Alkaline phosphatase reactivity in rabbit airway epithelium: a potentially useful marker for airway basal cells

The purpose of this study was to investigate alkaline phosphatase (ALPase) reactivity in rabbit airway epithelial cells. Acetone-fixed, methyl benzoate and xylene-cleared (AMeX-treated) paraffin sections of trachea, bronchus, and lung tissue were stained by an azo dye coupling method for ALPase and examined by light microscopy. Electron histochemical staining was also performed in order to study the sensitivity and specificity of reactivity in each cell type. ALPase reactivity at the light microscopic level was observed exclusively in trachco-bronchial basal cells, and not in bronchiolar basal cells. By electron microscopy, ALPase reactivity was noted in 97.9% of basal cells in the trachea, 97.0% of basal cells in the bronchus, and 94.5% of basal cells and 15.4% of Clara cells in the bronchiole. This was also true for dispersed tracheal epithelial cells. Reactivity was rarely observed in ciliated cells, non-goblet-type secretory cells, and undetermined cells. The reactivity was heatlabile, levamisole-sensitive, and of a non-specific type. These findings indicate that basal cells of rabbit trachea and bonchus have fairly high specificity for ALPase of a non-specific isozyme (92.2% and 95.6%, respectively). Therefore, ALPase is considered to be a useful marker for these cells.     

Oligo-glycine synthesis in an aqueous solution of glycine under oxidative conditions

Di-and tri-glycine were synthesized in 1M aqueous solution of glycine by bubbling for 90 hr with oxygen discharged in the path from an oxygen cylinder. The peptides were also produced by an incubation at 37°C of 2M glycine solution prepared with 75% hydrogen peroxide, and the yields were traced for 200 days. The final yields were about 0.25% and 0.01% for di-and tri-glycine, respectively. The solution at 166 days of incubation was applied to a Sephadex G 10 column, and the fractions around the top of the chromatogram were found to increase the intensity of ninhydrin color about 45 times after hydrolysis, indicating an existence of oligo-glycine. The solutions of 1M glycine and 0.5M diglycine prepared with 30% hydrogen peroxide were incubated at 37°C for 38 days, and di-and tetra-glycine were detected in the yields of 0.12% and 0.33%, respectively.     

Lipase modification by various synthetic polymers for use in chloroform

Summary Lipase was modified with several hydrophilic and hydrophobic synthetic polymers. The modified lipase was solubilized into chloroform by. The catalytic esterification activity of modified lipase increased linearly with the increase of its solubility in chloroform.     

Kinetics of the Unrolling of the Second Leaves Detached from Rice Seedlings by Blue Light

Unrolling due to blue light (B) irradiation of the second leavesdetached from 8-day-old rice (Oryza saliva L.) seedlings wassimilar to that reported previously for nondetached leaves.The effect of B was counteracted by irradiation with red light(R). The counteracting effect of R was reversed by subsequentirradiation with far-red light (FR). When the detached leaf was irradiated with B passed througha 1-mm-wide slit 5, 8, 10, 12 or 15 mm from the leaf tip, irradiation10 mm from the leaf tip was the most effective. The effect of a 1 mm-wide-B irradiation 10 mm from the leaftip was counteracted by a 1 mm-wide-R irradiation at the sameposition, but not by irradiations at the other points. The counteractingeffect of R was reversed by a 1 mm-wide-FR irradiation at thesame position. This suggests that the excitation or the reactionof the B photoreceptor(s) is affected directly by the PFR formof phytochrome. The dose-response curve for the unrolling caused by B showeda simple Bunsen-Roscoe relation without two peaks, which differsfrom that for the phototropism in Avena caused by B. (Received August 21, 1980; Accepted December 20, 1980)     

