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M Imai

Publications and source records attributed to M Imai.

At least 415 records · Page 23Linked to original sources
PubMed1987

Site-directed mutagenesis of hepatitis B surface antigen sequence at codon 160 from arginine to lysine for conversion of subtypic determinant from r to w.

Site-directed mutagenesis from G to A was induced at nucleotide 479 in the S gene of hepatitis B virus DNA, cloned from an individual carrying the surface antigen of subtype ayr. HepG2 cells were transfected with the plasmid DNA containing the mutant. They produced surface antigen of subtype ayw, unlike HepG2 cells harboring the parent viral DNA that produced surface antigen of subtype ayr. These results indicate that a point mutation from G to A at nucleotide 479 in the S gene, changing codon 160 for arginine to that for lysine, can convert the subtypic determinant of hepatitis B surface antigen from r to its allelic determinant w.

Amino Acid Sequence↗
PubMed1987

Preparation and characterization of polyclonal and monoclonal antibodies against human interleukin 1 alpha (IL 1 alpha).

Polyclonal and monoclonal anti-human IL 1 alpha antibodies (Ab) have been established. These Ab neutralized human recombinant IL 1 alpha (rIL 1 alpha) activity effectively, but did not interfere with human rIL 1 beta, murine rIL 1 alpha, or human rIL 2 activity. Fifty percent of rIL 1 alpha activity (25 U/ml, or 2.5 ng/ml) was neutralized by less than 0.06 microgram/ml of rabbit anti-IL 1 alpha Ab (R-38.3G) and by less than 0.13 microgram/ml of monoclonal Ab (clone 28(3B1], respectively. In other experiments, 10 micrograms/ml of rabbit anti-IL 1 alpha Ab could effectively neutralize 50% of 2000 U of rIL 1 alpha activity, and the same amount of monoclonal Ab neutralized 50% of 500 U/ml of rIL 1 alpha activity. Not only IL 1 alpha activity in the thymocyte costimulator assay, but also IL 1-dependent IL 2 production by a human leukemic cell line, HSB.2 subclone, were blocked by these polyclonal or monoclonal Ab. In addition, pI 4.9 IL 1 activity produced by the myelomonocytic cell line THP-1 and by the Epstein-Barr virus-transformed B cell lines, were neutralized by these Ab, suggesting that these cell lines also produce IL 1 alpha. The specificity of these polyclonal and monoclonal Ab was further confirmed by immunochemical method (Western blotting), in which anti-IL 1 alpha Ab reacted with rIL 1 alpha in a specific manner. Furthermore, an enzyme-linked immunosorbent assay system has been developed that can detect low levels of IL 1 alpha activity (less than 0.3 ng/ml or less than 3 U/ml), which is still less sensitive than thymocyte comitogenic assay and considerably less sensitive than the D10 assay. Finally, anti-IL 1 alpha Ab-conjugated affinity columns were prepared, by which IL 1 alpha activity, but not IL 1 beta activity, was specifically adsorbed and eluted effectively.

Antibodies, Monoclonal↗
PubMed1987

Domain mapping of chicken gizzard caldesmon.

Limited proteolysis, affinity chromatography, and immunoblotting have been used to define the domains of chicken gizzard caldesmon, caldesmon120, that interact with calmodulin, F-actin, and a monoclonal antibody prepared using human platelet caldesmon. Treatment of caldesmon120 with chymotrypsin produces groups of fragments near 100, 80, 60, 38, and 20 kDa. Further digestion produces peptides between 40 and 50 kDa. The 100- and 80-kDa peptides cross-react with the monoclonal antibody; the smaller polypeptides do not. The kinetics of cleavage and the antibody studies indicate that the 38- and 80-kDa fragments are the two major pieces of the 120-kDa protein. The 38-kDa fragment, purified by high performance liquid chromatography, and several of its subfragments at 21 and 25 kDa sediment with F-actin, bind to calmodulin-Sepharose in the presence of Ca2+, and are displaced from F-actin by Ca2+-calmodulin. The 80-kDa fragments did not interact with F-actin or calmodulin. We have tentatively placed the 38-kDa fragment at the C-terminal using polyclonal antibodies selected against a beta-galactosidase-caldesmon120 fusion protein produced by a lambda gt11 lysogen. The 38-, 25-, and 21-kDa fragments cross-react with these antibodies; the 80- and 60-kDa fragments do not. Caldesmon77 from human platelets also cross-reacts with these selected antibodies. The results suggest that interacting calmodulin and F-actin binding sites are localized on a 38-kDa C-terminal fragment of caldesmon. The smallest subfragment of this peptide that binds to both F-actin and calmodulin-Sepharose is about 21 kDa. The monoclonal antibody epitope is tentatively localized near the N-terminal of caldesmon77 and must be within 50 kDa of the N-terminal on caldesmon120.

