Biomedical subjects

T Xia

Publications and source records attributed to T Xia.

69 records · Page 4Linked to original sources

PubMed1994

[MR lymphography: first application to human].

First clinical trial of MR lymphography is described. The subjects were two normal volunteers. The contrast agent was cideferron, which was an iron colloid. We obtained MR images of the inguinal lymph nodes before and after subcutaneous injection of the agent in the dorsal feet. As a result, the signal intensities on T2 weighted images remarkably decreased at two hours after injection and kept low until three months after. Although pigmentation around the injection site was observed, it was gradually fading within six months. In conclusion, MR lymphography with cideferron can be applied clinically.

Colloids↗

PubMed1994

Pseudomonas aeruginosa possesses homologues of mammalian phenylalanine hydroxylase and 4 alpha-carbinolamine dehydratase/DCoH as part of a three-component gene cluster.

Pseudomonas aeruginosa possesses a multigene operon that includes phenylalanine hydroxylase (PhhA; phenylalanine 4-monooxygenase, EC 1.14.16.1). phhA encodes PhhA (M(r) = 30,288), phhB (M(r) = 13,333) encodes a homologue of mammalian 4 alpha-carbinolamine dehydratase/homeodomain protein transregulator, and phhC encodes an aromatic aminotransferase (M(r) = 43,237). The reading frames specifying phhB and phhC overlap by 2 bases. The P. aeruginosa PhhA appears to contain iron and is pterin dependent. Unlike the multimeric mammalian hydroxylase, the native P. aeruginosa enzyme is a monomer. The P. aeruginosa PhhA is homologous with mammalian PhhA, tryptophan hydroxylase, and tyrosine hydroxylase. Expression of PhhA from its native promoter required phhB. This may suggest a positive regulatory role for phhB, consistent with the dual catalytic and regulatory roles of the corresponding mammalian homologue.

Amino Acid Sequence↗

PubMed1994

Inhalation MR lymphography: a new method for selective enhancement of the lung hilar and mediastinal lymph nodes.

On MR lymphography, a new approach to the lung hilar and mediastinal nodes was developed in an animal model. Five rabbits were made to inhale iron colloid (cideferron) that was nebulized to aerosol. Two days after inhalation of the agent, the mediastinal lymph nodes decreased in signal on SE 2000/30 and SE 2000/60 images and proved to have iron on histological evaluation, whereas the popliteal nodes did not have any iron. Experimental results indicate that inhalational administration can deliver the agent to the pulmonary lymphatic system and has the potential of lung hilar and mediastinal MR lymphography.

Administration, Inhalation↗

PubMed1994

[Influence of combined Salvia miltiorrhiza and Ligusticum wallichii on pharmacokinetics of tetramethylpyrazine in rats].

Influence of Salvia miltiorrhiza (SM) and/or Ligusticum wallichii (LW) on pharmacokinetics of tetramethylpyrazine (TMP) was observed in rats. The content of TMP in LW and LW-SM decoctions were 375.8 and 236.2 micrograms/ml respectively. The rat serum components after oral administration of LW were analyzed by modified HPLC. One of 3 compounds was detected, which was identified as TMP by the comparison of the parameters of 4 spectrums (UV, IR, MS and NMR) with those of TMP in literature. Pharmacokinetics of TMP in rats after the oral administration of LW and LW-SM decoction respectively showed that: (1) both of the data fitted adequately to two-compartment open model; (2) Ka, AUC and serum concentration were higher (P < 0.05-0.01) for LW decoction than that for LW-SM decoction, indicating a higher bioavailability. It demonstrated that the absorption of TMP of LW-SM decoction was slower and the bioavailability of TMP of LW-SM decoction reduced. Contents of TMP in LW and LW-SM decoction and serum concentration of TMP were determined by HPLC method.

Animals↗

PubMed1993

Preliminary crystallographic study of cyclohexadienyl dehydratase from Pseudomonas aeruginosa.

