Assay of alpha-cysteine proteinase inhibitor in serum or plasma

A new proteinase inhibitor has recently been found in human serum or plasma which specifically inhibits cysteine proteinases such as ficin, papain, bromelain and cathepsin B. However, serum contains alpha 2-macroglobulin which also inhibits these cysteine proteinases and, consequently, interferes with the assay of the new alpha-cysteine proteinase inhibitor. Therefore, assay of the inhibitor in serum has not been established previously. In the present method, the alpha 2-macroglobulin is inactivated by preincubating the serum in methylamine solution at 55 degrees C, while the alpha-cysteine proteinase inhibitor retains its activity. The inhibitory power against cysteine proteinases is found to be due mainly to this protein in human serum. This inhibitor is also found in mammals such as cows, pigs and rats. Vitamin E deficient rats show a very high inhibitor level. Therefore, the present method will enable us to investigate the relation between diseases and the activity of the alpha-cysteine proteinase inhibitor. Also, this method is simple and inexpensive. The necessary amount of serum is only 10 microliter.     

Changes in the levels of prostaglandins and thromboxane and their roles in the accumulation of exudate in rat carrageenin-induced pleurisy — a profile analysis using gas chromatography-mass spectrometry —

Injection of γ-carrageenin into t he pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B₂ were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethyllisopropylsilyl (DMiPS) ether or ME-methoxine-DMiPS ether derivatives, 6-keto-PGF_1α reached the maximum at 1 hr after carrageenin, then PGE₂ and TXB₂ showed peaks at 3 hr and waned off before 9 hr. he PGF_2α level was kept low, but PGD₂, PGE₁ and PGF_1α were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB₂ levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE₂, definitely play an important role in the exudation during the first 5 hr.     

Effects of IAA, Zeatin, Ammonium Nitrate and Sucrose on the Initiation and Development of Floral Buds in Torenia Stem Segments Cultured In Vitro

In Torenia stem segments cultured on a defined medium from whichammonium nitrate and growth regulators were omitted, adventitiousbuds were readily formed from epidermal tissue, with subsequentdifferentiation of floral buds. Using this plant material, thecorrelation between the time of application of various chemicalsand the time-course of floral bud differentiation was investigated.Histological examination showed that adventitious buds werevegetative during the first two weeks of the culture, and floralprimordia appeared after about three to four weeks of culture.We divided the flowering process in Torenia stem segments intothe following 3 phases: the first phase (first 2 weeks) duringwhich adventitious buds are formed, the second phase (3rd and4th weeks) during which floral buds are initiated and the thirdphase (5th to 12th weeks) during which floral buds develop.Then we added IAA, zeatin, ammonium nitrate or a high concentrationof sucrose to the medium during one, two or three of these phases.Ammonium nitrate added during the third phase suppressed floralbud development, but the high concentration of sucrose givenduring this phase stimulated it. These two chemicals influencedonly the development of floral buds previously initiated. Theapplication of IAA during the first phase promoted both theinitiation and development of floral buds. However, its applicationafter 2 weeks of culture failed to promote floral bud formation.Zeatin inhibited floral bud formation in a manner similar toammonium nitrate, but if it was added to the medium only duringthe first phase, it slightly promoted the initiation and developmentof floral buds. (Received July 7, 1981; Accepted October 12, 1981)     

Electrophoretic and biochemical studies of human aldehyde dehydrogenase isozymes in various tissues

Four major ALDH isozymes have been identified in human tissues using starch gel electrophoresis and isoelectric focusing. The isozyme bands have been termed as ALDH I, II, III and IV according to their decreasing electrophoretic migration and increasing isoelectric point. The isozymes have been partially purified via preparative isoelectric focusing. Kinetic characteristics of ALDH I and II were found to be quite similar to ALDH enzyme 2 and enzyme 1 described earlier by Greenfield and Pietruszko (Biochem Biophys Acta, $\text{483}$ 35–45 1977). ALDH III and IV showed a very high Km for propionaldehyde (1.0–1.5 mM at pH 9.5) and were not inhibited by disulfiram at pH 9.5. A variant phenotype of ALDH which lacked in isozyme I was detected in various tissues from Japanese individuals. Comparative kinetic properties of normal and variant enzyme are given.     

Non-specific antagonism against thromboxane A2 of 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) in contraction of isolated smooth muscles

7-Ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) was reported as an antagonist of thromboxane (Tx) A2 in the contraction of rabbit aorta. It was, however, observed that EG-626 did inhibit the contraction of superfused rabbit aorta, but also did inhibit that of rabbit coeliac artery, rat stomach strip and rat colon induced by TxA2, PG endoperoxides, angiotensin II and PGF2 alpha in non-specific manner. EG-626 had no effect on the biosynthesis of PG endoperoxides as well as TxA2. These results indicate that EG-626 is not a TxA2 antagonist, but has a general inhibitory effect on the smooth muscles. This inhibitory effect of EG-626 may be explained by the inhibition of phosphodiesterase.     

Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques

Summary Feulgen nuclear staining with pararosanilin-SO₂ was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell.The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality beween the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by Böhm et al. (1968) and a reaction with ninhydrin at 37° C for 10 h.The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure.Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.Partly supported by Deutsche Forschungsgemeinschaft (DFG), grant Nr. Bo 395/4     

Blocked 5′ Termini in Brome Mosaic Virus RNA

All four components of brome mosaic virus RNA have m(7)G(5') ppp (5')Gp as their 5' terminus. The m(7)G can be removed by beta-elimination, resulting in the conversion to pppGp.     

Specific replacement of Q base in the anticodon of tRNA by guanine catalyzed by a cell-free extract of rabbit reticulocytes.

Guanylation of tRNA by a lysate of rabbit reticulocytes was reported previously by Farkas and Singh. This reaction was investigated further using 18 purified E. coli tRNAs as acceptors.Results showed that only tRNATyr, tRNAHis, tRNAAsn and tRNAAsp which contain the modified nucleoside Q in the anticodon acted as acceptors. Analysis of the nucleotide sequences in the guanylated tRNA showed that guanine specifically replaced Q base in these tRNAs.     

The effect of nasal obstruction on the susceptibility of mice to noxious gases (author's transl)]

The effect of nasal obstruction on mice--the maice were forced to breathe through the mouth--was studied when the animals were exposed to noxious gases. It was noted that the forced mouth breathing made the mice less vulnerable to hydrogen sulfide, carbon monoxide and toluen vapor when expressed by the time interval before they lost righting reflex or showed respiratory arrest. The levels of the gases which could induce these sings within 5-10 minutes were significantly elevated with the "mouth breathing" mice. Possible mechanisms for this phenomenon and its application for men were discussed.