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Biomedical subjects

K Nitta

Publications and source records attributed to K Nitta.

At least 343 records · Page 19Linked to original sources
PubMed1981

Effect of heat shock on protein synthesis by normal and malignant human lung cells in tissue culture.

Heat shock proteins were found in cultured human lung cells by sodium dodecyl sulfate:gel electrophoresis and autoradiography using [35S]methionine. The synthesis of a M.W. 70,000 protein was markedly stimulated in normal, malignant, and SV40-transformed cells, and that of M.W. 90,000 and M.W. 100,000 proteins was stimulated only in malignant and SV40-transformed cells after heat shock treatment. In contrast, the synthesis of M.W. 200,000 and M.W. 250,000 proteins observed in the unheated normal cells was diminished after the same procedure. The heat shock proteins were induced within a given range of temperature and duration of treatment, the conditions for normal and malignant cells being clearly different, i.e., optima of 43 degrees for 1 hr in the normal cells and 41 degrees for 1 hr in the malignant cells. The results suggest that the analysis of heat shock protein is very useful for identifying the differential heat susceptibilities of normal and malignant cells and for elucidating their bases and mechanisms.

Cells, Cultured↗
PubMed1980

Intramolecular perturbation of tryptophans induced by the protonation of ionizable groups in goat alpha-lactalbumin.

Goat alpha-lactalbumin shows two septral changes at acid pH. One of these corresponds to the acid denaturation, while the other has been regarded as a result of the intramolecular perturbation of tryptophans induced by the protonation of ionizable groups in the native molecule. In order to distinguish between these spectral changes and to investigate the perturbation caused by the ionizable groups, the absorption changes with a change in pH have been followed by the stopped-flow pH-jump method. Only the acid denaturation can be observed kinetically while the perturbation is too rapid to follow by the kinetic method. Extrapolations of the time-dependent absorption changes to zero time in the unfolding and refolding pH jumps give the apparent titration curves of the ionizable groups that participate in the perturbations in the native and in the acid-denatured molecule. The perturbation of the native protein is induced by the protonation of at least two ionizable groups, while the acid-denatured molecule does not show the perturbation because of the loss of the unique spatial arrangement of the ionizable groups around tryptophans. The results of circular dichroism measurements and the comparison with known results of the charge perturbation of lysozyme are also discussed.

Animals↗
PubMed1980

A cytotoxicity assay using 3H-uridine and emulsions scintillator.

In immunological studies, cytotoxicity assay are extremely important. The assay method was modified so that the radioactivity of beta-decaying isotopes in a large volume of aqueous sample could be counted. Through tests of various scintillators, it was found that emulsion scintillators had a higher counting efficiency than toluene- or dioxane-based scintillators. The results obtained by this method using an emulsion scintillator agreed well with those obtained by the 51Cr release method. The advantages of the emulsion scintillator method are as follows: (a) the activity of a beta-decaying isotope can be counted in a large volume of aqueous sample, (b) a long half-life beta-decaying isotope can be used, (c) the effects of free labeled materials can be eliminated by simple procedures, and (d) no gamma counter is needed.

Animals↗
PubMed1979

Impairment of the catalytic activity of Escherichia coli ribonucleic acid polymerase by a unique reaction of 4-chloro-7-nitrobenzofurazan.

Escherichia coli RNA polymerase loses 55-65% of its catalytic activity on reaction with Nbf-Cl (4-choro-7-nitrobenzofurazan). This partial inactivation was shown to be the result of specific impairment of RNA-chain elongation, since initiation of RNA chains was not altered after treatment with Nbf-Cl. The site of reaction was shown to be a unique thiol on the beta-subunit. This thiol is not accessible to reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). No protection of the enzyme against reaction with Nbf-Cl could be obtained with the inhibitor rifamycin nor with calf thymus DNA, GTP or 1,10-phenanthroline, indicating that the unique thiol is probably not within the active site. The specific impairment of RNA-chain elongation thus appears to be the result of a local conformational change which leaves chain initiation unimpaired. Changes observed in the tryptophan fluorescence spectrum of the enzyme or reaction with Nbf-Cl are consistent with formation of a Meisenheimer complex of the reagent with a nucleophilic group on the enzyme near the reactive thiol. It is proposed that formation of such a complex and a subsequent conformational change renders this thiol unusually susceptible to reaction with Nbf-Cl.