Single crystals of cyclohexadienyl dehydratase from Pseudomonas aeruginosa have been obtained by vapour diffusion from ammonium sulphate solution (pH 6.0) at 4 degrees C. The crystals belong to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2 with a = b = 105.5 A and c = 165.0 A. The asymmetric unit contains at least one dimeric protein molecule with M(r) = 72 kDa. The crystals diffract to 3 A resolution and are suitable for an X-ray analysis.

Prephenate Dehydratase↗

PubMed1993

An allosterically insensitive class of cyclohexadienyl dehydrogenase from Zymomonas mobilis.

The key enzyme of tyrosine biosynthesis in many Gram-negative prokaryotes is cyclohexadienyl dehydrogenase. The Zymomonas mobilis gene (tyrC) coding for this enzyme was cloned in Escherichia coli by complementation of a tyrosine auxotroph. The tyrC gene was 882 bp long, encoding a protein with a calculated molecular mass of 32086 Da. The Z. mobilis cyclohexadienyl dehydrogenase expressed in E. coli was purified to electrophoretic homogeneity. The subunit molecular mass of the purified enzyme was 32 kDa as determined by SDS/PAGE. The ratio of the activity of arogenate dehydrogenase to that of prephenate dehydrogenase (approximately 3:1) remained constant throughout purification, and the two activities were therefore inseparable. The genetic and biochemical data obtained demonstrated a single enzyme protein capable of catalyzing either of two reactions. Km values of 0.25 mM and 0.18 mM were obtained from prephenate and L-arogenate, respectively. The Km value obtained for NAD+ (0.09 mM) was the same regardless of whether the enzyme was assayed as arogenate dehydrogenase or as prephenate dehydrogenase. Unlike the corresponding enzyme of Pseudomonas aeruginosa or E. coli, the cyclohexadienyl dehydrogenase of Z. mobilis lacks sensitivity to feedback inhibition by L-tyrosine. A typical NAD(+)-binding domain was found to be located at the N-terminus of the protein. Although the deduced amino-acid sequence of the Z. mobilis cyclohexadienyl dehydrogenase showed relatively low identity (19-32%) with the prephenate dehydrogenases of Bacillus subtilis and Saccharomyces cerevisiae, as well as with the cyclohexadienyl dehydrogenase components of the bifunctional T-proteins of E. coli and Erwinia herbicola, a presumptive motif was identified which may correspond to critical residues of the binding site for cyclohexadienyl substrate molecules. Immediately upstream of tryC a portion of a gene was sequenced and found to exhibit clearcut homology of the deduced amino-acid sequence with the B. subtilis hisH gene product. Thus, the Zymomonas gene organization is reminiscent of the linkage of genes encoding a tryosine-pathway dehydrogenase and a histidine-pathway aminotransferase in B. subtilis.

Amino Acid Sequence↗

PubMed1993

The pheA/tyrA/aroF region from Erwinia herbicola: an emerging comparative basis for analysis of gene organization and regulation in enteric bacteria.

