Biomedical subjects
Publications and source records attributed to T Ohta.
Pit-1 is a pituitary-specific transcription factor that binds to and transactivates promoters of growth hormone- and prolactin-encoding genes. A chromosomal gene related to human Pit-1 isolated from human gene libraries was over 14 kb long and split into six exons. All of the splice donor and acceptor sites conformed to the GT/AG rule. The gene was mapped to human chromosome region 3p11.
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Two species of lipoprotein containing apoA-I, one containing only apoA-I (LpA-I), and the other containing apoA-I and apoA-II (LpA-I/A-II), were tested for their effects on macrophage foam cells. Rat macrophages were converted to foam cells by incubation with radiolabeled acetylated LDL. Incubation with LpA-I or LpA-I/A-II decreased the cellular cholesteryl esters (CE) mass. However, the free cholesterol (FC) mass was only reduced by LpA-I. All the radioactivity excreted into the medium was associated with LpA-I or LpA-I/A-II; 39% of the excreted radioactivity was esterified in LpA-I and 10% in LpA-I/A-II. Upon complete inactivation of lecithin: cholesterol acyltransferase (LCAT) activity with dithiobisnitrobenzoic acid, the cholesterol reducing capacity of LpA-I was weakened significantly. However, the CE mass reducing capacity of LpA-I/A-II was not affected. When LpA-I and LpA-I/A-II were combined, the cholesterol reducing capacity of the mixture was similar to that of LpA-I alone. However, LpA-I re-isolated from the medium showed a lower esterification rate than did the re-isolated LpA-I/A-II, thereby indicating that the cholesterol esterified in LpA-I was transferred to LpA-I/A-II. These results suggest that (i) the function of LpA-I is closely linked to the LCAT activity while that of LpA-I/A-II is not, and (ii) LpA-I in concert with LpA-I/A-II induces a series of extracellular events; LCAT-mediated esterification of excreted FC by LpA-I and a subsequent CE transfer to LpA-I/A-II. These mechanisms might be important for net cholesterol efflux from macrophage foam cells in physiological states.
In Lactobacillus plantarum non-allosteric L-lactate dehydrogenase (L-LDH), the highly conserved His188 residue, which is involved in the binding of an allosteric effector, fructose 1,6-bisphosphate [Fru(1,6)P2], in allosteric L-LDH is uniquely substituted by an Asp. The mutant L. plantarum L-LDH, in which Asp188 is replaced by a His, showed essentially the same Fru(1,6)P2-independent catalytic activity as the wild-type enzyme, except that the Km and Vmax values were slightly decreased. However, the addition of Fru(1,6)P2 induced significant thermostabilization of the mutant enzyme, as in the case of many allosteric L-LDHs, while Fru(1,6)P2 showed no significant effect on the stability of the wild-type enzyme, indicating that only the single-point mutation, G-->C, sufficiently induces the Fru(1,6)P2-binding ability of L. plantarum L-LDH. The mutant enzyme showed higher thermostability than the wild-type enzyme in the presence of Fru(1,6)P2. In the absence of Fru(1,6)P2, on the other hand, the mutant enzyme was more labile below 65 degrees C but more stable above 70 degrees C.
Purified alanine carrier proteins were cleaved into peptides either chemically after solubilization in 1,1,1,3,3,3-hexafluoro-2-propanol or proteolytically with lysylendopeptidase. From the amino acid sequence analyses of these peptides, we synthesized a DNA probe and utilized it for successful cloning of a gene encoding the alanine carrier protein (acp gene). The 5'-flanking region was determined by an inverse polymerase chain reaction, and an open reading frame consisting of 1,335 nucleotides was found. The amino acid sequence deduced from the open reading frame consists of 445 amino acids, and all the partial amino acid sequences determined are included in the sequence. Although the calculated M(r) of 47,803 is significantly larger than the apparent M(r) of 42,500 as reported previously (Hirata, H., Kambe, T., and Kagawa, Y. (1984) J. Biol. Chem. 259, 10653-10656), an in vitro translation experiment revealed that the product of the acp gene migrates at a position coinciding with that of the purified alanine carrier. Hydropathy analysis suggests that the protein contains at least 8 hydrophobic segments presumably spanning membrane. A homology search on a database reveals relatively high scores of homology with either the Escherichia coli melibiose carrier or the human Na+/glucose symporter, particularly in the region from Leu246 to Glu286. Furthermore, the region also reveals low but significant similarities to other Na(+)-coupled symporters.