4-Chloro-7-nitrobenzofurazan↗
PubMed1979

Anomalous reaction of 4-chloro-7-nitrobenzofurazan with thiol compounds.

The kinetics of reaction of 4-chloro-7-nitrobenzofurazan with thiol groups at pH values above 5 cannot be accounted for solely on the basis of formation of a single product, the 4-thio derivative. Spectroscopic observations indicate that, in addition to the 4-thio derivative, at least two other products are formed. One of these, referred to as P1, is most likely a reversible complex of thiol compound and 4-chloro-7-nitrobenzofurazan of the Meisenheimer type. The other product, P2, which forms primarily when thiol compound is in a large excess, does not appear to result from direct reaction of thiol group and 4-chloro-7-nitrobenzofurazan, but may be a reaction of product P1 and thiol compound. The coloured product, P2, will react further with proteins, such as bovine serum albumin and Escherichia coli RNA polymerase. This reaction irreversibly destroys the catalytic activity of RNA polymerase. The implications of these observations for utilization of 4-chloro-7-nitrobenzofurazan as a protein-modifying agent are discussed.

4-Chloro-7-nitrobenzofurazan↗
PubMed1978

Detection and characterization of the intermediate on the folding pathway of human alpha-lactalbumin.

To discuss the relation between the folding mechanism and the chemical structure of proteins, the reversible unfolding reactions of human alpha-lactalbumin by acidification and by guanidine hydrochloride at 25 degrees C are studied by means of circular dichroism, difference spectra and pH-jump measurements and are compared with those for bovine alpha-lactalbumin. As shown previously for bovine alpha-lactalbumin, the folding process at neutral pH is not explained by a simple two-state mechanism but involves an intermediate form that has the same amount of helical structures as the native form. The transition between the intermediate and the fully denatured states is too rapid to be measured and corresponds to the helix-coil transition of the backbone. One of the differences of human alpha-lactalbumin from the bovine protein is the remarkable stability of the intermediate at neutral pH, which can be explained by differences in the primary chemical structure. Another difference is the existence at acid pH of an additional helical form, which is more helical than the native form. The transition from this to the intermediate or to the fully denatured one also is shown to resemble the helix-coil transition. The following folding scheme of human alpha-lactalbumin is proposed: formula: (see text). Here N is the native form, and the intermediate is a macroscopic state distributed around the state A3 at neutral pH, while the distribution in the acid and fully denautured states shifts toward Am and A-n, respectively.

Circular Dichroism↗
PubMed1978

Ultrasono-cardiographic diagnosis of the prolapsed mitral valve.

The characteristic ultrasono-cardiographic (UCG) findings of prolapsed mitral valve were studied in 9 cases by means of ultrasonocardiotomography (UCT). The changes in degree and shape of the prolapsed valve were examined and the leaflet involved was identified. Of the 9 cases, 5 had prolapse of anterior, 3 had that of posterior leaflet and 1 had both. In all cases with midsystolic click and late systolic murmur or pansystolic murmur with late systolic accentuation, the prolapse of anterior leaflet was observed on UCT, and though UCG disclosed midsystolic buckling it was difficult to determine the exact timing of the prolapse by UCG alone. In the cases with rheumatic mitral regurgitation, the prolapse of posterior leaflet and the ballooning of anterior leaflet could easily be found on UCT, but the detection of the above-mentioned 2 findings was difficult by UCG alone. Three of the 9 cases were found to have prolapse of posterior leaflet by left ventriculography, whereas these 3 had prolapse of anterior leaflet on UCT. Hence, the diagnosis of prolapsed mitral valve based on the cineangiography may require re-evaluation.

Adolescent↗
PubMed1978

Susceptibilities of normal and malignant human lung cells in culture to the cytocidal action of antitumor agents.

Cultured normal and malignant human lung cells were examined for their different sensitivities to the cytocidal action of nine antitumor and three non-antitumor agents. The malignant cells were killed preferentially at lower concentrations and in a shorter time with each antitumor agent, Adriamycin, neocarzinostatin, bleomycin, actinomycin D, mitomycin C, carboquone, 1-beta-D-arabinofuranosylcytosine, 5-fluorouracil, and 5-fluorodeoxyuridine. Among these, Adriamycin, 5-fluorouracil, and 5-fluoro-deoxyuridine exhibited a higher differential lethal action on the malignant cells than did the other antitumor agents. The two cell lines exhibited little difference in cytolytic sensitivity to non-antitumor cytotoxic agents such as amphotericin B, G-strophanthin, and alph-amanitin.

