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Biomedical subjects

T Beppu

Publications and source records attributed to T Beppu.

At least 199 records · Page 11Linked to original sources
PubMed1991

A new approach to chemoembolization for unresectable hepatocellular carcinoma using aclarubicin microspheres in combination with cisplatin suspended in iodized oil.

Sixty-six consecutive patients with unresectable hepatocellular carcinoma (HCC) were treated with transcatheter arterial chemoembolization (TACE) using aclarubicin microspheres (ACRms) in combination with cisplatin suspended in iodized oil (Lipiodol, Laboratoire Guerbert, Paris, France) (CSL). The stages of the disease were as follows: Stage I (n = 1), Stage II (n = 10), Stage III (n = 26), and Stage IV (n = 29). The effectiveness of TACE was assessed by comparing ACRms with CSL with ACRms without CSL. Of 66 patients treated with ACRms and CSL, 62 (93.9%) could be examined for response. According to response criteria, there were 31 (50.0%) partial responses and 17 (27.4%) minor responses. In 13 cases (21.0%) there was no change and in 1 case (1.6%) there was progressive disease. The cumulative survival rate was 80.7% at 1 year, 64.2% at 2 years, and 50.6% at 3 years. The rates were significantly higher than those of the group treated with ACRms. Eleven patients in the ACRms and CSL group experienced clinical complications: cholecystitis (4.5%), pancreatitis (3.0%), liver abscess (3.0%), hepatic failure (3.0%), gastrointestinal bleeding (1.5%), and renal failure (1.5%). No lethal side effects related to the therapy were observed. TACE using ACRms in combination with CSL prolongs the survival of patients with unresectable HCC.

Aclarubicin↗
PubMed1991

Cloning, nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1.

Rhodococcus rhodochrous J1 produces two kinds of cobalt-containing nitrile hydratases (NHases); one is a high molecular mass-NHase (H-NHase) and the other is a low molecular mass-NHase (L-NHase). Both NHases are composed of two subunits of different sizes (alpha and beta subunits). The H- and L-NHase genes were cloned into Escherichia coli by a DNA-probing method using the NHase gene of Rhodococcus sp. N-774, a ferric ion-containing NHase producing strain, as the hybridization probe and their nucleotide sequences were determined. In each of the H- and L-NHase genes, the open reading frame for the beta subunit was located just upstream of that for the alpha subunit, which probably belongs to the same operon. The amino acid sequences of each subunit of the H- and L-NHases from R. rhodochrous J1 showed generally significant similarities to those from Rhodococcus sp. N-774, but the arrangement of the coding sequences for two subunits is reverse of the order found in the NHase gene of Rhodococcus sp. N-774. Each of the NHase genes was expressed in E. coli cells under the control of lac promoter, only when they were cultured in the medium supplemented with CoCl2.

Amino Acid Sequence↗
PubMed1991

Resonance Raman spectra of plastocyanin and pseudoazurin: evidence for conserved cysteine ligand conformations in cupredoxins (blue copper proteins).

New resonance Raman (RR) spectra at 15 K are reported for poplar (Populus nigra) and oleander (Oleander nerium) plastocyanins and for Alcaligenes faecalis pseudoazurin. The spectra are compared with those of other blue copper proteins (cupredoxins). In all cases, nine or more vibrational modes between 330 and 460 cm-1 can be assigned to a coupling of the Cu-S(Cys) stretch with Cys ligand deformations. The fact that these vibrations occur at a relatively constant set of frequencies is testimony to the highly conserved ground-state structure of the Cu-Cys moiety. Shifts of the vibrational modes by 1-3 cm-1 upon deuterium exchange can be correlated with N-H...S hydrogen bonds from the protein backbone to the sulfur of the Cys ligand. There is marked variability in the intensities of these Cys-related vibrations, such that each class of cupredoxin has its own pattern of RR intensities. For example, plastocyanins from poplar, oleander, French bean, and spinach have their most intense feature at approximately 425 cm-1; azurins show greatest intensity at approximately 410 cm-1, stellacyanin and ascorbate oxidase at approximately 385 cm-1, and nitrite reductase at approximately 360 cm-1. These variable intensity patterns are related to differences in the electronic excited-state structures. We propose that they have a basis in the protein environment of the copper-cysteinate chromophore. A further insight into the vibrational spectra is provided by the structures of the six cupredoxins for which crystallographic refinements at high resolution are available (plastocyanins from P. nigra, O. nerium, and Enteromorpha prolifera, pseudoazurin from A. faecalis, azurin from Alcaligenes denitrificans, and cucumber basic blue protein). The average of the Cu-S(Cys) bond lengths is 2.12 +/- 0.05 A. Since the observed range of bond lengths falls within the precision of the determinations, this variation is considered insignificant. The Cys ligand dihedral angles are also highly conserved. Cu-S gamma-C beta-C alpha is always near -170 degrees and S gamma-C beta-C alpha-N near 170 degrees. As a result, the Cu-S gamma bond is coplanar with the Cys side-chain atoms and part of the polypeptide backbone. The coplanarity accounts for the extensive coupling of Cu-S stretching and Cys deformation modes as seen in the RR spectrum. The conservation of this copper-cysteinate conformation in cupredoxins may indicate a favored pathway for electron transfer.