Animals↗
PubMed1987

The use of operon fusions in studies of the heat-shock response: effects of altered sigma 32 on heat-shock promoter function in Escherichia coli.

Derivatives of lambda pF13 phage in which lacZ expression (beta-galactosidase synthesis) is directed by transcription initiated at a heat-shock promoter (PrpoDhs or PgroE) were constructed and used for analysis of the heat-shock response in Escherichia coli. A wild-type strain (MC4100) lysogenic for either of these phages exhibited typical transient induction of beta-galactosidase synthesis upon a temperature shift from 30 degrees to 42 degrees C or after addition of ethanol to the medium (4% to 5%) at 30 degrees C. In contrast, most amber rpoH (htpR) mutants tested (in a Su- background) failed to respond to a temperature shift, though some mutants affected in the carboxy-terminal region exhibited a partial response. All rpoH mutants tested showed a weak but significant response to ethanol. F' plasmids carrying each of six known nonsense suppressors were then introduced into each of four rpoH amber mutants lysogenic for lambda pF13-(Phs-lacZ), creating a set of F' strains that produce sigma 32 protein with a specific amino acid substitution at a known site. Some of these strains showed an essentially normal heat-shock response while others showed little response with either or both of the promoters. In some instances, the response was significantly delayed. These results point to the usefulness of the lambda pF13-derivative phages for quantitative and systematic analysis of heat-shock response in E. coli.

Bacterial Proteins↗
PubMed1987

Functional heterogeneity in the hamster medullary thick ascending limb of Henle's loop.

Cellular heterogeneity was examined in the hamster medullary thick ascending limb (MAL) perfused in vitro by electrophysiological measurements with an intracellular microelectrode. Random measurements of fractional resistance of basolateral membrane (RfB) revealed two cell populations, high basolateral conductance (HBC) cells having RfB of 0.05 +/- 0.01 (n = 24) and low basolateral conductance (LBC) cells having RfB of 0.80 +/- 0.03 (n = 32). Basolateral membrane potentials (VB) were not different between HBC cells and LBC cells (-72.6 +/- 1.2, n = 43 vs. -70.0 +/- 1.2, n = 35). Addition of 2 mmol/l Ba2+ to the bath depolarized the basolateral membrane in the HBC cells from -70.4 +/- 3.2 to -20.9 +/- 5.9 mV (n = 8) but not in the LBC cells (from -74.4 +/- 1.9 to -72.0 +/- 2.1 mV). Increasing K+ or decreasing Cl- in the bathing solution caused marked positive deflection of VB in the HBC cells but little or no change in VB in the LBC cells. Elimination of Cl- from the lumen or addition of furosemide to the lumen enhanced the potential response of the HBC cells to basolateral application of Ba2+. Accordingly, with Ba2+ present in the bath, the potential response of the HBC cells to a decrease in bath Cl- concentration was enhanced. These observations suggest that a K+ conductance exists in the basolateral membrane of HBC cells in parallel with a Cl- conductance. The basolateral cell membrane of LBC cells also contains a Cl- conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Effects of glutaraldehyde fixation on renal tubular function. I. Preservation of vasopressin-stimulated water and urea pathways in rat papillary collecting duct.

