Cold adaptation of the thermophilic enzyme 3-isopropylmalate dehydrogenase.

We have performed random mutagenesis coupled with selection to isolate mutant enzymes with high catalytic activities at low temperature from thermophilic 3-isopropylmalate dehydrogenase (IPMDH) originally isolated from Thermus thermophilus. Five cold-adapted mutant IPMDHs with single-amino-acid substitutions were obtained and analyzed. Kinetic analysis revealed that there are two types of cold-adapted mutant IPMDH: k(cat)-improved (improved in k(cat)) and K(m)-improved (improved in k(cat)/K(m)) types. To determine the mechanisms of cold adaptation of these mutants, thermodynamic parameters were estimated and compared with those of the Escherichia coli wild-type IPMDH. The Delta G(m) values for Michaelis intermediate formation of the k(cat)-improved-type enzymes were larger than that of the T. thermophilus wild-type IPMDH and similar to that of the E. coli wild-type IPMDH. The Delta G(m) values of K(m)-improved-type enzymes were smaller than that of the T. thermophilus wild-type IPMDH. Fitting of NAD(+) binding was improved in the K(m)-improved-type enzymes. The two types of cold-adapted mutants employed one of the two strategies of E. coli wild-type IPMDH: relative destabilization of the Michaelis complex in k(cat)-improved-type, and destabilization of the rate-limiting step in K(m)-improved type mutants. Some cold-adapted mutant IPMDHs retained thermostability similar to that of the T. thermophilus wild-type IPMDH.

Clinical experience of a vacuum-assisted nonroller extra-corporeal circulation system.

We designed a nonroller extra-corporeal circulation system (NRECC) to minimize space requirements and to improve the safety and ease of cardiopulmonary bypass. The NRECC includes a circuit, a centrifugal pump, and a vacuum regulator. The vacuum regulator has five channels, each of which can control negative pressure independently. Negative pressure is applied to suctions, a vent, and a venous reservoir. Auto Vent-SV and one-way valves were placed between the suctions/vent and the reservoir. The total space required for the NRECC with a heat exchanger was 0.49 m2, which is much smaller than that of our current roller pump system. The maximum volume through the suction/vent was 2.5 L/min at -100 mmHg measured in a simulated system. We used the NRECC for six CABG cases and compared results with those of five CABG cases managed by traditional roller pump extra-corporeal circulation (RECC). The two systems were compared in terms of set-up time and stability/accuracy of the suction/vent. The set-up time of the NRECC (5.3 +/- 1.9 min) was significantly shorter than that of RECC (29 +/- 3.4 min) (p < .01). Stable and accurate blood flow were achieved with the NRECC operating between -15 mmHg and -25 mmHg. In conclusion, NRECC can run a bypass easily and safely and has the benefit of reducing space requirements and shortening set-up time.

[Study on the most appropriate time for Romurtide administration (Nopia) in radiotherapy patients].

The most appropriate time for administration was studied based on examination of the total number of leukocytes, differential white blood count, and the number of platelets in 30 cases in which 200 micrograms Romurtide (Nopia) was injected subcutaneously on the day of radiotherapy, 5 times a week, for 2 weeks, totally 10 times because leukopenia was caused during treatment with radiotherapy. It was recognized that the number of leukocytes, mainly neutrophils, increased from 1 week after starting administration of Romurtide to after the completion of administration (2 weeks after administration), except 1 week after completion of administration. The increasing effect in the number of platelets was not recognized, and it had decreased from at the completion of administration to 1 week after the completion of administration of Romurtide. The rate of completion of radiotherapy was 100% without any serious adverse events, but there were 4 cases in which fever was observed. Therefore, it is not very favorable to administer Romurtide immediately after radiation, taking account of the treatment period of radiotherapy. It is also considered that it would be necessary to start radiation after sufficient recovery of the number of leukocytes or increase in their number by using G-CSF preparation in cases in which the leukocyte count has fallen since starting radiation due to the influence of the preceding chemotherapy.