Azurin↗
PubMed1991

Preliminary X-ray diffraction analysis of a crystallizable mutant of malate dehydrogenase from the thermophile Thermus flavus.

Malate dehydrogenase from mutant strain F428 of the thermophilic bacterium Thermus flavus has now been crystallized from polyethylene glycol 8000 in a form suitable for diffraction studies. The protein crystallizes in the orthorhombic P2(1)2(1)2(1) space group with unit cell dimensions a = 71.8 A, b = 88.6 A, c = 119.0 A. The asymmetric unit consists of one homodimer of molecular mass 67,000 Da. The X-ray diffraction extends beyond 1.7 A and a full data set to 1.9 A has been collected.

Crystallization↗
PubMed1991

Role of threonine 190 in modulating the catalytic function of malate dehydrogenase from a thermophile Thermus flavus.

Random mutagenesis of malate dehydrogenase from a thermophilic bacterium, Thermus flavus AT-62, had revealed that a Thr190----Ile replacement near the essential catalytic residue His187 caused marked modulation of the catalytic properties. For further exploration of a role of the residue at this position, this residue was substituted with each of the other amino acids by site-directed mutagenesis. Most of the mutations except for substitution with Ser caused increases in Km for oxaloacetate and increases Ki for oxaloacetate of 2-110 times. Substitution with His or Pro was characterized by the complete loss of substrate inhibition, along with a marked increase in Km for oxaloacetate. Kinetic analyses of the native and altered malate dehydrogenases at various pHs revealed that both Km and Ki for oxaloacetate decreased proportionally to the decrease in pH from 8.40 to 5.75, whereas kcat was nearly constant within the pH range. Apparent shifts of the optimum pH values toward acidity observed with most of the altered malate dehydrogenases were attributed to the increase in Ki, which facilitated the release from the substrate inhibition at a lower pH. Replacement of Thr310, a possible counterpart with which Thr190 forms a hydrogen bond, by Ile caused changes in the catalytic properties similar to those of the Thr190-substituted enzymes. These results suggest that not only the loss of the hydrogen bond between Thr190 and Thr310 but also properties of the residues introduced at position 190 cause modulation of the catalytic properties, probably through dislocation of the loop structure that contains the catalytic residue His187.

Base Sequence↗
PubMed1991

Isolation and characterization of human pro-urokinase and its mutants accumulated within the yeast secretory pathway.

Human pro-urokinase (pro-UK) and two pro-UK deletion mutants, one lacking the epidermal growth factor(EGF)-like domain, and the other lacking both the EGF-like domain and the kringle domain, were produced in Saccharomyces cerevisiae. This was done using the yeast GAL7 promoter and the prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). Although biologically active and heavily glycosylated pro-UKs were secreted into the culture medium, the amounts were extremely small. On the other hand, large amounts of pro-UKs of a single-chain form were accumulated inside the cells, exceeding 3-4% of total cellular proteins. The intracellular pro-UKs were N-glycosylated, probably with a single core carbohydrate unit, and amino acid sequencing of their N termini revealed that the secretion signal of MPR was correctly processed. Biologically active pro-UKs were recovered in high yields by means of solubilization with 4.5 M guanidine.HCl and subsequent dialysis for refolding. The refolded yeast pro-UK was indistinguishable from human kidney-derived pro-UK in terms of specific enzymatic activity and its secondary structure, as determined by circular dichroism spectroscopy.