Using the in vitro microperfusion technique on isolated rat papillary collecting duct (PCD), we examined whether the glutaraldehyde-fixation method can be also applied to the mammalian collecting duct for preservation of the vasopressin-stimulated water and urea transport. Arginine vasopressin (AVP) at 10(-9) mol/l increased diffusional water permeability (Pdw) from 101.9 +/- 10.76 to 283.3 +/- 16.67 X 10(-7) cm2 s-1 (n = 8, P less than 0.01) and urea permeability (Purea) from 30.3 +/- 2.24 to 83.5 +/- 7.80 X 10(-7) cm2 s-1 (n = 8, P less than 0.01). Both parameters remained elevated after fixation with 0.1 mol/l glutaraldehyde even in the absence of AVP, with the values being 265.0 +/- 14.47 and 74.5 +/- 7.15 X 10(-7) cm2 s-1, respectively. Glutaraldehyde fixation did not affect the basal levels of Pdw or Purea. Phloretin at 2.5 X 10(-4) mol/l decreased glutaraldehyde-fixed AVP-stimulated Purea from 79.0 +/- 7.96 to 29.7 +/- 3.66 X 10(-7) cm2 s-1 (n = 4, P less than 0.01) and from 73.2 +/- 7.05 to 38.7 +/- 3.53 X 10(-7) cm2 s-1 (n = 4, P less than 0.01) when the drug was added to the lumen or to the bath, respectively. Phloretin also decreased glutaraldehyde-fixed non-stimulated Purea by 25-40%. However, this drug did not affect glutaraldehyde-fixed Pdw. These findings indicate that the glutaraldehyde fixation method can be applied to mammalian collecting tubules for studying vasopressin stimulated Pdw and Purea. Purea fixed by glutaraldehyde is functionally flexible and may be distinct from the water pathway.

Aldehydes↗
PubMed1987

Effect of glutaraldehyde on renal tubular function. II. Selective inhibition of Cl- transport in the hamster thin ascending limb of Henle's loop.

In order to further characterize Cl- transport of the thin ascending limb of Henle's loop (TAL), we observed the effects of glutaraldehyde on Na+ and Cl- transport in hamster TAL perfused in vitro. We found that 0.1 mol/l glutaraldehyde added either to the lumen or to the bath caused a rapid irreversible reversal of the NaCl diffusion potential. This was mainly accounted for by an inhibition of Cl- permeability (10(-7) cm2 s-1) from 93.51 +/- 8.39 to 14.89 +/- 3.91 (P less than 0.01, n = 9). By contrast, Na+ permeability changed little from 34.18 +/- 3.27 to 26.56 +/- 2.74 (P less than 0.01, n = 6). Glutaraldehyde treatment abolished the halogen-permselectivity of the TAL as determined by the voltage deflection seen upon ionic substitution. Permeabilities for Cl-, Br-, I-, and SCN- relative to Na+ were changed from 3.16 +/- 0.20, 3.22 +/- 0.19, 2.97 +/- 0.26 and 4.36 +/- 0.36 to 0.38 +/- 0.07, 0.35 +/- 0.06, 0.36 +/- 0.07 and 0.58 +/- 0.05, respectively. The effect of glutaraldehyde on the NaCl diffusion potential was dose-dependent in the range from 10(-5) to 10(-1) M. The effect was reversible at concentrations lower than 10(-3) M. Glutaraldehyde did not affect the NaCl diffusion potential of the long-loop descending limb. These observations constitute additional evidence that the mechanism of Cl- transport across the TAL is different from that of Na+ transport. Glutaraldehyde might inhibit Cl- transport in the TAL by cross-linking amino acid residues of the proteins essential for halogen transport across this segment.

Aldehydes↗
PubMed1987

cDNA probes of individual genes of human rotavirus distinguish viral subgroups and serotypes.

The use of cDNA probes for detection of rotaviruses has been investigated using plasmids containing inserts specific for each of the eleven genes of human rotavirus strain Wa. In a dot-blot detection system in which radioactive DNA probes were hybridized to viral RNA extracted from cultivatable rotavirus strains, cDNAs of genes 7, 8, 10 and 11, were found to be the most reliable probes for detecting a range of rotavirus strains. Unexpectedly, rotaviruses could be distinguished with respect to subgroup and subtype specificities when cDNAs of genes 6 and 9, which encode the immunologically relevant proteins VP6 (group-specific antigen) and VP7 (type-specific antigen), were used as probe, even though the nucleic acid sequences of these genes are known to have a high degree of sequence homology.