Extensive knowledge exists in Escherichia coli about the contiguous pheA and aroF-tyrA operons which have opposite transcription orientations and are separated by a bidirectional transcription terminator. The corresponding structural genes and individual components of the terminator and attenuator from Erwinia herbicola have been analyzed from an evolutionary vantage point. A 7.5-kb DNA fragment from E. herbicola carrying the linked pheA, tyrA, and aroF genes was cloned by functional complementation of E. coli auxotrophic requirements. A 3,433-bp segment of DNA consisting of more than half of aroF, all of tyrA, and the entire phenylalanine operon (promoter, leader region encoding the leader peptide and containing the phe attenuator, and pheA) was sequenced. A bidirectional transcription terminator was positioned between the divergently transcribed pheA and tyrA. The adjacent aroF and tyrA genes share a common transcription orientation, consistent with their probable coexistence within an operon. However, tyrA can be expressed efficiently from an internal promoter which appears to lie within the 3' portion of aroF. The gene order is pheA tyrA aroF in E. herbicola, with the same tail-to-tail arrangement of transcription known to exist in E. coli. The pheL coding region of the phe operon was dominated by phenylalanine codons, seven of the 15 amino acid residues of the leader peptide being L-phenylalanine. The E. herbicola pheA and tyrA genes were 1,161 bp and 1,119 bp in length, respectively, and corresponded to deduced gene products having subunit molecular weights of 43,182 and 41,847. The deduced amino acid sequences of PheA and TyrA were homologous at their N-termini, consistent with a common evolutionary origin of the chorismate mutase domains present at the amino terminus of both PheA and TyrA. A detailed comparison of the E. coli and E. herbicola sequences was made. The pheA, tyrA, and aroF genes of E. herbicola exhibited high overall identity with the counterpart E. coli genes. Within the leader region of the phe operon, the leader peptide coding region was highly conserved. Although the 1:2 and 2':3' stems defining the pause structure and the antiterminator, respectively, were also highly conserved, RNA segment 4 of the attenuator terminator exhibited considerable divergence, as did the distal portion of the attenuator region. Within the span of attenuator region encoding the three stem-loop structures of mRNA secondary configuration, hot spots of base-residue divergence were localized to looped-out regions. No changes occurred which would simultaneously disrupt alternative pairing relationships of secondary configuration. The bidirectional terminator between pheA and tyrA has diverged very substantially.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed1993

Cyclohexadienyl dehydratase from Pseudomonas aeruginosa is a periplasmic protein.

The gene encoding cyclohexadienyl dehydratase from Pseudomonas aeruginosa, designated pheC, was cloned in Escherichia coli and sequenced recently by Zhao et al. (Journal of Biological Chemistry 267, 2487-2493, 1992). N-Terminal sequencing of the purified cyclohexadienyl dehydratase yielded a run of 11 residues which matched perfectly with the deduced amino acid residues 26-36. This showed that a 25 residue peptide was cleaved from the N-terminus of a preprotein formed in E. coli. The amino acid composition of the 25 residue peptide was typical of signal sequences for periplasmic proteins. Most or all of the cyclohexadienyl dehydratase was released from P. aeruginosa and E. coli carrying the pheC gene following spheroplast formation, osmotic shock or chloroform treatment. The location of the enzyme in the periplasm of both E. coli and P. aeruginosa was confirmed by Western blotting analysis using antibody prepared against PheC. Electron microscopy using immunogold labelling showed an apparent localization of cyclohexadienyl dehydratase at the polar regions of the periplasmic space in E. coli.

Amino Acid Sequence↗

PubMed1993

The aroQ-encoded monofunctional chorismate mutase (CM-F) protein is a periplasmic enzyme in Erwinia herbicola.

Enteric bacteria possess two species of chorismate mutase which exist as catalytic domains on the amino termini of the bifunctional PheA and TyrA proteins. In addition, some of these organisms possess a third chorismate mutase, CM-F, which exists as a small monofunctional protein. The CM-F gene (denoted aroQ) from Erwinia herbicola was cloned and sequenced for the first time. A strategy for selection by functional complementation in a chorismate mutase-free Escherichia coli background was devised by using a recombinant plasmid derivative of pUC18 carrying a Zymomonas mobilis tyrC insert which encodes cyclohexadienyl dehydrogenase. The aroQ gene is 543 bp in length, predicting a 181-residue protein product having a calculated molecular mass of 20,299 Da. The E. herbicola aroQ promoter is recognized by E. coli, and a putative sigma-70 promoter region was identified. N-terminal amino acid sequencing of the purified CM-F protein indicated cleavage of a 20-residue signal peptide. This was consistent with the monomeric molecular mass determined for the enzyme of about 18,000 Da. The native enzyme is a homodimer. The implied translocation of CM-F was confirmed by osmotic shock experiments which demonstrated a periplasmic location. Immunogold electron microscopy indicated a polar localization within the periplasm. Polyclonal antibody raised against E. herbicola CM-F did not cross-react with the CM-F protein from the closely related Serratia rubidaea, as well as from a number of other gram-negative bacteria. Furthermore, when the E. herbicola aroQ gene was used as a probe in Southern blot hybridizations with EcroRI digests of chromosomal DNA from S. rubidaea and other enteric organisms, no hybridization was detected at low stringency. Thus, the aroQ gene appears to be unusually divergent among closely related organisms. The deduced CM-F amino acid sequence did not exhibit compelling evidence for homology with the monofunctional chorismate mutase protein of Bacillus subtilis.

