Biomedical subjects
T Beppu
Publications and source records attributed to T Beppu.
Regulation of the aortic valve opening. In vivo dynamic measurement of aortic valve orifice area.
Aortic valve orifice area was dynamically measured in anesthetized dogs with a new measuring system involving electromagnetic induction. This system permits us real-time measurement of the valve orifice area in beating hearts in situ. The aortic valve was already open before aortic pressure started to increase without detectable antegrade aortic flow. Maximum opening area was achieved while flow was still accelerating at a mean of 20 to 35 msec before peak blood flow. Maximum opening area was affected by not only aortic blood flow but also aortic pressure, which produced aortic root expansion. The aortic valve orifice area's decreasing curve (corresponding to valve closure) was composed of two phases: the initial decrease and late decrease. The initial decrease in aortic valve orifice area was slower (4.1 cm2/sec) than the late decrease (28.5 cm2/sec). Aortic valve orifice area was reduced from maximum to 40% of maximum (in a triangular open position) during the initial slow closing. These measurements showed that (1) initial slow closure of the aortic valve is evoked by leaflet tension which is produced by the aortic root expansion (the leaflet tension tended to make the shape of the aortic orifice triangular) and (2) late rapid closure is induced by backflow of blood into the sinus of Valsalva. Thus, cusp expansion owing to intraaortic pressure plays an important role in the opening and closing of the aortic valve and aortic blood flow.
A computerized control system for cardiopulmonary bypass.
A pump control system using a microcomputer for cardiopulmonary bypass has been developed. The parameters monitored by the computer are central venous pressure, aortic pressure, blood volume in a reservoir, and collapsing of a small vinyl bag in a venous withdrawal tube. Both pumps in the arterial infusion and the venous withdrawal lines are automatically controlled through an interface unit throughout cardiopulmonary bypass. The system regulates central venous pressure with a proportional-integral control algorithm to maintain intravascular volume constant. A screening algorithm is devised to remove artifacts introduced to the pressure measurement. During the weaning period, a perfusionist can select either the central venous pressure control or a reservoir blood volume control. Computerized pump perfusion was applied on 15 children during cardiac operations. Perfusion flow and central venous pressure were controlled satisfactorily with stable operation. Compared with conventional manually controlled pump perfusion, no significant difference was noted in mean aortic pressure, central venous pressure, net fluid balance, total urination, blood chemistry, and urinalysis. This system is useful and is expected to improve the safety of pump perfusion.
Signal transduction and secondary metabolism: prospects for controlling productivity.
Evidence is accumulating that demonstrates the key roles played by diffusible molecules in regulating cellular differentiation, even among prokaryotic microorganisms. This is exemplified by A-factor and its analogues, which act as autoregulators for morphological differentiation and secondary metabolism in Streptomyces. The identification of a specific receptor for A-factor and an A-factor-controlled promoter sequence in S. griseus indicate the close similarity of this system to eukaryotic hormonal control. The involvement of prokaryotic homologues of the eukaryotic Ser/Thr-kinases in the regulation of differentiation processes seems to be another characteristic feature of this group of bacteria. Recent evidence for the presence of these molecular signalling systems in Streptomyces is reviewed, along with the inherent implications.
Specific depletion of spermidine and spermine in HTC cells treated with inhibitors of aminopropyltransferases.
