Determination of molecular species composition of C80 or longer-chain alpha-mycolic acids in Mycobacterium spp. by gas chromatography-mass spectrometry and mass chromatography

The molecular species composition of alpha-mycolic acids ranging from C68 to C86 in 13 rapidly growing and 12 slowly growing mycobacterial species was determined by gas chromatography, gas chromatography-mass spectrometry, and mass chromatography. In gas chromatographic analysis, the molecular species of alpha-mycolic acids were well separated as trimethylsilyl ether derivatives of the methyl esters, according to their total carbon numbers. The total carbon and double-bond numbers of mycolic acids at each peak on gas chromatograms were determined from the [M]+, [M - 15]+, and [M - 90]+ ions on the mass spectrum, and straight and branched chain structures were identified by the mass fragment ions [A]+, due to C2--C3 cleavage [R-CH-O-Si(CH3)3]+, and [B]+, due to C3--C4 cleavage [(CH3)3-Si-O-CH-CH(R')-COOCH3]+. The concentration of odd- and even-carbon-numbered mycolic acids, which often overlap each other on gas chromatograms, and the composition of three homologous mycolic acids with different alpha units (C22:0, C24:0, and C26:0) were clearly determined by mass chromatography monitoring [M - 15]+ ions and [B - 29]+ ions, respectively. The molecular species composition of alpha-mycolic acids and their average carbon numbers (av. cn.) as a simple expression of the composition were calculated from the mass chromatograms. Each mycobacterial species examined was demonstrated to possess a characteristic profile of alpha-mycolic acid composition, and based on this the species were classified approximately into eight groups: C68 to C76 (av. cn. 72), dienoic, possessing a C20 alkyl branch at the 2 position (C22 alpha-unit) for Mycobacterium diernhoferi and Mycobacterium sp. strain 3707, a chromogenic rapid grower; C72 to C78 (av. cn. 75), dienoic with both C22 and C24 alpha units, containing a small or a large amount of odd-carbon-numbered molecules, for M. vaccae, M. rhodesiae, and M. phlei (chromogenic rapid growers); C72 to C80 (av. cn. 75 to 77), dienoic with C24 alpha-unit, containing a moderate or a large amount of odd-carbon-numbered molecules, for M. smegmatis, M. chitae, M. chelonae (M. chelonei), and M. fortuitum (nonchromogenic rapid growers); C78 to C82 (av. cn. 80), even-carbon-numbered dienoic with C24 alpha unit for M. agri and M. thermoresistible (rapid growers); C75 to C81 (av. cn. 77 to 79), odd-carbon-numbered dienoic with C24 alpha unit for M. nonchromogenicum complex (M. nonchromogenicum, M. terrae, and "M. novum") (slow growers); (vi) C76 to C84 (av. cn. 79 to 81), even-carbon-numbered dienoic with C24 alpha unit for MAIS complex including M. scrofulaceum, M. avium, and M. intracellulare (slow growers); (vii) C72 to C80 (av. cn. 77 to 79), even-carbon-numbered dienoic with C24 alpha unit for M. szulgai, M. gordonae, and M. kansasii (chromogenic slow growers); and (viii) C76 to C86 (av. cn. 79 to 81), even-carbon-numbered dienoic with C26 alpha unit M. bovis Ravenol and BCG and M. tuberculosis H37Rv. This study demonstrated that gas chromatography-mass spectrometric analysis of the molecular species composition of alpha-mycolic acid can give rapid, important, and very precise information for the identification of pathogenic and nonpathogenic mycobacterial species.     

Glutaminase-like immunoreactivity in the lower brainstem and cerebellum of the adult rat

Distribution of putative glutamatergic neurons in the lower brainstem and cerebellum of the rat was examined immunocytochemically by using a monoclonal antibody against phosphate-activated glutaminase, which has been proposed to be a major synthetic enzyme of transmitter glutamate and so may serve as a marker for glutamatergic neurons in the central nervous system. Intensely-immunolabeled neuronal cell bodies were densely distributed in the main precerebellar nuclei sending mossy fibers to the cerebellum; in the pontine nuclei, pontine tegmental reticular nucleus of Bechterew, external cuneate nucleus, and lateral reticular nucleus of the medulla oblongata. Phosphate-activated glutaminase-immunoreactive granular deposits were densely seen in the brachium pontis and restiform body, suggesting the immunolabeling of mossy fibers of passage. In the cerebellum, neuropil within the granule cell layer of the cerebellar cortex displayed intense phosphate-activated glutaminase-immunoreactivity, and that within the deep cerebellar nuclei showed moderate immunoreactivity. These results indicate that many mossy fiber terminals originate from phosphate-activated glutaminase-containing neurons and utilize phosphate-activated glutaminase for the synthesis of transmitter glutamate. Intensely-immunostained neuronal cell bodies were further observed in other regions which have been reported to contain neurons sending mossy fibers to the cerebellum; in the dorsal part of the principal sensory trigeminal nucleus, dorsomedial part of the oral subnucleus of the spinal trigeminal nucleus, interpolar subnucleus of the spinal trigeminal nucleus, paratrigeminal nucleus, supragenual nucleus, regions dorsal to the abducens nucleus and genu of the facial nerve, superior and medial vestibular nuclei, cell groups f, x and y, hypoglossal prepositus nucleus, intercalated nucleus, nucleus of Roller, reticular regions intercalated between the motor trigeminal and principal sensory trigeminal nuclei, linear nucleus, and gigantocellular and paramedian reticular formation. Neuronal cell bodies with intense phosphate-activated glutaminase-immunoreactivity were also found in other brainstem regions, such as the paracochlear glial substance, posterior ventral cochlear nucleus, and cell group e. Although it is still controversial whether all glutamatergic neurons use phosphate-activated glutaminase in a transmitter-related process and whether phosphate-activated glutaminase is involved in other metabolism-related processes, the neurons showing intense phosphate-activated glutaminase-immunoreactivity in the present study were suggested to be putative glutamatergic neurons.     