The lymphatic pathway from the head of the pancreas to the para-aortic lymph nodes was examined on the basis of the frequency of lymph node involvements. Forty-four patients were examined. All patients had extended radical operations. Thirty-one of 44 (70.5%) patients had lymph node involvement. The lymph nodes that had a high metastatic rate included the following: (1) lymph nodes around the common hepatic artery (number 8 lymph node); (2) lymph nodes of the hepatoduodenal ligament (number 12 lymph node); (3) the posterior pancreaticoduodenal lymph node (number 13 lymph node); (4) lymph nodes around the superior mesenteric artery (number 14 lymph node); (5) para-aortic lymph nodes (number 16 lymph node); and (6) the anterior pancreaticoduodenal lymph node (number 17 lymph node). Twenty-eight of these 31 patients had disease in the posterior pancreaticoduodenal lymph node. The patterns of lymph node involvement consisted of four combinations: number 13-number 17, number 13-number 14, number 14-number 16, and number 17-number 8. All of the patients with number 16 nodal involvement had number 14 lymph node metastasis. However, there was no relationship between tumor size and lymph node involvement. Based on these results, the main lymphatic pathway from the head of the pancreas to the para-aortic lymph nodes was thought to be via the lymph nodes around the superior mesenteric artery, assuming that lymphatic flow is anterograde. In addition, this study demonstrates that it is necessary to perform an extensive lymph node dissection, including the para-aortic lymph node, even in patients with small tumors.
BACKGROUND: The efficiency of anti-CD3 antibody (OKT3) for adoptive immunotherapy using lymphokine-activated killer (LAK) cells generated from tumor-infiltrating lymphocytes (TIL), regional lymph node lymphocytes (RLNL), and peripheral blood lymphocytes (PBL) was investigated. METHODS: TIL, RLNL, and PBL derived from 39 patients with gastrointestinal cancers (16 gastric cancers, 17 colorectal cancers, and 6 esophageal cancers) were cultured for 4 weeks with 200 U/ml of recombinant interleukin-2. To one group, solid-phase 10 micrograms/ml OKT3 was added during the initial culture period (day 2 or 4). Cytotoxicity against K562 cells (NK-like activity) and Daudi cells (LAK activity) and the phenotypes of effector cells generated after culturing for 2-3 weeks were studied. RESULTS: Proliferative responses were significantly increased by OKT3 in each type of effector cell (P less than 0.01); in particular, TIL expanded more by OKT3 than PBL and RLNL (P less than 0.01). The population of CD8+ CD11b- cytotoxic T-cells in OKT3-stimulated groups was significantly larger than that in unstimulated groups (P less than 0.01), whereas no differences were observed with CD4+ cells (helper/inducer T-cells) and CD8+ CD11b+ cells (suppressor T-cells). OKT3 enhanced the NK-like activity of TIL and PBL but did not affect their LAK activity. OKT3 suppressed the NK and LAK activity of RLNL. CONCLUSIONS: OKT3 stimulation did not significantly enhance the LAK activity, but the authors propose that OKT3 could be an effective addition to adoptive immunotherapy using TIL due to an increased proliferation and generation of a large cytotoxic T-cell population.