The effects of a potent spermidine synthase inhibitor, trans-4-methylcyclohexylamine (4MCHA), and a spermine synthase inhibitor, N-(3-aminopropyl)cyclohexylamine (APCHA), on polyamine biosynthesis and cell growth have been studied in rat hepatoma cells (HTC cells) in culture. Treatment of HTC cells with 4MCHA or APCHA caused a marked decrease of spermidine or spermine with a compensatory increase of putrescine and spermine or spermidine, respectively, in a dose-dependent manner, suggesting specific and potent inhibition of each target enzyme. When 250 microM 4MCHA or APCHA was administered to the cells for 8 days, spermidine was decreased to 2% of control culture or spermine below 1%, respectively, while total polyamine (sum of putrescine, spermidine, and spermine) remained almost unchanged during the culture. There were no significant changes in the growth rate during treatment with the inhibitors at 250 microM concentration. The results suggest that in the growth of HTC cells, putrescine and spermine can be substituted for most of the fraction of cellular spermidine, and spermidine for most of the fraction of cellular spermine. Of five enzymatic activities involved in polyamine biosynthesis and interconversion, S-adenosylmethionine decarboxylase activity increased 8-fold with 250 microM 4MCHA, and 3-fold with 250 microM APCHA during the treatment. This increase was partially due to the increase of half-life of the enzyme. Separate roles for spermidine and spermine in the biosynthesis of the enzyme protein were also suggested.
Morphology of ras-transformed cells becomes apparently normal again with tyrosine kinase inhibitors without a decrease in the ras-GTP complex.
Radicicol, an inhibitor of protein-tyrosine kinase, was found to cause morphological reversion of v-Ha-ras-transformed NIH3T3 fibroblasts and T24 human urinary bladder carcinoma cells that contain an activated ras mutation. The network of actin stress fibers was restored during the treatment with radicicol. A similar morphological change was observed with another protein-tyrosine kinase inhibitor, herbimycin A. Radicicol did not cause any changes in the proportion of the active GTP binding form of p21ras or its subcellular localization. These results rule out the possibility that the morphological reversion by radicicol is due to direct or indirect inhibition of the p21ras function. Cycloheximide and actinomycin D inhibited the morphological change by radicicol, suggesting that the induced transcription of a gene(s) followed by de novo protein synthesis is required for suppression of the transformed phenotype in ras-transformed cells by tyrosine kinase inhibitors.
Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6.
Pseudoazurin, a low molecular weight protein containing a single type I copper, functions as an electron donor to a copper-containing nitrite reductase (NIR) in a denitrifying bacterium Alcaligenes faecalis S-6. To elucidate the protein-protein interaction between these two copper-containing proteins, each of nine out of 13 lysine residues on the surface of pseudoazurin were independently replaced by alanine or aspartate, and the effects of the mutations on the interaction with NIR, as well as the physicochemical properties of pseudoazurin, were analyzed. All of the mutated pseudoazurins showed optical spectra and oxidation-reduction potentials almost identical to those of wild-type pseudoazurin, suggesting that none of the replacements of these lysine residues affected the environment around the type I copper site. Kinetic analysis of electron transfer between mutated pseudoazurins and NIR reveals that the lysine mutations have very little effect on the rate of electron transfer to NIR, but substitution at residues 10, 38, 57 and 77, all close to the copper site, substantially decreases the affinity of pseudoazurin for NIR. This suggests that pseudoazurin interacts with NIR through the region close to the type I copper site. The refined X-ray structures of Lys38Asp and Lys10Asp/Lys38Asp show that the molecular structure has indeed changed little. A new space group is observed for the Lys109Ala mutant crystal. Crystal packing interactions change for the Lys10Asp/Lys38Asp mutant but remain the same for Lys38Asp and Lys59Ala mutants.
Development of an autoflow cruise control system for a centrifugal pump.
To improve the ease of driving a centrifugal pump that is afterload dependent, we have developed an automatic flow control system for the Terumo Capiox centrifugal pump system. This system consists of an autoflow cruise control system with a safety cutoff. The Capiox Pump Console 3000 was controlled by a personal computer through a serial communication line. In the usual manual mode, the motor speed knob works as a pump speed control, and in the autoflow mode, the same knob works as a blood flow rate control. After selecting and obtaining the desired flow rate, the mode was changed from manual to autoflow mode. In the autoflow mode, the computer compares the desired flow rate with the actual flow measured by an ultrasonic Doppler flowmeter and adjusts the motor rotational speed accordingly. During both in vivo and in vitro testing, this autoflow mode was able to return the changed flow that was disrupted by either clamping and declamping of the tubing or by the bolus injection of a vasomotor drug to the selected flow rate within 10 s without any significant fluctuation. In conclusion, the newly developed computer controlled autoflow system was able to produce a reliable and effective flow regulation for a centrifugal pump.
Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.
The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH2-terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.
Cloning and characterization of the A-factor receptor gene from Streptomyces griseus.
A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein control streptomycin production, streptomycin resistance, and aerial mycelium formation in Streptomyces griseus. The A-factor receptor protein (ArpA) was purified from a cell lysate of S. griseus IFO 13350. The NH2-terminal amino acid sequences of ArpA and lysyl endopeptidase-generated fragments were determined for the purpose of preparing oligonucleotide primers for cloning arpA by the PCR method. The arpA gene cloned in this way directed the synthesis of a protein having A-factor-specific binding activity when expressed in Escherichia coli under the control of the T7 promoter. The arpA gene was thus concluded to encode a 276-amino-acid protein with a calculated molecular mass of 29.1 kDa, as determined by nucleotide sequencing. The A-factor-binding activity was observed with a homodimer of ArpA. The NH2-terminal portion of ArpA contained an alpha-helix-turn-alpha-helix DNA-binding motif that showed great similarity to those of many DNA-binding proteins, which suggests that it exerts its regulatory function for the various phenotypes by directly binding to a certain key gene(s). Although a mutant strain deficient in both the ArpA protein and A-factor production overproduces streptomycin and forms aerial mycelium and spores earlier than the wild-type strain because of repressor-like behavior of ArpA, introduction of arpA into this mutant abolished simultaneously its streptomycin production and aerial mycelium formation. All of these data are consistent with the idea that ArpA acts as a repressor-type regulator for secondary metabolite formation and morphogenesis during the early growth phase and A-factor at a certain critical intracellular concentration releases the derepression, thus leading to the onset of secondary metabolism and aerial mycelium formation. The presence of ArpA-like proteins among Streptomyces spp., as revealed by PCR, together with the presence of A-factor-like compounds, suggests that a hormonal control similar to the A-factor system exists in many species of this genus.
Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus.
A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essentially required for aerial mycelium formation and streptomycin production in Streptomyces griseus. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this strain on a high-copy-number plasmid. Subcloning and nucleotide sequencing revealed that one open reading frame with 218 amino acids, named AmfC, served as a multicopy suppressor of the aerial mycelium-defective phenotype of the A-factor-deficient strain. The amfC gene did not restore A-factor or streptomycin production, indicating that amfC is involved in aerial mycelium formation independently of secondary metabolic function. Disruption of the chromosomal amfC gene in the wild-type S. griseus strain caused a severe reduction in the abundance of spores but no effect on the shape or size of the spores. The infrequent sporulation of the amfC disruptant was reversed by introduction of amfC on a plasmid. The amfC-defective phenotype was also restored by the orf1590 gene but not by the amfR-amfA-amfB gene cluster. Nucleotide sequences homologous to the amfC gene were distributed in all of 12 Streptomyces species tested, including Streptomyces coelicolor A3(2). The amfC homolog of S. coelicolor A3(2) was cloned and its nucleotide sequence was determined. The AmfC products of S. griseus and S. coelicolor A3(2) showed a 60% identity in their amino acid sequences. Introduction of the amfC gene of S. coelicolor A3(2) into strain HH1 induced aerial mycelium formation and sporulation, which suggests that both play the same functional role in morphogenesis in the strains.
Double-exponential curve fitting of isometric relaxation: a new measure for myocardial lusitropism.