The effects of prostaglandin E1 on the pepsin activities in gastric mucosa and juice.

Prostaglandin E1 elevated pepsin activity in gastric mucosa but lowered pepsin activity in the gastric juice of rats treated by pylorus ligation and intragastric administration of hydrochloric acid. In these animals zymogen granules with low electron density were numerous in the gastric chief cells following prostaglandin E1 treatment. The prostaglandin E1-induced increase in mucosal pepsin activity was slightly inhibited by actinomycin D and there was no apparent increase in 3H-thymidine incorporation into gastric mucosa following treatment with prostaglandin E1. It is suggested that prostaglandin E1 causes an elevation of pepsin activity in the gastric mucosa by stimulating pepsin synthesis and perhaps also by facilitating pepsin release from zymogen granules. However, it also appears to inhibit pepsin release from the mucosa into the gastric cavity judging by the decrease of pepsin activity in gastric juice. The reduced pepsin activity in gastric juice may account, in part, for the reported anti-ulcerative action of prostaglandin.     

M pathway and areas 44 and 45 are involved in stereoscopic recognition based on binocular disparity

We characterized the visual pathways involved in the stereoscopic recognition of the random dot stereogram based on the binocular disparity employing a functional magnetic resonance imaging (fMRI). The V2, V3, V4, V5, intraparietal sulcus (IPS) and the superior temporal sulcus (STS) were significantly activated during the binocular stereopsis, but the inferotemporal gyrus (ITG) was not activated. Thus a human M pathway may be part of a network involved in the stereoscopic processing based on the binocular disparity. It is intriguing that areas 44 (Broca's area) and 45 in the left hemisphere were also active during the binocular stereopsis. However, it was reported that these regions were inactive during the monocular stereopsis. To separate the specific responses directly caused by the stereoscopic recognition process from the nonspecific ones caused by the memory load or the intention, we designed a novel frequency labeled tasks (FLT) sequence. The functional MRI using the FLT indicated that the activation of areas 44 and 45 is correlated with the stereoscopic recognition based on the binocular disparity but not with the intention artifacts, suggesting that areas 44 and 45 play an essential role in the binocular disparity.     

Determination of the depth of 50% of maximum ionization, I50, for electron beams by the divided difference method

A new characterization of depth-ionization parameters for electron beams is empirically deduced from our data analysis based on the divided difference method (the DD method), which employs the numerical differential of an ionization curve. The important feature of the present method is that it does not necessarily require normalized percent depth-ionization (NPDI) data. The depth of 50% of maximum ionization, I50, which is an important parameter for electron beam dosimetry, can be deduced from the analysis of an unnormalized (or partial) depth-ionization (UDI) curve obtained over a short interval of depth. The values of I50 determined by the DD method are in agreement to within 0.1 mm for energies of 4, 6, and 9 MeV, compared with the ones determined by the TG-51 protocol method (or the conventional method), and the difference was 0.9 mm for 12 and 15 MeV. The dose at the reference depth, dref, calculated from I50 by the DD method, is found to be in agreement with TG-51 to within 0.1%. The field size dependence of the DD method using UDI data was studied for three field sizes: 6 x 6, 10 x 10, and 20 x 20 cm2. For all energies, the discrepancies of I50 as determined by both methods were 0.9 mm on average for the 6 x 6 cm2 fields and 0.6 mm for the other two field sizes. This dependence was remarkable for 6 x 6 cm2 fields for 12 and 15 MeV, and the discrepancies shown by the DD method were 1.2 mm for 12 MeV and 1.8 mm for 15 MeV, respectively. Since the reference field size in clinical dosimetry is usually 10 x 10 cm2, this dependence will not affect clinical dosimetry. The DD method could be an alternative option for checking beam quality in dose calibration.     

