five species for deON and ten species for PS were clearly identified on the SIM chromatogram on the RPIP-HPLC/ESI-Q-MS. Accordingly, the present method allows for the effective separation and inline identification of the species corresponding to the diverse non-stoichiometric substitutions in LPS related compounds.

Development of acidophils was investigated electron microscopically in pituitaries from the rat embryos on the 15th, 17th, 18th and 20th day of gestation and from the neonatal and postnatal rats on the 1st, 3rd, 4th, 6th, 12th and 18th day of birth. Pituitary agranular primordial cells are the stem cells from which acidophils develop. During embryonic life, the male is more advanced in differentiation of primordial cells than the female. Initial granulation begins to take place in “ambiguous cells” (Yoshimura et al., 1970) on the 17th day of gestation as a row of minute granules about 50 mμ along the plasmamembrane. Incidence of ambiguous cells with various shapes is higher in the male than in the female. During embryonic period, only a small number of immature acidophils have developed from ambiguous cells despite spherical enlargement of cell-bodies where dense granules maintain their size within the proximity of 250 mμ. Most of ambiguous cells tend to transform into immature basophils, and even gonadotroph-like basophils have accumulated during the latest pregnancy. On the other hand many immature acidophils emerge directly from primordial cells without the mediation of ambiguous cells. In a certain primordial cells, a row of dense granules as large as 130-200 mμ in diameter suddenly appear along the plasmamembrane on the 18th day of gestation. In equilibrium with the prompt increase in the cell number, their granules rapidly increase in dimension and their maximal size reaches 250 mμ towards the end of fetal period. Postnatal development of acidophils is quite quick as to the population and granulation in both sexes. Numerous immature acidophils, not always spherical but somewhat elongated, develop appositionally from primordial cells. Appearance of a row of large granules of 200-250 mμ along the plasmamebrane of primordial cells seems to be related to morphological representation for the main developmental process of acidophils. Granules in immature acidophils increase in number, their size being persistently less than 200 mμ up to the 6th postnatal day. Enlarged spherical premature acidophils containing numerous granules of 200-300mμ in diameter begin to appear on the 12th day. So-called somatotrophs identical with the fully developed acidophils filled with large granules of about 350 mμ start to appear on the 18th day of age.

A marked decrease in parts per billion levels of mercury from distilled and natural waters during storage is discussed. Radioactive mercury ( 203 Hg) was added to distilled water, pond water, and seawater, and the loss of activity from the solutions was measured and compared to that of 65 Zn added to the same media. Behavior of inorganic and organic mercury is also examined using 203 HgCl 2 , and CH 3 203 HgCl. The specific behavior of Hg is mainly ascribed to three factors: vaporization, adsorption to the surface of containers and/or penetration through the walls, and susceptibility to biological activities in natural waters. Highly sensitive Hg analysis was therefore required for determining the low levels of Hg in natural waters. It was achieved by adopting the long quartz absorption cell to the cold-vapor flameless atomic absorption method. The sensitivity increases linearly with increasing cell length and the highest detection limit, 0.02 ng of Hg, was obtained when a 150-cm cell was used. Some precautions to eliminate a loss or change in the concentration of Hg in natural waters are stated.

Chromophobes isolated by the original method from the rat anterior pituitaries morphologically differentiated into acidophils when cultured in a chemically defined medium, NCTC-109, supplemented with various amounts of CRF (0.15 to 15μg/2ml) and colchicine. An electrophoresis and a paper chromatography revealed that ACTH was a main hormone produced by the cells maintained in the supplemented medium for as long as 12 days. The addition of larger amounts of CRF shortened a duration to reach to a certain maximal level of ACTH concentration in the cells, and this maximal level was equivalent in the cases of the variable amounts of CRF. A maximal release of the ACTH from the cells into the medium took place always 3 days after reaching to a maximal concentration of ACTH in the cells. The chromophobes produced α-MSH only in an earlier phase of differentiation without discharging it into the medium. The intracellular granules isolated from the cells kept in the control medium were equally 100 to 150 mp in diameter and regarded as the common storage carriers of six trophic hormones because they showed commonly all their low activities. Three different granules were successfully isolated from the cells differentiating in the CRF-medium from the chromophobes into the acidophils with negligible cross-contaminations. They were small (100 to 200mμ in diameter), large (300 to 450 mμ) and huge and irregularly shaped granules (450 to 1000 mμ) respectively. All the granules were confirmed by bio-and immuno-assays to contain ACTH activity alone regardless of their dimension.

The six kinds of cells, viz. chromophobes, ambiguous cells, acidophilic and basophilic intermediate cells, typical acidophils and basophils, were isolated from the rat fresh anterior pituitaries by the procedure of Ishikawa (1969) with some modifications. Bioassays of ACTH, prolactin, and a-MSH, and radioimmunoassay of GH were carried out for a mass of each isolated cell type. The chromophobes showed no hormonal activity. The ambiguous cells contained only α-MSH. Both ACTH and GH were localized predominantly in the acidophilic intermediate cells and typical acidophils, but prolactin was found only in the latter. Only a trace amount of ACTH, GH, and prolactin was found in the basophilic intermediate cells and typical basophils. It was elucidated that the cells on the acidophilic axis were involved in the producing cells of ACTH.