Child, Preschool↗
PubMed1987

Genetic resistance to mammary tumorigenesis in a mouse strain with high murine mammary tumor virus expression.

Although II-TES mice release large amounts of murine mammary tumor virus (MMTV) in milk, they are resistant to mammary tumorigenesis. High mammary tumor incidence was observed in (BALB/ca X II-TES)F1 and (C57BL/6N X II-TES)F1, whereas no mammary tumors developed in BALB/ca X OZ-F)F1. Mammary tumors developed in 68% of (OZ-F X (OZ-F X II-TES)F1 and 45% of (II-TES X (OZ-F X II-TES)F1). These results suggest that the II-TES mouse carries a recessive gene for mammary tumor resistance which does not inhibit MMTV release, and two independent dominant mammary tumor promoting genes which are inhibited by the resistant gene.

Animals↗
PubMed1987

Construction of a new plasmid vector that can express cloned cDNA in all translational reading frames.

Construction of a bacterial expression vector, pSI4001, is described. The vector contains the lac promoter-operator and three sets of ribosome-binding sites (RBSs) tandemly arranged in all possible reading frames. cDNA can be directly cloned downstream from these translational start points in the fixed and proper orientation by using the method of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280-289]. The open reading frame of any cDNA inserted may be automatically aligned in phase with either of the three ATG start codons, thus enabling its expression with a maximum theoretical probability of unity. Fusion with the lacZ gene (coding for beta-galactosidase) has shown that at least two of the three translation initiation sites exhibit high expression capacities and the remaining one can also function at a lower but significant rate. We used the vector to construct a bovine pituitary cDNA library, from which clones coding for prolactin were detected by immunological screening with an efficiency as high as two in three clones. The construction with triple RBSs should also provide a unique experimental model to study the regulation of overlapping translations.

Animals↗
PubMed1987

Construction and characterization of plasmid and lambda phage vector systems for study of transcriptional control in Escherichia coli.

We constructed a family of lambda phage and plasmid vectors which facilitate cloning and quantitative analysis of transcriptional regulator in both single and multiple copies. Their expression system was modified from the ara-trp-lac fusion operon of plasmid pMC81 [Casadaban and Cohen, J. Mol. Biol. 138 (1980) 179-207], which is designed to assay both promoters and terminators with a single vehicle. To eliminate transcriptional and translational polar effects liable to occur in the original fusion operon upon insertion of a foreign nucleotide sequence, intracistronic Rho-dependent terminators, that are present within the trpB gene and distal to the cloning site were deleted, and DNA spacers containing stop codons were introduced immediately before and after the cloning site. In analysis of the cloned trp regulatory region, the lambda phage system faithfully reproduced the tight regulation by tryptophan characteristic to the natural trp operon on the E. coli chromosome, whereas the plasmid counterpart exhibited a substantially relaxed response. Comparative studies on the relative strengths of various promoters and terminators have further demonstrated that the lambda phage vector system permits accurate assays of exceptionally strong promoters like Ptrp and lambda pL without disturbing the bacterial growth, while being sensitive enough for detecting low-level transcription under the control of weak promoters or potent terminators. Cloning with the lambda phage vector can be greatly facilitated by transferring the target regulatory site precloned with the plasmid onto the phage genome through in vivo recombination.

Bacteriophage lambda↗
PubMed1987

Host control of plasmid replication: requirement for the sigma factor sigma 32 in transcription of mini-F replication initiator gene.