Amino Acid Sequence↗

PubMed1993

Selective retention of essential fatty acids: the role of hepatic monoacylglycerol acyltransferase.

In the suckling rat, chick embryo, and hibernating marmot, fatty acids provide the major source of energy, and despite the high rate of hepatic beta-oxidation, these animals selectively retain long-chain polyunsaturated derivatives of C18:2n-6 and C18:3n-3. To determine whether the hepatic microsomal activity monoacylglycerol acyltransferase (MGAT) (EC 2.3.1.22) could provide a mechanism to selectively acylate monoacylglycerols that contain essential fatty acids, we tested the ability of MGAT activity from each of the three species to acylate sn-2-monoC18:1-, sn-2-monoC18:2-, sn-2-monoC18:3-, and sn-2-monoC20:4-glycerols. Hepatic MGAT activity acylated sn-2-monoC18:3-glycerol and sn-2-monoC18:2-glycerol in preference to sn-2-monoC18:1-glycerol in each of the three different lipolytic animals. MGAT's acyl group specificity could not be explained by invoking differences in membrane fluidity because the apparent affinity for sn-2-monoC20:4-glycerol was not increased. Further, sn-2-monoC18:3-glycerol remained a preferred substrate under assay conditions when both the C18:3 and C18:1 species were present in equal amounts. As would be predicted in the presence of high activity of a selective MGAT, the hepatic glycerolipids from neonatal rats showed increases in dienoic, trienoic, and C22:6 fatty acids and relative decreases in monoenoic, saturated, and C20:4 fatty acids. We hypothesize that, during lipolysis, the reacylation of sn-2-monoacylglycerols by MGAT may provide a mechanism by which essential fatty acids are retained within specific tissues.

Acylation↗

PubMed1992

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.

Aging↗

PubMed1992

Monofunctional chorismate mutase from Serratia rubidaea: a paradigm system for the three-isozyme gene family of enteric bacteria.

Serratia rubidaea (ATCC 27614) typifies a substantial number of enteric bacteria which, unlike Escherichia coli, possess a monofunctional species of chorismate mutase (denoted CM-F). CM-F coexists with two additional species of chorismate mutase, each of the latter being one catalytic domain of a bifunctional protein. The two bifunctional proteins are utilized for phenylalanine (CM-P/prephenate dehydratase) and tyrosine (CM-T/cyclohexadienyl dehydrogenase) biosynthesis in all enteric bacteria. S. rubidaea was selected as the organism of choice for purification of CM-F because of the relatively abundant level of expression found for this enzyme. The monofunctional CM-F enzyme was purified about 1600-fold with a yield of about 16%. This is the first monofunctional chorismate mutase to be purified from any gram-negative prokaryote. The CM-F enzyme is a positively charged homodimer made up of 20-kDa subunits. It has a pH optimum of 5.5, exhibits a Km value of 0.33 mM for chorismate, and is sensitive to product inhibition by prephenate that is competitive with respect to chorismate. It is insensitive to feedback inhibition by any of the aromatic amino acids. Partial purification of the bifunctional P-protein and the bifunctional T-protein was also carried out in order to compare the properties of CM-F, CM-P, and CM-T in a common organism. The most striking differential properties of the three isozymes were those of pH optimum and degree of protection conferred by dithiothreitol.

Binding, Competitive↗

PubMed1992

A monofunctional prephenate dehydrogenase created by cleavage of the 5' 109 bp of the tyrA gene from Erwinia herbicola.

