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Helicobacter pylori (HP) may transform from helical bacillary forms to coccoid forms after several days' in vitro incubation. The authors examined 111 consecutive gastrectomy specimens for the presence of coccoid forms of H pylori. Tissues from 64 stomachs (57.7%) showed colonization by H pylori, including 49 cases (76.6%) of adenocarcinoma, 14 cases (21.9%) of benign peptic ulcer, and 1 case (1.6%) of malignant lymphoma. Of these, coccoid forms of H pylori were identified in 53 cases (82.8%). In hematoxylin-and-eosin-stained sections coccoid forms of H pylori appeared as solid, round, basophilic dotlike structures. Under an electron microscope, coccoid forms of H pylori appeared as U-shaped bacilli, with the ends of the two arms joined by a membranous structure. Ultrastructural findings were identical to those from cultures of H pylori. With anti-Helicobacter antibody, coccoid forms of H pylori were positively stained by immunoperoxidase. Helical bacillary forms of H pylori invariably coexisted with the coccoid forms. By semiquantitative analysis, the number of coccoid forms in adenocarcinoma was significantly (P > .01) greater than that in benign peptic ulcers. This study confirms that H pylori can exist in coccoid forms in the human stomach. Coccoid forms should be distinguished from the pathogenic or nonpathogenic bacterial cocci, fungal spores, and cryptosporidia that may colonize the human stomach.

The three-dimensional structures of brain pyridoxal kinase and its complex with the nucleotide ATP have been elucidated in the dimeric form at 2.1 and 2.6 Å, respectively. Results have shown that pyridoxal kinase, as an enzyme obeying random sequential kinetics in catalysis, does not possess a lid shape structure common to all kinases in the ribokinase superfamily. This finding has been shown to be in line with the condition that pyridoxal kinase binds substrates with variable sizes of chemical groups at position 4 of vitamin B 6 and its derivatives. In addition, the enzyme contains a 12-residue peptide loop in the active site for the prevention of premature hydrolysis of ATP. Conserved amino acid residues Asp 118 and Tyr 127 in the peptide loop could be moved to a position covering the nucleotide after its binding so that its chance to hydrolyze in the aqueous environment of the active site was reduced. With respect to the evolutionary trend of kinase enzymes, the existence of this loop in pyridoxal kinase could be classified as an independent category in the ribokinase superfamily according to the structural feature found and mechanism followed in catalysis.

To understand the processes involved in the catalytic mechanism of pyridoxal kinase (PLK), 1 we determined the crystal structures of PLK⅐AMP-PCP-pyridoxamine, PLK⅐ADP⅐PLP, and PLK⅐ADP complexes. Comparisons of these structures have revealed that PLK exhibits different conformations during its catalytic process. After the binding of AMP-PCP (an analogue that replaced ATP) and pyridoxamine to PLK, this enzyme retains a conformation similar to that of the PLK⅐ATP complex. The distance between the reacting groups of the two substrates is 5.8 Å apart, indicating that the position of ATP is not favorable to spontaneous transfer of its phosphate group. However, the structure of PLK⅐ADP⅐PLP complex exhibited significant changes in both the conformation of the enzyme and the location of the ligands at the active site. Therefore, it appears that after binding of both substrates, the enzyme-substrate complex requires changes in the protein structure to enable the transfer of the phosphate group from ATP to vitamin B 6 . Furthermore, a conformation of the enzyme-substrate complex before the transition state of the enzymatic reaction was also hypothesized. Pyridoxal kinase (PLK)1 catalyzes the phosphorylation of vitamin B6 (including pyridoxal, pyridoxine, and pyridoxamine) in the presence of ATP and Zn 2ϩ , which is an essential step in the synthesis of pyridoxal 5Ј-phosphate (PLP), an active form of the vitamin in mammals (1-3). PLK is expressed in all mammalian tissues because of the fact that PLP cannot cross cell membranes, and PLK is required for the activation process inside cells (4). Genes encoding PLK have been cloned from both mammalian and plant cells. PLK activity has also been detected in bacteria, because PLP can be synthesized through the PLP salvage pathway (5, 6).Recently, the three-dimensional structures of PLK from sheep brain and its complex with ATP were determined (7). Although structural analyses have shown that PLK exhibits a folding pattern similar to the core structure of enzymes in the ribokinase superfamily (8 -13), low sequence homology between the two types of enzymes has been found. Despite kinetic studies that have shown that ribokinase and adenosine kinase both follow an ordered substrate-binding mechanism, PLK binds ATP and pyridoxal randomly (8,10,14). During the binding of ATP, a flexible loop containing 12 amino acid residues in the active site of PLK was responsible for triggering a major conformational change of the protein structure by interacting with the bound ATP. It has been suggested that the purpose for the inability of ATP to interact with this loop before catalysis is to prevent the nucleotide from hydrolysis, which is an essential feature in the random substrate binding mechanism.Further interest in crystallographic studies of PLK in the presence of substrates has arisen from several considerations. First, the structure of PLK complexes in the presence of pyridoxal has never been revealed. Thus, the exact interactions between molecules in the active site of PLK are unknown. S...