Replication of F factor or mini-F plasmid is strongly inhibited in the rpoH (htpR) mutants of Escherichia coli deficient in the sigma factor (sigma 32) known to be required for heat shock gene expression. Transcription of the mini-F repE gene encoding a replication initiator protein (E protein) was examined by operon fusion and by direct determination of repE mRNA. The synthesis rate and the level of repE mRNA were found to increase transiently upon temperature upshift (30 degrees C to 42 degrees C) in wild-type cells but to decrease rapidly in the rpoH mutants. Thus sigma 32 appeared to be directly involved in transcription of repE whose product, E protein, in turn activates DNA replication from the mini-F ori2 region. This scheme of host-controlled plasmid replication is further supported by the analysis of transcription in vitro: RNA synthesis can be initiated from the repE promoter by a minor form of RNA polymerase containing sigma 32 but not by the major polymerase containing the normal sigma factor sigma 70. The sigma 32-mediated transcription from the repE promoter is strongly inhibited by the E protein. We conclude that transcription of the mini-F repE gene is mediated by the host transcription factor sigma 32 and is negatively controlled by its own product.

Bacterial Proteins↗
PubMed1987

The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma.

Nineteen DNA samples that carry integrated hepatitis B virus (HBV) DNA were isolated from seven independent human hepatomas by molecular cloning, and their structures were determined. The results, combined with reported data, were analyzed so that one can obtain insights into the mechanisms of integration of this virus DNA and possible rearrangements that occur subsequently. The distribution of DNA junctions along the virus genome suggests that there are recombination-proficient regions. Thus, about half of the integrants were the Coh type, viz., one of their virus-cell DNA junctions fell within the so-called cohesive end region that lies between two 11-bp direct repeats (DR1 and DR2) in the virus genome where transcription and replication of the genome are initiated. All the integrated virus genomes were defective at least in one site around the cohesive end region, particularly within the X gene. The recombination-proficient regions are used not only for formation of virus-cell but also of virus-virus junctions. Neither virus nor cell DNA show unique sequences at the junctions, and targets for integration lie on many different chromosomes.

Base Sequence↗
PubMed1987

Point mutation in the S gene of hepatitis B virus for a d/y or w/r subtypic change in two blood donors carrying a surface antigen of compound subtype adyr or adwr.

Genomes of hepatitis B virus (HBV) were cloned from the plasma of a blood donor who carried subviral particles of three distinct subtypes in the following proportions: adr, 25%; ayr, 63%; and adyr, 12%. HBV DNA clones were classified into two groups based on a difference at only one nucleotide in the S gene. Two clones had A as nucleotide 365 that formed part of the codon for lysine as amino acid residue 122 and produced a surface antigen of subtype adr in transfected NIH 3T3 cells. The remaining four clones had G determining the codon for arginine and produced a surface antigen of subtype ayr in transfected cells. Similarly, HBV genomes were cloned from the plasma of an individual who carried subviral particles of subtypes adr (71%) and adwr (29%). Two clones had T and A as nucleotides 476 and 479, respectively. The other seven clones had C and G as the respective nucleotides. Based on a comparison with previously reported HBV genomes of various subtypes, the mutation of nucleotide 479, forming part of the codon for lysine or arginine as amino acid residue 160, was deduced to determine the w or r subtype, respectively. When NIH 3T3 cells were transfected separately with the genome of subtype adw or adr, derived from plasma containing a surface antigen of subtype adwr, and then cocultured, they produced subviral particles of either subtype adw or adr. When cells were transfected with the genomes of subtypes adw and adr simultaneously, however, subviral particles were produced that possessed w and r determinants on the selfsame particles. These results attributed the d/y or w/r subtypic change to a point mutation in the S gene and favored coinfection of hepatocytes with an HBV genome and its mutant as the mechanism of compound subtypes.

Base Sequence↗
PubMed1987

Free and integrated forms of hepatitis B virus DNA in human hepatocellular carcinoma cells (PLC/342) propagated in nude mice.