Antineoplastic Agents↗
PubMed1978

Dependence on chain length of antitumor activity of (1 leads to 3)-beta-D-glucan from Alcaligenes faecalis var. myxogenes, IFO 13140, and its acid-degraded products.

The well-defined (1 leads to 3)-beta-D-glucan with (DPn) 540, produced by cultivation of Alcaligenes faecalis var. Myxogenes (IFO 13140), a mutant of a soil bacterium, had marked inhibitory activity against the s.c.-implanted Sarcoma 180 at 5 to 50 mg/kg for 10 days. It also exhibited very high activity in doses of 60 and 100 mg/kg i.p. by a single injection at 7 days after the initial s.c. transplantation of Sarcoma 180. The inhibition ratios observed with Ehrlich carcinoma, NTF (Nakahara-Tokuzen-Fukuoka) reticulum cell sarcoma, and CCM adenocarcinoma were somewhat less but were still significant. On the other hand the treatment failed to inhibit the growth of ascites Sarcoma 180 or to induce prolongation of life span. The mechanism of action of this glucan was considered to be host mediated because of a lack of effect in vitro and also because of the effectiveness of pretreatment of animals by injection before transplantation of a tumor. The results from the bioassay study of the low-molecular-weight (1 leads to 3)-beta-D-glucans prepared from the glucan with number-average degrees of polymerization (DPn) 540 by hydrolysis with formic acid or sulfuric acid showed that the glucans with DPn greater than 50 inhibited the growth of Sarcoma 180 implanted s.c. in mice, whereas the result of the schedule-dependent effect was obtained in the pretreatment of animals by the glucan with DPn 50. By i.v. administration, even the glucan with DPn 16 showed a strong antitumor effect comparable to that of the glucan with DPn 540.

Alcaligenes↗
PubMed1977

Thermodynamic characterization of partially denatured states in the denaturation process of bovine alpha-lactalbumin by inorganic denaturants.

In an attempt to understand the specific effect of inorganic protein denaturants, lithium cation and perchlorate anion, upon the molecular conformation of bovine alpha-lactalbumin and to characterize the denatured states of the protein and the denaturation processes, themodynamic studies on the reversible unfolding of the protein in the presence of lithium chloride, lithium perchlorate and sodium perchlorate were made by means of circular dichroism and ultraviolet absorption measurements. The denaturation reaction caused by lithium chloride was found to take place in a three-state type, while that caused by the two perchlorates in a two-state type. The latter produces the same denatured state as the acid does on the protein, the state where the helical structures remain unchanged. The former produces two kinds of the denatured state, one being a less unfolded state than the acid denatured one and the other a fully unfolded state which is identical with the finally denatured state induced by organic denaturants such as guanidinium chloride, guanidinium thiocyanate and urea.

Animals↗
PubMed1977

Equilibrium and kinetics of the unfolding of alpha-lactalbumin by guanidine hydrochloride (IV): dependence of the N equilibrium A transconformation on the temperature.

The reversible unfolding from the native (N) state to the acid (A) state of alpha-lactalbumin by guanidine-HCl (0.8-2.0 M) was studied at 10-35 degrees C by means of difference spectra and pH-jump measurements. At each temperature, all points plotted as the logarithmic equilibrium constant log KNA of the N equilibrium A process against pH could fall on a curve independent of the denaturant concentration by shifting each point along the log KNA axis, where the shift factor f did not depend on temperature. Moreover, by shifting the points at each temperature along the log (KNA/f) axis, a master curve, independent of both temperature and the denaturant concentration, could be obtained for the pH-dependence of log KNA. From the dependence of the logarithmic rate constants on pH, master curves independent of both temperature and the denaturant concentration could also be made for the N leads to A and the A leads to A processes, where A mean the activated state. The results show the two-state character of the N equilibrium A process. The enthalpy changes and the differences in heat capacity for the N equilibrium A, N equilibrium A and A equilibrium A processes were determined from the accurate measurements of temperature dependence of the unfolding at pH 4.3 and 1.0 M guanidine-HCl. The results show that the disruption of hydrophobic interaction is caused mainly in the A leads to A process, while most of the changes in the pK values of the ionizing groups are caused in the N leads to A process.

Guanidines↗