A new pseudo-peptide of Arg-Gly-Asp (RGD) with inhibitory effect on tumor metastasis and enzymatic degradation of extracellular matrix

A series of pseudo-peptide analogs of the Arg-Gly-Asp (RGD) sequence of fibronectin have been synthe-sized, and their anti-metastatic effects in mice and inhibitory effects on tumor cell invasion in vitro have been examined. The partially modified retro pseudo-peptide of RGD, R_rev-COCH₂CO-D (FC-63), was more effective in inhibiting tumor metastasis than the original RGDS peptide. Replacement of the malonyl moiety of FC-63 with a carboxyethylene linkage (R_rev-COCH₂CH₂-D, FC-303 ) achieved more potent inhibition of lung metastasis of melanoma cells than FC-63. Among the analogs, FC-336, a p-xylylendiamine derivative having two FC-303 moieties, showed the most potent inhibitory effect on experimental lung metastasis produced by i.v. co-injection with B16-BL6 melanoma or colon 26 M3.1 cells in a dose-dependent manner. Multiple administrations of FC-336 after tumor inoculation also showed efficient therapeutic potency against spontaneous lung metastasis of B16-BL6 melanoma in mice. Furthermore, FC-336 effectively inhibited the invasion, migration and adhesion of tumor cells in vitro, but its inhibitory effects were not more than those of RGDS peptide. Zymography analysis revealed that FC-336 inhibited the degradation of gelatin substrate by matrix metalloproteinases (MMPs) produced by tumor cells, while the RGDS peptide did not affect the enzymatic degradation. These findings indicate that the pseudo-peptides of the RGD sequence, possessing the inhibitory property of the degradation by MMPs differently from original RGD-containing peptides, may be advantageous and useful in preventing tumor metastasis. © Rapid Science 1998     

Characterization of DNA sequences constituting the terminal heterochromatin of the chicken Z chromosome

Two clones, pCZTH5-8 and pCZTH12-8, were isolated from a female chicken genomic library by screening with sequences obtained from genomic libraries which had been constructed from a terminal region of a single Z chromosome of chicken utilizing laser microbeam irradiation and PCR amplification. Fluorescencein situ hybridization to the mitotic Z chromosome and the lampbrush ZW bivalent of chicken demonstrated that both the cloned sequences are located in the heterochromatic region of the Z chromosome at the end opposite to the pairing region with the W chromosome. The sequences pCZTH5-8 and pCZTH12-8 are distributed widely on both the telomeric bow-like loops (TBL) and the region I (short loops region) of the Z lampbrush chromosome. These clones, pCZTH12-8 particularly notably, hybridized also to the TBLs of lampbrush bivalents 1–4 of chicken. Both sequence are transcribed in the lampbrush stage oocytes on the Z chromosome and on other macrobivalents. The subfragment of pCZTH5-8 which hybridizes to the TBLs and the insert of pCZTH12-8 contain regions that are closely similar in sequence. The pCZTH5-8 sequence has no internal repeats and may be part of the 24-kb macrosatellite repeating unit that is evident afterNhel digestion of the genomic DNA. A cloned 24-kb unit, pFN-1, does not show significant DNA curvature, but cytosines of its CpG dinucleotides may be highly methylatedin vivo. This contrasts with the repeat sequences of the W heterochromatin which not only have highly methylated CpG but are also strongly curved. The 24-kb unit is repeated about 830 times in the diploid genome of a female chicken, suggesting that nearly the entire terminal heterochromatin on the Z chromosome consists of this macrosatellite family. Sequences of the greater part of the pCZTH5-8 are restricted to the genusGallus but the sequence of one subregion which hybridizes to TBLs is present in the genomes of the order Galliformes.accepted for publication by M. Schmid     

Isolation and Hypoglycemic Activity of Ephedrans A, B, C, D and E, Glycans of Ephedra distachya Herbs1.

Administration of an aqueous methanol/water extract of the Oriental crude drug "maō" (Ephedra), EPHEDRA DISTACHYA herbs, caused transient hyperglycemia followed by long lasting hypoglycemia in mice. Activity-guided fractionation of the extract led to isolation of five glycans, ephedrans A, B, C, D and E, which exhibited significant hypoglycemic effects in normal and alloxan-induced hyperglycemic mice.     

Isolation and Hypoglycemic Activity of Lithospermans A, B and C, Glycans of Lithospermum erythrorhizon Roots1.

A water extract of the Oriental crude drug "shikon", LITHOSPERMUM ERYTHRORHIZON roots, remarkably diminished the plasma sugar level in mice. Fractionation of the extract by monitoring the activity yielded three glycans, lithospermans A, B and C. These glycans exerted marked hypoglycemic effects in normal and alloxan-induced hyperglycemic mice.