High-performance liquid chromatographic determination of primary aromatic amines in urine after derivatization to an azo dye with 2-aminoanthracene.

A sensitive HPLC method for the determination of primary aromatic amines (anilino compounds) is described. Samples were prepared by derivatization of the substrate to an azo dye with 2-aminoanthracene (2-AA). 2-AA was found to react with the diazonium salts prepared from substituted anilines such as 4-halo, -sulfonyl, -carboxyl, -nitro or -acetyl derivatives, but not 4-hydroxy or -alkyl derivatives. In this work, three model compounds [sulfanilamide, 4-aminobenzoyl-beta-alanine and 4-aminobenzoic acid (PABA)] were used to test the linearity and accuracy of the method. Chromatographic separation was carried out using a reversed-phase column (ODS) and ultraviolet detection at 279 nm. Good linearity for the three compounds was found within the range 50-2000 ng/ml. The intra-day coefficient of variation for the three compounds (at 100, 500, 1000 ng/ml) was below 10%. Using this method, the urinary excretion of PABA and its metabolites was studied after oral administration of PABA to rats.

Enhancement of anti-inflammatory effects of biphenylylacetic acid by its incorporation into lipid microspheres.

Using lipid microspheres (LM), average diameter 0.2 micron, and containing methyl and ethyl esters of biphenylylacetic acid (BPAA) in the carrageenan paw oedema tests in rats, it was found that their anti-inflammatory activities were enhanced 3 to 8 times over that of free BPAA. By electron microscopy, LM were seen to be taken up into the endothelial cells of the blood vessels and macrophages at the inflamed sites. In a study using dogs, after an intravenous injection of BPAA-methyl ester incorporated into LM (lipo-BPAA-Me), lipo-BPAA-Me rapidly disappeared from the blood and the BPAA serum level was gradually elevated. These results, together with previous findings, suggest that part of lipo-BPAA-Me was immediately transferred to the inflamed site and taken up by prostaglandin (PG)-producing cells as macrophages. It is considered that the anti-inflammatory effects of BPAA are enhanced by incorporating it into LM.

[Studies on the effect of lentinan on human immune system. II. In vivo effect on NK activity, MLR induced killer activity and PHA induced blastic response of lymphocytes in cancer patients].

Immunological activities of lymphocytes were studied in patients with cancer of digestive organs and mamma after administration of lentinan. When 11 cancer patients received lentinan i.v. 2 mg, NK activity of lymphocytes was increased in 4 patients on the following day. With the same condition, MLR induced killer activity against Raji cell of lymphocytes was increased in 3/7 patients and PHA response was also increased in 3/7 patients. These activities were returned to values before the administration of lentinan. Then each activity was examined after administration of lentinan twice a week. NK activity, MLR induced killer activity and PHA response were increased in 4/15, 8/12 and 5/11 patients, respectively.

Changes of metabolism and substrate-binding spectrum of emorfazone between immature and mature guinea-pigs.

Km values for oxidative metabolism at two types of carbon atoms adjacent to nitrogen and oxygen of the morpholino moiety of emorfazone (alpha- and beta-oxygenation) were 1.2 X 10(-2) and 3.0 X 10(-4)M in immature male guinea-pigs, and 8.6 X 10(-3) and 2.5 X 10(-4) M in mature male guinea-pigs, respectively. Vmax values of alpha- and beta-oxygenation were 0.409 and 0.229 mumol/4 nmol cytochrome P-450 in immature male guinea-pigs, and 0.391 and 0.061 mumol/4 nmol cytochrome P-450 in mature male guinea-pigs, respectively. Substrate-binding difference spectrum was composed of type I and reverse type I(RI) spectra both in immature and mature animals, but the spectrum pattern was different. The type RI spectrum was predominant in immature animals, but the spectral pattern in mature animals gradually changed from type RI to type I with increase in substrate concentration. alpha- and beta-Oxygenation may be attributed to two kinds of cytochrome P-450 with different affinities, and the proportions of these cytochrome P-450 species and their spinstate change with maturation of guinea-pigs.