Clostridium sp. G0005 produces a cell-bound glucoamylase (CGA). The gene encoding CGA has been sequenced. The deduced amino acid sequence begins with a putative 21-residue signal sequence for secretion of bacterial lipoproteins, which suggests that a putative CGA precursor is modified and secreted like other bacterial lipoproteins in Clostridium sp. G0005, and that the modified residue is important in the cell-bound form of mature CGA. Comparison of the amino acid sequence of the CGA precursor with known eukaryotic enzymes showed several regions of high similarity in spite of low similarity throughout the overall primary structure. CGA is the first bacterial glucoamylase to be cloned. The CGA gene was expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5' non-coding region and the N-terminal coding region of the gene were replaced with the lac promoter. Kinetic studies of the cloned enzyme purified from E. coli were performed with a set of linear malto-oligosaccharides as substrates, and the subsite affinity was calculated from the kinetic parameters. CGA had typical kinetic properties for a glucoamylase, but this bacterial enzyme had higher isomaltose-hydrolyzing activity than other eukaryotic glucoamylases.
The dynamic equilibrium of a catalytic site between active and inactive conformations, the missing link between the structure and function of allosteric enzymes, was identified using protein engineering and NMR techniques. Kinetic analyses of the wild-type and three mutants of Thermus L-lactate dehydrogenase established that the allosteric property of the enzyme is associated with a concerted transition between the high-affinity (R) and low-affinity (T) states. By introducing mutations, we prepared an enzyme in which the R and T states were balanced. The conformation of the enzyme-bound coenzyme, NAD+, which interacts directly with the substrate, was analyzed using NMR spectroscopy. NAD+ bound to the mutant enzyme was in a conformational mixture of the active and inactive forms, while NAD+ took on predominantly one of the two forms when it was bound to the other enzymes we had analyzed. We interpret this to mean that the catalytic site is in equilibrium between the two conformations. The ratio of the conformers of each enzyme agreed with the [T]/[R] ratio as determined by kinetic analyses. Therefore, it is the identified conformational equilibrium of the catalytic site that governs the allosteric regulation of the enzyme activity.
To elucidate an anti-atherogenic nature of high density lipoprotein (HDL) at cellular level, its in vitro effect on macrophage foam cells was examined. Rat peritoneal macrophages were converted to foam cells by incubation with [3H]cholesterol-labeled acetylated LDL. HDL addition to these foam cells resulted in a reduction in cellular radioactive cholesteryl esters (CE) as well as its CE mass. The radioactive free cholesterol (FC) was similarly reduced with time, whereas its FC mass level was unaltered. Other lipoproteins such as very low density lipoprotein and low density lipoprotein also reduced the radioactive FC. However, their CE-reducing capacity was negligibly weak. These results suggest that (i) CE reduction is selective to HDL, (ii) FC transfer from plasma membrane to lipoprotein (cholesterol efflux) expressed by reduction in radioactive FC is not selective to HDL but occurs to other lipoproteins, (iii) the CE-reducing capacity of HDL became weaker when cellular binding of HDL was reduced by chemical modification with tetranitromethane or a chemical cross-linker, dithiobis-succinimidylpropionate, suggesting an importance of the specific binding in the HDL-mediated CE reduction. These in vitro results gave an experimental support to a definite role of HDL as an anti-atherogenic lipoprotein in vivo.
The precursor of aqualysin I, an extracellular protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, the protease domain and a C-terminal pro-sequence. In an Escherichia coli expression system, mature and active aqualysin I is formed by treatment at 65 degrees C and the N-pro-sequence is required for its production. Complete deletion of the C-pro-sequence did not affect the production of active aqualysin I, indicating that the C-pro-sequence is not essential. A non-covalent N-pro-region was separately synthesized from the protease domain with or without the C-pro-sequence. In this system, mature and active aqualysin I was detected only when the C-pro-sequence was deleted.
The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase. Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53,901). Codon usage in the T. thermophilus Glu-tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T. thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNA(Glu) are highly conserved. The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography. Single crystals of T. thermophilus Glu-tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor-diffusion technique. The crystals diffract X-rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm.