Amino Acid Sequence↗
PubMed1991

Cloning and characterization of an amidase gene from Rhodococcus species N-774 and its expression in Escherichia coli.

For investigation of an unknown open reading frame which is present upstream of the nitrile hydratase (NHase) gene from Rhodococcus sp. N-774, a longer DNA fragment covering the entire gene was cloned in Escherichia coli. Nucleotide sequencing and detailed subcloning experiments predicted a single open reading frame consisting of 521 amino acid residues of Mr 54,671. The amino acid sequence, especially its NH2-terminal portion, showed significant homology with those of indoleacetamide hydrolases from Pseudomonas savastanoi and Agrobacterium tumefaciens, and acetamidase from Aspergillus nidulans. The 521-amino acid coding region was therefore expressed by use of the E. coli lac promoter in E. coli, and was found to direct a considerable amidase activity. This amidase hydrolyzed propionamide efficiently, and also hydrolyzed, at a lower efficiency, acetamide, acrylamide and indoleacetamide. These data clearly show that the unknown open reading frame present upstream of the NHase coding region encodes an amidase. Because the TAG translational stop codon of the amidase is located only 75 base pairs apart from the ATG start codon of the alpha-subunit of NHase, these genes are probably translated in a polycistronic manner.

Amidohydrolases↗
PubMed1991

Cloning and sequencing of the gene cluster encoding two subunits of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes.

The membrane-bound alcohol dehydrogenase (ADH) from Acetobacter polyoxogenes NBI1028 is composed of a 72 kDa subunit and a 44 kDa cytochrome c subunit. The amino acid sequences of the two regions of the 72 kDa subunit were determined to prepare oligonucleotides for the purpose of amplification of a DNA fragment corresponding to the intermediate region by the polymerase chain reaction. A 0.5 kb DNA fragment thus amplified was used as the probe to clone a 7.0 kb PstI fragment coding for the whole 72 kDa subunit. Nucleotide sequencing and immunoblot analysis revealed that the cloned fragment contained the full structural genes for the 72 kDa and the 44 kDa subunits and they were clustered with the same transcription polarity. The predicted amino acid sequence of the gene for the 72 kDa subunit showed homology with that of the 72 kDa subunit from ADH of A. aceti and those of methanol dehydrogenase from methylotrophic bacteria. The 72 and 44 kDa subunits contained one and three typical haem binding sequences, respectively.

Acetobacter↗
PubMed1991

Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase.

The amino acid sequences of cellulase from Bacillus subtilis (BSC) and that from an alkalophilic Bacillus sp. N-4 (NK1) show significant homology in most parts except for the C-terminal portions. Despite the high homology, the pH activity profiles of the two enzymes are quite different; BSC has its optimum pH at 6-6.5, whereas NK1 is active over a broad pH range from 6 to 10.5. In order to identify the structural features which determine such pH activity profiles, chimeric cellulases between BSC and NK1 were constructed using four restriction sites commonly present within the homologous coding sequences, and were produced in Escherichia coli. The chimeric cellulases showed various chromatographic behaviors, reflecting the origins of their C-terminal regions. The pH activity profiles of the chimeric enzymes in the alkaline range could be classified into either the BSC or NK1 type mainly depending on the origins of the fifth C-terminal regions. In the acidic range, the profile was determined only by the origin of the fourth enzyme region from the N terminus. Comparison of the kinetic parameters between pH 5 and 6 using p-nitrophenyl cellobioside as a substrate indicated that the fourth region is responsible for the pH-dependent change of the kcat value. Only a limited number of amino acids in the fourth region may affect on deprotonation of catalytic residues of the cellulases and modulate the catalytic activity in the acidic pH values.