A cohesive phylogenetic cluster that is limited to enteric bacteria and a few closely related genera possesses a bifunctional protein that is known as the T-protein and is encoded by tyrA. The T-protein carries catalytic domains for chorismate mutase and for cyclohexadienyl dehydrogenase. Cyclohexadienyl dehydrogenase can utilize prephenate or L-arogenate as alternative substrates. A portion of the tyr A gene cloned from Erwinia herbicola was deleted in vitro with exonuclease III and fused in-frame with a 5' portion of lacZ to yield a new gene, denoted tyrA*, in which 37 N-terminal amino acids of the T-protein are replaced by 18 amino acids encoded by the polycloning site/5' portion of the lacZ alpha-peptide of pUC19. The TyrA* protein retained dehydrogenase activity but lacked mutase activity, thus demonstrating the separability of the two catalytic domains. While the Km of the TyrA* dehydrogenase for NAD+ remained unaltered, the Km for prephenate was fourfold greater and the Vmax was almost twofold greater than observed for the parental T-protein dehydrogenase. Activity with L-arogenate, normally a relatively poor substrate, was reduced to a negligible level. The prephenate dehydrogenase activity encoded by tyrA* was hypersensitive to feedback inhibition by L-tyrosine (a competitive inhibitor with respect to prephenate), partly because the affinity for prephenate was reduced and partly because the Ki value for L-tyrosine was decreased from 66 microM to 14 microM. Thus, excision of a portion of the chorismate mutase domain is shown to result in multiple extra-domain effects upon the cyclohexadienyl dehydrogenase domain of the bifunctional protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Loss of allosteric control but retention of the bifunctional catalytic competence of a fusion protein formed by excision of 260 base pairs from the 3' terminus of pheA from Erwinia herbicola.

A bifunctional protein denoted as the P protein and encoded by pheA is widely present in purple gram-negative bacteria. This P protein carries catalytic domains that specify chorismate mutase (CM-P) and prephenate dehydratase. The instability of a recombinant plasmid carrying a pheA insert cloned from Erwinia herbicola resulted in a loss of 260 bp plus the TAA stop codon from the 3' terminus of pheA. The plasmid carrying the truncated pheA gene (denoted pheA*) was able to complement an Escherichia coli pheA auxotroph. pheA* was shown to be a chimera composed of the residual 5' part of pheA (901 bp) and a 5-bp fragment from the pUC18 vector. The new fusion protein (PheA*) retained both chorismate mutase and prephenate dehydratase activities. PheA* had a calculated subunit molecular weight of 33,574, in comparison to the 43,182-molecular-weight subunit size of PheA. The deletion did not affect the ability of PheA* to assume the native dimeric configuration of PheA. Both the CM-P and prephenate dehydratase components of PheA* were insensitive to L-phenylalanine inhibition, in contrast to the corresponding components of PheA. L-Phenylalanine protected both catalytic activities of PheA from thermal inactivation, and this protective effect of L-phenylalanine upon the PheA* activities was lost. PheA* was more stable than PheA to thermal inactivation; this was more pronounced for prephenate dehydratase than for CM-P. In the presence of dithiothreitol, the differential resistance of PheA* prephenate dehydratase to thermal inactivation was particularly striking.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗

PubMed1990

Fetal prostate growth and development.

Normal fetal and newborn prostates were studied to evaluate growth patterns, histogenesis, and secretory activity. Whole cross-sectioned prostates harvested from 107 necropsies of fetuses and newborns ages 20 weeks gestation to 1 month of age were used. Development of the prostate occurred in three stages: bud stage (20-30 weeks gestation), bud-tubule stage (31-36 weeks gestation), and acinotubular stage (37-42 weeks gestation). Squamous metaplasia often appeared in the urethra, utricle, and primary lobular ducts, and occasional microcysts were noted. PAS and alcian blue-PAS positive secretion were present in 65% of the specimens by 20-30 weeks gestation and in over 87% of the specimens by 37 or more weeks gestation. Secretory activity was most prominent in the lateral regions of the peripheral zone. Prostate-specific antigen was only weakly detected throughout the prenatal period and was not related to secretory activity as evidenced by the PAS technique.

Antigens, Neoplasm↗