Thirty-four patients who had resection of cancer of the pancreatic head were examined clinicopathologically to elucidate neural invasion of cancer of the pancreatic head to the extrapancreatic nerve plexus. Invasion of cancer to the retropancreatic tissue (rp+) was observed in 29 (85%) of the 34 patients, and neural invasion to the extrapancreatic nerve plexus was observed in 21 (72%) of the 29 patients with rp+. The incidence of invasion to the second region of the nerve plexus of the pancreatic head was high (14 patients; 67%). The degree of the neural invasion tended to increase as the intrapancreatic neural invasion became more severe and lymph vessel invasion more marked. Based on these findings, en bloc resection of the retropancreatic tissue involving the nerve plexus and fat tissue is necessary in the surgical treatment of cancer of the pancreatic head.
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By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge.
The cloning of five DNA segments carrying at least seven genes (fms1, fms3, fms4, fms5, fms7, fms11, and fms12) that participate in fortimicin A (astromicin) biosynthesis was described previously. These DNA fragments were used to screen a cosmid library of genomic DNA in order to examine if these biosynthetic genes are clustered in Micromonospora olivasterospora. One cosmid clone (pGLM990) was obtained, which hybridized to all the probes. Complementation analysis, using mutants blocked at various steps and chimeric plasmids subcloned from pGLM990, showed that three additional genes (fms8, fms10, and fms13) are present in pGLM990. A gene conferring self-resistance to the antibiotic, which was independently cloned in Streptomyces lividans, using the plasmid vector pIJ702 was also found to be linked to the cluster of biosynthetic genes. Thus, at least ten biosynthetic genes and a self-defense gene are clustered in a chromosomal region of about 27 kb in M. olivasterospora. Interestingly, the fms8 gene which participates in the dehydroxylation step of fortimicin A biosynthesis was found to have homology with a neomycin resistance gene nmrA from the neomycin-producing Micromonospora sp. MK50. Studies using a cell-free extract of the fms8 mutant and its parent strain showed that the enzyme encoded by fms8 phosphorylates a biosynthetic precursor, fortimicin KK1, in the presence of ATP. Thus the dehydroxylation reaction is suggested to occur via the phosphorylation of the target hydroxyl group. DNA regions homologous to fms genes were found in Micromonospora sp. SF-2098 and Dactylosporangium matsuzakiense, both producers of fortimicin group antibiotics.
We have cloned the seven genes that are responsible for biosynthesis of the antibiotic fortimicin A (FTM A) using a recently developed self-cloning system that employes the plasmid vector pMO116 for Micromonospora olivasterospora. Five chimeric plasmids that restored FTM A production in M. olivasterospora mutants blocked at different biosynthetic steps were isolated by shotgun cloning. Secondary transformation using other non-producing mutants showed that two additional FTM A biosynthetic genes were included on these plasmids, and that at least four of the genes were clustered. Interestingly AN38-1, a non-producing mutant that had a defect in dehydroxylation of a precursor of FTM A, was complemented by the DNA fragment containing a neomycin resistance gene that had been cloned from a neomycin-producing strain (Micromonospora sp. FTM A non-producing strain) in the course of constructing the plasmid vector pMO116. These results clearly show that this novel gene cloning system in Micromonospora is of practical use.
A case of intraductal papillary adenocarcinoma of the pancreas associated with mass forming chronic pancreatitis without calcifications is described. Pancreatolithiasis, or calcified pancreas, is recognized as a high risk factor for pancreatic cancer. However, epidemiologic studies have found that carcinoma of the pancreas associated with chronic pancreatitis was rare. The question is whether chronic pancreatitis without calcifications is actually a precancerous background lesion or not. This case suggests that hyperplasia of the pancreatic ductal epithelium may be a precancerous lesion for pancreatic cancer in some patients with chronic pancreatitis.