Amino Acid Sequence↗
PubMed1991

Identification of an A-factor-dependent promoter in the streptomycin biosynthetic gene cluster of Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is a microbial hormone controlling streptomycin (Sm) production, Sm resistance and sporulation in Streptomyces griseus. In order to identify A-factor-dependent promoters in the Sm biosynthetic gene cluster, a new promoter-probe plasmid with a low copy number was constructed by using an extremely thermostable malate dehydrogenase gene as the reporter. Of the three promoters in the Sm production region that includes strR, aphD and strB, only the promoter of strR, which codes for a putative regulatory protein, was found to be directly controlled by A-factor. This was also confirmed by S1 nuclease mapping. The region essential for its A-factor-dependence was determined to be located 430-330 base pairs upstream of the transcriptional start point. Increase in the copy number of the strR promoter region did not lead to a corresponding increase in the total promoter activity, probably due to titration of a putative activator which binds to the enhancer-like region and controls the expression of the strR promoter. This putative activator is apparently distinct from the A-factor-receptor protein. The aphD gene, which encodes the major Sm resistance determinant, Sm-6-phosphotransferase, was transcribed mainly by read-through from the A-factor-dependent strR promoter; this accounts for the prompt induction of Sm resistance by A-factor.

4-Butyrolactone↗
PubMed1991

Characterization of an operon encoding succinyl-CoA synthetase and malate dehydrogenase from Thermus flavus AT-62 and its expression in Escherichia coli.

An open reading frame (ORF) was found upstream of the mdh gene in Thermus flavus by computer-aided analysis. It was identified as the gene encoding the alpha subunit of succinyl-CoA synthetase (SCS) and termed scsA. Nucleotide sequencing of a further upstream region revealed the presence of another ORF, corresponding to the sequence of the beta subunit of SCS. The latter gene was termed scsB. The scsB gene was found to contain an unusual translational initiation codon, TTG. S1 nuclease mapping indicates that transcription starts at the nucleotide at position--31 upstream of the initiation codon of the beta gene. The scsB and scsA genes along with the mdh gene appear to form an operon and are most likely co-transcribed in this order, because the intercistronic regions between them are very short; in fact, the termination codon of scsB overlaps the initiation codon of scsA. A stretch characteristic of the--10 region of a typical prokaryotic promoter was found upstream of scsB, whereas no sequence characteristic of a typical--35 region was present. Escherichia coli harboring a plasmid containing scsA and scsB did not produce thermostable SCS activity, even when a synthetic promoter for E. coli was attached. However, when an inverted repeat present in front of scsB, which covers the putative ribosome-binding site and is capable of forming a stable stem-loop structure, was altered by site-directed mutagenesis, overproduction of heat-stable SCS was observed.

Amino Acid Sequence↗
PubMed1991

Highly synchronous culture of fibroblasts from G2 block caused by staurosporine, a potent inhibitor of protein kinases.

The effect of staurosporine, a potent microbial inhibitor of protein kinases, on the cell cycle of cultured fibroblast cells was investigated. A low concentration of staurosporine (1-10 ng/ml) blocked the cell cycle of rat 3Y1 fibroblasts at the early G1 phase within 2 h after serum stimulation. On the other hand, a higher concentration of the drug (100 ng/ml) caused the specific G2 block. Both of these blocks were reversible. After release from the G2 block, highly synchronous transition to M phase was observed and both nuclear and cell divisions were completed within 180 min. This reversible G2 block showed a clear contrast to those by the other G2 arresters, trichostatin A and leptomycin B, which formed proliferative tetraploid cells after release by entering the cells into a new S phase without passage through M phase. The presence of trichostatin A or leptomycin B did not interfere with this synchronous progression through G2/M phases, suggesting that the arrest point of staurosporine was present in late G2 phase following those of trichostatin A and leptomycin B.

Alkaloids↗
PubMed1991

Molecular mechanisms of the a-factor-dependent control of secondary metabolism in streptomyces.

