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Biomedical subjects

M Imai

Publications and source records attributed to M Imai.

At least 505 records · Page 28Linked to original sources
PubMed1983

Three-site sandwich radioimmunoassay with monoclonal antibodies for a sensitive determination of human alpha-fetoprotein.

Utilizing monoclonal antibodies against human alpha-fetoprotein, 3 distinct antigenic determinants were identified. These antigenic determinants, provisionally designated a, b and c, were arranged in such a manner that the binding of one determinant with the corresponding antibody did not inhibit, or only barely inhibited the binding of antibodies directed to the other 2 determinants. Monoclonal antibodies with 3 different specificities were, therefore, applied to develop a sandwich-type solid-phase radioimmunoassay of the antigen in which wells were coated with anti-a, and radiolabeled anti-b together with radiolabeled anti-c was employed to detect the bound antigen. The 3-site sandwich radioimmunoassay involving 3 different determinants gave a higher sensitivity than 2-site assays in which only anti-b or anti-c was employed as a radiolabeled reagent, because the radioactivity of the 2 labeled antibodies was added on the antigen bound to immobilized anti-a.

Antibodies, Monoclonal↗
PubMed1983

Biotransformation of nitric oxide, nitrite and nitrate.

Biotransformation of NO, nitrite and nitrate was investigated in rats and mice in a 15NO inhalation experiment and intraperitoneal injection experiments of 15N-nitrite and 15N-nitrate, and the following results were obtained: (1) Rats were forced to inhale 15NO (145 ppm, 123 minutes) or were given an intraperitoneal injection of 15N-nitrite (2 mg animal-1 as 15N) or 15N-nitrate (2mg animal-1 as 15N), and determination of 15N recovery in urine was made up to 48 h later. The results were 55, 53 and 78% of the inhaled or injected 15N, respectively. (2) 15N-nitrate in the urine was converted into a 6-nitro derivative of 3,4-xylenol and its identification and quantitative determination were made by the GC-MS method. As to 15N-urea in the urine, identification and quantitative determination were made by the urease method. 15N was present in the urine of rats after 15NO inhalation in the form of NO3- and urea. 75 and 24% respectively. In the urine of rats injected with 15N-nitrite, about 20% of unidentified 15N-compounds not discovered in the inhalation experiment was found. The content of 15N-urea in the urine after injection with 15N-nitrate was lower than that after injection with 15N-nitrite. (3) When 15N-nitrite (0.617 mg animal-1 as 15N) was injected intraperitoneally in mice, 60.7, 7.8 and 0.3% of the injected 15N were found in the urine, feces and exhaled gas (NO, NO2 and NH3 in the gas were caught) up to 48 h after injection respectively, and 1.6% was found in the body 48 h after injection, but the remaining 30% of 15N could not be recovered.

Animals↗
PubMed1983

A pitfall in two-site sandwich 'one-step' immunoassay with monoclonal antibodies for the determination of human alpha-fetoprotein.

Utilizing monoclonal antibodies directed to 2 distinct antigenic determinants of the human alpha-fetoprotein (AFP), Uotila, Ruoslahti and Engvall developed the 2-site sandwich immunoassay. They found that due to the different specificities of monoclonal antibodies, 2 antigen-antibody reactions, AFP with immobilized antibody and AFP with labeled antibody, could be accomplished in a single step. We have found, however, that above a certain concentration, AFP detectable by their method decreased because the labeled antibody tended to bind with AFP that failed to react with the immobilized antibody. Consequently, 2 different AFP concentrations produced the same result, and an extremely high, still clinically expectable concentration gave a false negative result. Such an inhibition in high AFP concentrations was not observed in the conventional '2-step' immunoassay within the range of concentrations tested (0.1 ng-3 mg/ml). On the basis of these observations, the 2-site '1-step' immunoassay for AFP would have to be applied on multiple dilutions of the serum to avoid an erroneous interpretation of the results.

Animals↗
PubMed1983

Molecular cloning of double-stranded RNA virus genomes.

Genome double-stranded RNAs isolated from purified human reovirus (serotype 3) and rotavirus (Wa strain) were modified at the 3' termini by addition of oligo(C) approximately 15 with T4 RNA ligase. These RNAs were transcribed into cDNA by oligo(dG)-primed reverse transcriptase and cloned after insertion into pBR322 at the Pst I site. Hybridization of plasmid-transformed Escherichia coli RR1 colonies with 32P-labeled viral genome RNAs demonstrated the presence of DNA clones representative of each of the 10 reovirus RNAs and 10 of the 11 constituent segments of the rotavirus genome. Analyses of the size and terminal nucleotide sequences of insert DNAs indicated that some clones contained a full-length copy of the virus genome segment. The complete nucleotide sequence of rotavirus genome segment 11 double-stranded RNA was obtained by using this procedure. It provides a general method for cloning double-stranded RNAs and also nonpolyadenylylated single-stranded RNAs.