Pyridoxal kinase has been purified 9000-fold from sheep brain. The purification procedure involves ammonium sulphate fractionation, DEAE-cellulose chromatography, affinity chromatography and Sephadex G-1 00 gel filtration. The final chromatography step yields a homogeneous preparation of high specific activity with a PI of 5. The molecular mass of the native enzyme was estimated to be approximately 80 kDa by 10-25% gradient polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration. The subunit molecular mass was determined by sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis to be 40 kDa compared with a series of molecular mass standards. This indicates that pyridoxal kinase is a dimeric enzyme. Further results obtained from electron microscopy, using a negative staining technique, provide evidence that pyridoxal kinase exists as a dispherical subunit structure.Pyridoxal 5-phosphate (pyridoxal-5-P) is the cofactor required by mumerous enzymes catalysing transamination and carboxylation reactions. The formation of pyridoxal-5-P from ATP, pyridoxal and a divalent cation (Zn") is catalysed by pyridoxal kinase, an enzyme which has been detected in various rat and bovine tissues [l]. Procedures have been developed for the purification of pyridoxal kinase from porcine and rat brains [2,3]. A previous investigation has shown that the mechanism by which brain slices concentrate extracellular vitamin B-6 depend on pyridoxal kinase [4]. Other studies on vitamin B-6 metabolism have shown that the administration of pyridoxal kinase inhibitors to laboratory animals can induce neurological disorders, which may include convulsions In t h s paper we present a procedure for the purification of pyridoxal kinase from sheep brain. Investigation of the physical structure of the enzyme showed that pyridoxal kinase exists as a dimer. Under the examination by electron microscopy a 'cleft', which trapped negative-staining material, was observed running across the protein mass of one subunit of the enzyme, indicating the presence of two physical domains on a subunit.PI.

Oriental ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius) are two widely used valuable traditional Chinese medicines (TCM). Previously, the identification of ginseng was mainly performed by analyzing the ginsengnosides using high performance liquid chromatography and amplification of polymorphic DNA using polymerase chain reaction. However, these methods cannot be used to distinguish TCM samples which are from different parts (main root, lateral roots, rhizome head and skin) of ginseng and ginseng culture cells from wild-grown ginseng. The present study aimed to identify different species of ginseng, different parts of the same ginseng and cultured cells of ginseng using a proteomic approach. Two-dimensional electrophoresis (2-DE) maps were established from the American ginseng main root, different parts (main root, lateral roots, rhizome head and skins) of Oriental ginseng and Oriental ginseng culture cells. Our results show that the 2-DE maps of different ginseng samples contain sufficient differences to permit easy discrimination. We have also identified common and specific protein spots in the 2-DE maps of different ginseng samples. The use of these "marker proteins" may help to speed up the identification process.

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