Primary hepatocellular carcinoma cells (PLC/342) propagated in nude mice produce hepatitis B surface antigen of subtype adr, as well as core particles containing viral DNA and DNA polymerase. Free and integrated forms of hepatitis B virus (HBV) DNA in the tumor were isolated by molecular cloning, and their nucleotide sequences were determined. Both of the two representative clones of free HBV DNA had the same genomic length (3,158 base pairs) and had two stop codons as well as two deletions in the envelope gene. None of the seven distinct clones of integrated HBV DNA possessed the entire viral genome. The integrated clone sequences had deletions and rearrangements, and only two clones possessed the envelope gene including the promoter and enhancer sequences. The C gene, which codes for core protein, was preserved in the two free clones and one of the integrated clones. The P gene, which codes for DNA polymerase, had deletions at two positions of 21 and 36 base pairs in both free clones, but was carried in toto by one of the integrated clones. The nucleotide sequences of the S genes of two free and four integrated clones, as well as their two inverted repeats, were compared. All of the eight sequences of the S gene possessed two nucleotide substitutions in common that were not displayed by any of the reported HBV genomes. The sequences differed from one another by only 1.2%. They differed, however, from 11 reported HBV genomes of subtype adr by 2.4%, from an ayr genome by 1.9%, from 2 adw genomes by 6.9%, and from 2 ayw genomes by 5.9%. These results indicate that all free and integrated HBV DNA species in the PLC/342 tumor cell evolved from a common progenitor. The free HBV DNA underwent nucleotide substitutions during several integration events, resulting in integrated HBV DNA copies that were similar in sequence but distinct from the reported HBV genomes.

Animals↗
PubMed1987

K transport in upper portion of descending limbs of long-loop nephron from hamster.

By use of the in vitro microperfusion technique, we investigated potassium transport in the upper portion of the descending limb of the long-loop nephron isolated from hamster kidney. The net potassium flux determined by ultramicro-flame photometry was -0.63 +/- 1.84 pmol X mm-1 X min-1, a value that was not significantly different from zero. The salt permeability for KCl was calculated from the amount of potassium entering the tubular lumen when the concentration of potassium in the bath was increased by approximately 5 mM. The value was 38.9 +/- 1.9 X 10(-5) cm X s-1. The bidirectional fluxes of 86Rb were measured as indices of potassium fluxes. Flux coefficients from lumen-to-bath and bath-to-lumen were 51.2 +/- 9.2 and 48.8 +/- 13.5 X 10(-5) cm X s-1, respectively. These values were not significantly different, confirming that there was no net flux for 86Rb. In another series of five experiments, the lumen-to-bath 86Rb flux coefficients were 69.4 +/- 13.2 X 10(-5) cm X s-1 in the absence of unlabeled Rb, and 70.2 +/- 13.9 X 10(-5) cm X s-1 in the presence of 5 mM unlabeled Rb. The lumen-to-bath 42K flux coefficient measured in the same series of animals was 85.3 +/- 10.5 X 10(-5) cm X s-1 (n = 10), a value that is slightly higher than, but not significantly different from, that of 86Rb. These data show that active potassium transport may exist in this segment and that the passive permeability for potassium is very high.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Profiles of water and solute transport along long-loop descending limb: analysis by mathematical model.

We simulated profiles of water and solute transport along the descending limb of the long-loop nephron by a mathematical model based on mass balance equations for water, sodium, potassium, and urea, using phenomenological coefficients reported for hamsters. We assumed that interstitial concentration of sodium, potassium, and urea increased linearly along the descending limb from 150 to 350, from 5 to 50, and from 5 to 300 mM, respectively. Under this condition an increase in osmolality at the end-descending limb was mainly accounted for by the absorption of water. Considerable amounts of potassium and urea were secreted along the descending limb. Sodium was reabsorbed rather than secreted along the descending limb by both diffusion and solvent drag. The secreted amounts of urea and potassium were comparable to those observed by micropuncture studies. The sodium concentration in the lumen was higher than in the interstitium, with the transmural sodium gradient being 15 meq/liter at the hairpin turn. The potassium mass flow rate at the end-descending limb increased by 2.4 times. Large variations in potassium concentration of the delivered fluid scarcely changed the potassium mass flow rate at the end-descending limb. The secretion of urea and potassium and the reabsorption of sodium were increased as a function of delivered flow rate. An increase in corticomedullary urea gradient decreased the net potassium secretion along the descending limb. When the transport parameters for rabbits were used, both reabsorption of sodium and addition of urea were decreased, but a similar amount of potassium was secreted. These analyses indicate that the mathematical model that takes the species difference and internephron heterogeneity into consideration is useful in illustrating the transport processes along the descending limb of Henle's loop under various physiological and pathophysiological conditions.

Biological Transport, Active↗