Amino Acid Sequence↗
PubMed1983

Capped and conserved terminal structures in human rotavirus genome double-stranded RNA segments.

Both 3'- and 5'-terminal structures of human rotavirus genome double-stranded RNA segments were determined. RNAs were labeled at the 3'-termini with [32P]pCp by incubation with RNA ligase and at the 5'-termini with [32P]phosphate by polynucleotide kinase or, in the case of 5' caps, with 3H by chemical modification with [3H]NaBH4. Examination of radiolabeled termini released by digestion with several base-specific RNases revealed that rotavirus RNA segments are base paired end-to-end and contain the same terminal structures: (formula; see text)

Base Sequence↗
PubMed1983

Sodium chloride and water transport in the renal distal tubule of the rainbow trout.

Isolated segments of the renal tubules from the freshwater trout, Salmo gairdneri, were perfused in vitro to characterize ion and water transport. The distal tubule showed a transepithelial voltage (Vt) positive in the lumen (+17.8 +/- 1.4 mV). Furosemide added to the lumen and Na cyanide and ouabain added to the bath reduced the lumen-positive Vt of the distal tubule. Removal of either Cl- or Na+ from both perfusate and bathing medium abolished the lumen-positive Vt. When the distal tubule was perfused and bathed with isosmotic solution, net water flux (Jv) was nearly zero. Jv and hydraulic conductivity remained low when the osmolality of the bathing fluid was increased with raffinose. Neurohypophysial hormones added to the bath showed no effect. Chloride efflux (lumen to bath, 171.1 +/- 17.1 peq x mm-1. min-1) was significantly higher than chloride influx (bath to lumen, 105.6 +/- 12.3 peq x mm-1 x min-1), suggesting that net chloride reabsorption exists. These results suggest that in the freshwater trout, which lack the loop of Henle, the distal tubule acts as a diluting segment. The presence of sodium, in addition to chloride, is required to generate the lumen-positive Vt in the distal tubule.

Animals↗
PubMed1983

Flow-dependent water permeability of the rabbit descending limb of Henle's loop.

Because completely opposite results have been reported on the water permeability of the rabbit descending limbs of Henle's loop (DLH), we rigorously examined water permeability of the upper portion of the descending limb of the rabbit long-looped nephron. Even when the double-cannulation method was used in an attempt to reduce the resistance of tubular outflow, the collected fluid-to-perfusate inulin ratio was equal to or very close to the bathing fluid-to-perfusate osmolality ratio, indicating that osmotic equilibration occurred along the tubule by absorption of water. When perfusion rates were controlled by varying the height of the fluid reservoir connected to the perfusion pipette, osmotic (Pf) as well as diffusional (Pdw) water permeability was shown to be correlated with perfusion rate and/or perfusion pressure. Pf and Pdw at zero perfusion rate as determined from the values of the intercept of regression lines were 253 X 10(-3) and 4.54 X 10(-3) cm X s-1, respectively. The maximal values for Pf and Pdw were 737-1,098 X 10(-3) and 18.3 X 10(-3) cm X s-1, respectively. By changing the resistance to perfusion at the tubular outflow, it was shown that changes in Pf paralleled changes in perfusion rate rather than changes in perfusion pressure. Under stop-flow conditions the luminal fluid volume rapidly decreased after the osmolality of the bathing fluid was increased, suggesting that the segment is highly permeable to water even at zero flow rate. Reflection coefficients for urea and NaCl were 1.01 and 0.82, respectively. These data support the view that this segment is highly permeable to water and that increases in osmolality along the DLH in vivo may be accounted for mainly by abstraction of water rather than addition of solutes.

Animals↗
PubMed1983

Immunochemical structure of hepatitis B e antigen in the serum.

