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.     

A new series of RNAs associated with the genome of spleen focus forming virus (SFFV) and poly(A)-containing RNA from SFFV-infected cells.

A series of low molecular weight RNAs (4.5 to 5.5S) as well as other 4 to 7S RNAs were dissociated from genomic RNA of spleen focus forming virus (SFFV) by heating. On two dimensional polyacrylamide gel electrophoresis, this series of RNAs gave a series of more than thirty spots. RNase T1 fingerprints of these spots were identical except for differences in 3'-terminal oligonucleotides, which were mainly due to different numbers of uridylic acid residues, larger RNA-molecules containing poly(U)sequences at their 3'-termini. This series of RNAs is also associated with poly(A)-containing nuclear and cytoplasmic RNAs from SFFV-infected cells.     

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     

An increase of pituitary 3', 5' cyclic adenosine monophosphate produced by estradiol benzoate in vitro: possible implication of this increase in the secretion of luteinizing hormone.

In an attempt to study the site and mechanism of action of estrogen in producing positive feedback control, porcine anterior pituitary slices were incubated in vitro in the presence of estradiol benzoate (EB). EB elevated pituitary cyclic AMP concentration within 5 min and augmented pituitary release of luteinizing hormone (LH). The magnitude of increase of cyclic AMP and LH release was related to the doses of EB used. Also, luteinizing hormone releasing hormone (LH-RH) elevated pituitary cyclic AMP concentration and stimulated pituitary release of LH. The magnitude of increase of cyclic AMP and LH release was inversely related to the doses of LH-RH used. EB and LH-RH were additive in increasing cyclic AMP. Progesterone and clomiphene citrate interfered with an increase of pituitary cyclic AMP produced by EB, but did not significantly affect the basal level of pituitary cyclic AMP. Testosterone propionate, human chorionic gonadotropin and hexestrol were without effect on either basal or stimulated level of pituitary cyclic AMP. Since cyclic AMP and dibutyryl cyclic AMP (DBC) stimulated LH release, it is suggested that EB directly stimulates the release of LH by augmenting cyclic AMP synthesis in the anterior pituitary.     

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.     

A comparative study of the differential radiosensitivity of seeds, seedlings and tissue cultures of the Japanese morning glory (Pharbitis nil)

Radiobiological effects of gamma rays on seeds, seedlings andcallus tissues of Pharbitis nil were investigated. Strikingdifferences in radiosensitivity were observed among the threetypes. Seedlings treated with doses between 2 and 12 Kr showedsuch morphological effects as stunted growth of the hypocotyl,severe inhibition of root formation and of the development oftrue leaves. Low dose irradiation of seeds resulted in a positivestimulation in seedling height. Higher doses, however, provedlethal; seed germination was inhibited and in seedlings theleaves became chlorotic and development was arrested. Callustissues were more resistant to radiation and could withstanddoses up to 30 Kr. ¹Present address: Bio-Organic Division, Bhabha Atomic ResearchCentre, Bombay 400 085, India. ²Present address: Department of Biological Sciences, Universityof Tsukuba, Ibaraki, 300-31, Japan. (Received September 22, 1975; )     

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.     

Regulation of arylsulfatase synthesis by sulfur compounds in Klebsiella aerogenes.

In Klebsiella aerogenes, arylsulfatase synthesis was repressed by inorganic sulfate, sulfite, sulfide, thiosulfate, and cysteine, but not by methionine under normal growth conditions. We isolated cysteine-requiring mutants (Cys minus), and mutants (AtsS minus, AtsR minus) in which the regulation of arylsulfatase synthesis was altered. In the cysteine auxotroph, enzyme synthesis was also repressed by inorganic sulfate or cysteine. Kinetic studies on mutants of the cysteine auxotroph showed that inorganic sulfate repressed arylsulfatase synthesis and that this was not due to cysteine formed by reduction of sulfate. Arylsulfatase synthesis in the AtsS minus mutant was not repressed by inorganic sulfate but was repressed by cysteine. This mutant strain had a normal level of inorganic sulfate transport. Another mutant strain, defective in the inorganic sulfate transport system, synthesized arylsulfatase in the presence of inorganic sulfate but not in the presence of cysteine. The AtsS minus mutant could synthesize the enzyme in the presence of inorganic sulfate but not cysteine. The AtsR minus mutant could synthesize the enzyme in the presence of either inorganic sulfate or cysteine. These results suggest that there are two independent functional corepressors of arylsulfatase synthesis in K. aerogenes.