New indexes for evaluation of isometric myocardial relaxation were proposed. In fully isometric and physiologically sequenced twitches, the time course of isometric force decline fitted well with Gompertz's double-exponential curve (r > or = 0.9995). We conformed the original equation to suit myocardial mechanics, i.e., F(t) = gamma 0 - gamma.exp [-alpha.exp (-beta t)] (t = 1, 2, ..., n), where F(t) denotes force as a function of time t. The gamma 0 and gamma relate to upper asymptote and force amplitude, respectively. Phase-plane analysis of F(t) revealed that alpha [3.56 +/- 0.67 (SD)] related to the phasic delay of relaxation onset but did not affect the F(t) vs. dF(t)/dt relation. The beta (0.127 +/- 0.021) and gamma were linearly related to negative dF(t)/dtmax; however, the terminal slope of the phase-plane diagram was governed by beta alone. The tau beta (0.081 +/- 0.017 s), a reciprocal of beta multiplied by sampling time, was practically independent of preload, total load, and muscle shortening. In isometric twitches, tau beta was substantially decreased by global ischemia, isoproterenol, and CaCl2 but increased by reperfusion. The alpha was independent of inotropic interventions but fell significantly during ischemia and was increased by reperfusion.
Enzymatic aminopropylation of certain secondary amines.
Two unusual aminopropyl acceptors found in a survey of putrescine binding sites of mammalian spermidine synthase, N-methylputrescine (I) and 4-aminomethylpiperidine (II), were examined for their aminopropyl derivatives. Studies under in vitro incubation conditions suggested that the aminopropyl derivatives of the secondary amine of I and II, N4-methylspermidine (Is) and 1-N-(3-aminopropyl)-4-aminomethylpiperidine (IIs), and of the primary amine of I and II, N8-methylspermidine (Ip) and 4-[N-(3-aminopropyl)aminomethyl]piperidine (IIp), respectively, were biosynthesized by rat spermidine synthase. Studies on the cell culture system of cultured rat hepatoma (HTC) cells treated with alpha-difluoromethylornithine, an ornithine decarboxylase inhibitor, clearly showed the presence of Is and Ip when I was administered, and IIs and IIp when II was administered, with no detection of putrescine or spermidine. These results suggested that mammalian spermidine synthase can transfer the aminopropyl moiety of decarboxylated S-adenosylmethionine to certain secondary amines in living cells.
High level secretion by Saccharomyces cerevisiae of human apolipoprotein E as a fusion to Rhizomucor rennin.
As the first step for production of human apolipoprotein E (hApoE) in Saccharomyces cerevisiae, the hApoE cDNA was cloned in Escherichia coli, on the basis of the nucleotide sequence reported previously. When the hApoE cDNA including its pre-sequence-encoding region was expressed under the control of the GAL7 promoter, no protein immunoreactive with anti-hApoE antibody was detected either in the culture medium or inside the cells. For efficient production and secretion of hApoE in S. cerevisiae, the mature hApoE-encoding region was fused to the prepro-sequence region of Rhizomucor rennin (MPR) and to the whole MPR gene including its prepro- and mature-MPR regions. When the fusion gene consisting of the prepro-sequence-encoding region and hApoE regions was expressed in S. cerevisiae, no protein reactive with the anti-hApoE antibody was detected in any fraction of the yeast cells, probably due to rapid degradation of the hApoE protein by yeast proteases. On the other hand, when hApoE was expressed as a fusion to the whole MPR protein, a considerable amount of the fused protein was secreted into the medium. The prepro-sequence of MPR was correctly processed from the fused protein in the medium by autocatalytic activity of MPR and by a protease(s) of the host cell.(ABSTRACT TRUNCATED AT 250 WORDS)
Secretion by Saccharomyces cerevisiae of human apolipoprotein E as a fusion to serum albumin.
For secretion of human apolipoprotein E (hApoE) by Saccharomyces cerevisiae, the hApoE gene was fused to truncated human serum albumin (HSA)-encoding sequences and expressed under the control of the GAL7 promoter. When the mature region of the hApoE gene was fused to the HSA-encoding sequence without its pro-region and expressed in galactose-containing medium, the HSA-hApoE fusion protein was efficiently secreted into the medium at a maximum yield of 6.3 mg per liter.