Hepatitis B e antigen (HBeAg) constitutes the nucleocapsid of hepatitis B virus (HBV) and occurs in association with plasma proteins, particularly with IgG, in the serum of persons infected with the virus. A polypeptide with an approximate m.w. of 15,500 (P15.5) is obtained either from HBeAg in the serum or from the nucleocapsid of HBV. P15.5 preparations from serum and virus resembled closely each other in the amino acid composition. The C-terminus amino acid sequence of P15.5 from serum was determined to be -Thr-Thr-Val-Val, whereas that from the virus ended with -Thr-Thr. The same sequence of four amino acid residues was found on the gene coding for the nucleopeptide of hepatitis B virus with a molecular size of 19,000 daltons (P19). P15.5 identified on the nucleotide sequence of P19 was composed of 149 amino acid residues with a calculated molecular size of 16,770 daltons. The gene coding for P19 had two -Asp-Pro-connections. By splitting these connections in P19 and P15.5 preparations with formic acid, smaller polypeptides were obtained with sizes predicted from the nucleotide sequence and with the N-terminus amino acid of proline as expected. One of two monoclonal antibodies raised against the core of Dane particles (HBcAg) bound with P15.5 preparations purified from serum and HBV. The IgG fraction from a human serum containing antibodies to HBcAg but not to HBeAg bound with P15.5 also. On the basis of the results obtained, the IgG molecules associated with P15.5 in the serum of persons infected with HBV may well represent the antibodies against HBcAg with limited specificities.

Amino Acid Sequence↗
PubMed1983

Quantitative analysis of mouse mammary tumor virus in milk in two sublines of RIII/AnOk mice with low and high mammary tumor incidence.

Mammary tumors have ceased to develop in descendants of one female of the RIII/AnOk mouse strain which was brought from the Mouse Colony of Okayama University Medical School in 1975, while descendants of another female of this strain have maintained a high tumor incidence. The former and latter descendants were separated out as RIII/AnOk/2 and RIII/AnOk/1 sublines, respectively. The amount of mouse mammary tumor virus (MMTV) in the milk of individual females in these two sublines was determined by means of a sensitive enzyme immunoassay. No MMTV antigens were detected in 59 milk samples of RIII/AnOk/2 females collected during the 1st to 7th lactation periods. On the other hand, all the milk samples of RIII/AnOk/1 females contained MMTV antigens with concentrations ranging from 9 to 400 micrograms/ml. When RIII/AnOk/2 mice were foster-nursed by RIII/AnOk/1 mothers, the infected RIII/AnOk/2 females produced MMTV in the milk to almost the same degree as did the RIII/AnOk/1 females. No genetic differences between the two sublines were observed by the use of reciprocal skin grafting, tumor transplantation, and analysis of biochemical genetic markers. These results indicate that the arrest of mammary tumor development in the RIII/AnOk/2 subline was due to the disappearance of MMTV in the milk.

Animals↗
PubMed1982

Function of transcription termination factor rho in a model transcription system using synthetic deoxyribonucleic acid as template.

The function of a transcription termination factor, rho, has been studied by using several synthetic DNAs with simple repetitive base sequences as templates for transcription. rho actually exhibits various effects on transcription depending on the base sequence of the template: (1) rho terminates poly(A) synthesis with poly(dA) x poly(dT), poly(dT), or oligo(dT), leading to release of RNA from RNA polymerase. rho also inhibits the synthesis of other homoribopolymers such as poly(U) directed by poly(dA) x poly(dT) and poly(C) and poly(I) directed by poly(dG) x poly(dC), presumably by a similar mechanism. (2) rho inhibits the synthesis of another homoribopolymer, poly(G), directed by poly(dG) x poly(dC) at the step of initiation rather than propagation of transcription. (3) rho stimulates rather than inhibits the synthesis of poly(A-C) and poly(G-U) directed by poly[d(A-C)] x poly[d(G-T)], presumably by enhancing the dissociation of transcription complexes. (4) rho has no influence on the synthesis of poly(A-U) and poly(G-C) directed by poly[d(A-T)] and poly[d(G-C)], respectively. In the first case, but not otherwise, the effect of rho is coupled with its RNA-dependent nucleosidetriphosphate phosphohydrolase activity, as is rho-mediated transcription termination on natural templates. The implication of these results is discussed in reference to the current view that rho acts on transcription complexes that have ceased elongation and causes release of RNA in an energy-requiring reaction.

Base Sequence↗
PubMed1982

E. coli initiator tRNA analogs with different nucleotides in the discriminator base position.

The effect of base changes at the fourth position from the 3'-terminus of Escherichia coli initiator tRNAMet has been studied to test the 'discriminator hypothesis' which proposed that the nucleotide in this position might have a role in the specificity of the aminoacylation reaction. E. coli initiator tRNA lacking the 3'-terminal tetranucleotide was prepared by partial digestion with S1 nuclease. To construct tRNA analogs with different bases in the fourth position this truncated tRNA was joined by RNA ligase to each of four chemically synthesized 2',3'-ethoxy-methylidene tetranucleotides pACCA(em), pCCCA(em), pGCCA(em), and pUCCA(em). In vitro aminoacylation studies showed that all four molecules accepted methionine, albeit with different Vmax values.

Base Sequence↗