A-factor, 2-isocapryloyl-(3 R)-hydroxymethyl-gamma-butyrolactone, is a microbial hormone controlling secondary metabolism and morphogenesis of STREPTOMYCES GRISEUS. The A-factor-receptor protein present in the cytoplasm is a repressor-type regulator; it plays a role in repressing streptomycin production and sporulation while the binding of A-factor to the receptor protein releases its repression. Thus, secondary metabolism and sporulation of S. GRISEUS involve a step of hormonal regulation by A-factor. The AFSR gene codes for a global regulatory protein of 993 amino acids controlling positively secondary metabolic formation in STREPTOMYCES COELICOLOR A3(2), probably at the transcriptional level. This protein comprises two domains, i.e., an ATP-binding domain at its NH (2)-terminal portion and a DNA-binding domain with a helix-turn-helix motif at its COOH-terminal portion. The AfsR protein is found to be phosphorylated. It is assumed that the extent of phosphorylation-dephosphorylation of the AfsR protein modulates the expression of genes responsible for secondary metabolic formation.

Journal Article↗
PubMed1991

Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a.

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.

Animals↗
PubMed1991

An in vivo area meter for real-time measurement of cross-sectional area in the cardiovascular system.

A direct real-time recording of the cross-sectional area of the heart valve is useful for the fluid dynamic study of the cardiovascular system. Electronic circuitry is described that is capable of driving the transmitter coil assembly placed outside the animal and detecting an area-related signal induced in the one-turn coil in vivo. When a piece of fine pliable metal thread encircles the area of interest (e.g. the mitral/aortic valve orifice) so as to form a single loop, the electrical potential between the ends of the loop is linearly related to the size of the area irrespective of its shape. The principle of measurement, construction of transmitter coil assembly, and simple but accurate direct calibration are also described.

Animals↗
PubMed1991

Aneurysm of the distal anterior inferior cerebellar artery unrelated to the cerebellopontine angle: case report.

This is a case report of an unusual aneurysm of the distal anterior inferior cerebellar artery. A 44-year-old woman had a severe frontal headache and vomiting of sudden onset. On the day after admission, the patient began to demonstrate nuchal rigidity and difficulty with upward gaze bilaterally. There were no complaints at this time suggestive of a syndrome of the cerebellopontine angle. A computed tomographic scan revealed an intraventricular hemorrhage of the 3rd and 4th ventricles; however, multiple attempts at four-vessel angiography were required before an aneurysm could be demonstrated. On the 28th hospital day, a suboccipital craniectomy using the retromastoid approach in the lateral position was performed. A saccular aneurysm with a surrounding hematoma in the distal anterior inferior cerebellar artery was found and clipped. The operative findings revealed that the aneurysm was unrelated to the cerebellopontine angle. After surgery, the patient had an uneventful recovery and complete resolution of symptoms.

Adult↗
PubMed1991

Cloning, nucleotide sequence, and transcriptional analysis of the NAD(P)-dependent cholesterol dehydrogenase gene from a Nocardia sp. and its hyperexpression in Streptomyces spp.

NAD(P)-dependent cholesterol dehydrogenases [NAD(P)-CDH], which allow easier quantification of cholesterol by means of directly measuring the A340 of NAD(P)H, are useful for clinical purposes. The amino acid sequences of the NH2 terminus and the fragments obtained by CNBr decomposition of the NAD(P)-CDH from a Nocardia sp. were determined for preparation of synthetic oligonucleotides as hybridization probes. A 4.4-kbp BamHI fragment hybridizing to these probes was cloned on pUC19 in Escherichia coli. The nucleotide sequence together with the determined amino acid sequences revealed that this enzyme consists of 364 amino acids (Mr, 39,792) and contains an NAD(P)-binding consensus sequence at its NH2-terminal portion. High-resolution S1 nuclease mapping suggested that in NAD(P)-CDH of both Nocardia and Streptomyces spp. transcription initiates at the adenine residue, which is the first position of the translational initiation triplet (AUG) of this protein. The S1 mapping experiments also showed that cholesterol-dependent regulation in the Nocardia sp. occurred at the level of transcription. In Streptomyces lividans containing the cloned fragment, however, this promoter was expressed constitutively. DNA manipulation of the cloned gene in E. coli, including the generation of a ribosome-binding sequence at an appropriate position by oligonucleotide-directed mutagenesis, led to production of this protein in a very large amount but in the enzymatically inactive form of inclusion bodies. On the other hand, a Streptomyces host-vector system was successfully used for producing 40 times as much enzymatically active NAD(P)-CDH as that produced by the original Nocardia sp.

3-Hydroxysteroid Dehydrogenases↗