Overexpression of a gene cluster encoding a chalcone synthase-like protein confers redbrown pigment production in Streptomyces griseus.
A 7.0-kb DNA fragment that conferred redbrown pigment production on Streptomyces griseus was shotgun-cloned with a multicopy vector pIJ486 from this microorganism. By restriction endonuclease mapping and subcloning, a 1.5-kb fragment which is essential for the production of redbrown pigment was determined. The nucleotide sequence of this region revealed the presence of two open reading frames, ORF1 with 109 amino acids (named RppA) and ORF2 with 262 amino acids (RppB), in addition to a truncated ORF3. The termination codon of rppA and the initiation codon of rppB overlapped, sharing one common nucleotide, which strongly suggests that these two genes are cotranscribed. Both rppA and rppB were essentially required for the pigmentation. The RppB protein showed great similarity in amino acid sequence to a chalcone synthase, a key enzyme of central importance in the biosynthetic pathway of all classes of flavonoids in plants. Part of RppA showed sequence similarity to the 33kDa phosphoprotein of adenovirus. Nucleotide sequences homologous to rppA and rppB were widely distributed in Streptomyces species, as determined by Southern hybridization. Further nucleotide sequencing of the entire orf-3 gene showed that ORF3 with 403 amino acids was a cytochrome P-450 (named P-450RPP). These data suggested that the cloned fragment contained part of a gene cluster for the biosynthesis of a certain metabolite. Introduction of the subcloned 1.5-kb fragment into Streptomyces lividans as well as Escherichia coli also caused production of redbrown pigment, suggesting that RppA and RppB are capable of synthesizing the redbrown pigment from metabolites commonly present in bacteria.
Involvement of the COOH-terminal pro-sequence of Serratia marcescens serine protease in the folding of the mature enzyme.
The serine protease (SSP) from a Gram-negative organism, Serratia marcescens, is excreted even from Escherichia coli. The protease is synthesized as a 1045-amino-acid precursor (preproSSP) composed of three functional portions, a typical NH2-terminal signal peptide of 27 amino acids, the mature protease part of 618 amino acids, and a large COOH-terminal part of 400 amino acids. After the mature part (Ala28 to Asp645) has been liberated into the medium, most of the COOH-terminal part (Phe717 to Phe1045) remains stably in the outer membrane. When a mutated gene encoding the prepro-SSP with deletion of the junction region (Ser646 to Gly716) between the mature protease and the processed COOH-terminal protein was expressed in E. coli, the SSP protein was not detected in any fractions of the cells nor in the medium, whereas a processed COOH-terminal protein was found in the outer membrane. However, when the outer membrane fraction prepared from the transformant exhibiting the junction region (Ser646 to Gly716) probably on the cell surface was co-cultured with the transformant, SSP was found in the medium. Furthermore, a significant portion of the SSP protein denatured with guanidine hydrochloride was correctly refolded in vitro into the active protease, only in the presence of the outer membrane preparation from the transformant exhibiting the junction region. These results suggest that the junction region is exposed outside the cells, and it plays a role for guiding the folding of SSP in the correct conformation.
Characterization and expression of a P-450-like mycinamicin biosynthesis gene using a novel Micromonospora-Escherichia coli shuttle cosmid vector.
A 29 kb shuttle cosmid vector, pTYS507, was constructed from a cryptic Micromonospora griseorubida plasmid and the Escherichia coli cosmid pJB8. Subcloning of mycinamicin II biosynthesis genes in pTYS507 led to the identification of a DNA region that could complement a mutant of M. griseorubida that lacked both hydroxylase and epoxidase activities. Nucleotide sequence and mutational analysis suggested that a single P-450-like protein catalyzes both reactions.