Enzymes of purine catabolism in soybean plants

Remarkable formation and utilization of allantoin is observedin soybean (Glycine max variety A62-1). To study this, variousenzymes involved in purine catabolism (i.e., xanthine oxidase,uricase and allantoinase) were measured in different regionsof soybean plants during development. Uricase, which catalyzesthe direct formation of allantoin from uric acid, was studiedin detail. The activities of these three enzymes were highest in the rootnodules, indicating that the nodules are the major site of allantoinmetabolism. Radicles only showed appreciable activity about80 hr after the seeds were planted. Allantoinase activity wasdetected in all regions tested, showing that allantoin translocatedfrom the nodules can be metabolized in the roots, stem and leaves.In the nodules, xanthine oxidase was localized in the nuclearfraction, while uricase was mainly restricted to the mitochondrialfraction and allantoinase to the soluble fraction. Uricase was partially purified from the nodules and radicles,respectively. The pH optimum of enzyme from the nodules was9.5, whereas that of enzyme from the radicles was 7.0. The enzymefrom the nodules did not require a cofactor, while that fromthe radicles showed an absolute requirement for a cofactor,which was a low molecular substance easily separable from theapoprotein. Thus, the uricase in nodules differs in chemicalproperties from that in the host plant. The results are discussedin relation to change in the allantoin level in soybean tissues. (Received November 1, 1974; )     

Replication of the simian virus 40 chromosome with purified proteins.

SV40 chromosomes prepared from infected CV-1 cells were replicated with the purified proteins of SV40 T antigen, HeLa DNA polymerase alpha-primase complex, single-stranded DNA-binding protein, and topoisomerases I and II, all of which have been shown to be essential for SV40 DNA replication in vitro. Replication started near the origin and proceeded bidirectionally. The maximum speed of replication fork movement was 200-300 nucleotides/min, which was similar to the rate of SV40 DNA replication with the same set of proteins. When replication products were digested with micrococcal nuclease, DNA fragments of 160-180 base pairs, which is the typical size of mononucleosomal DNA, were protected. This result indicates that replicated DNA was reconstructed into the nucleosome structure, complexed with parental histones.     

Comparison of the properties of detergent-solubilized NADPH-cytochrome P-450 reductases from pig liver and kidney immunochemical,kinetic, and reconstitutive properties

NADPH-cytochrome P-450 reductases from pig liver and kidney and rabbit liver microsomes were purified to a specific activity of 50–62 μmol cytochrome c reduced/min/mg. All reductase preparations were separated into one major and one minor fraction on Sephadex G-200 columns. The molecular weights of the major fractions of the reductases were estimated to be 74,000, 75,000, and 75,500 for rabbit liver, pig kidney, and liver reductases, respectively, whereas the molecular weight of the minor fractions of these reductases, 67,000, was the same as that of the steapsin-solubilized pig liver reductase on SDS-polyacrylamide gel electrophoresis. K_m values for NADPH and cytochrome c were: 20 and 29 μm or 14 and 28 μm for the pig kidney or liver reductase, respectively. Immunochemical studies, including Ouchterlony double diffusion experiments and inhibition of benzphetamine N-demethylation activity in microsomes by antibody against pig liver NADPH-cytochrome P-450 reductase, indicated the similarity of the purified liver and kidney reductases. There were no differences in the ability to reconstitute NADPH-mediated benzphetamine N-demethylation and laurate hydroxylation in reconstituted systems between the pig liver and kidney reductases, indicating that the reductase did not determine substrate specificity in these systems.     

Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03

A lipolytic enzyme gene (lip8) was cloned from organic solvent-tolerant Pseudomonas aeruginosa LST-03 and sequenced. In the sequenced nucleotides, an open reading frame consisting of 1,173 nucleotides and encoding 391 amino acids was found. Lip8 is considered to belong to the family VIII of lipolytic enzymes whose serine in the consensus sequence of -Ser-Xaa-Xaa-Lys- acts as catalytic nucleophile. The gene was expressed in Escherichia coli and purified by a combination of ammonium sulfate fractionation and hydrophobic interaction and ion-exchange chromatographies to homogeneity on SDS-PAGE analysis. The optimum temperature and heat stability of Lip8 were not as high as those of Lip3 and LST-03 lipase, two other lipolytic enzymes from the same strain. Addition of glycerol to a solution containing Lip8 stabilized this enzyme. By measuring the activities against various triacylglycerols and fatty acid methyl esters having carbon chains of different lengths, Lip8 was categorized as an esterase which has higher activities against fatty acid methyl esters with short-chain fatty acids.     

Seroprevalence of Toxoplasma gondii in wild boars (Sus scrofa leucomystax) and wild sika deer (Cervus nippon) in Gunma Prefecture, Japan

The ingestion of undercooked meat from wild animals can be a source of Toxoplasma gondii infection in humans and other animals. In this study, we determined the seroprevalence of T. gondii infection in 175 wild boars (Sus scrofa leucomystax) and 107 wild sika deer (Cervus nippon) hunted in 2004–2007 in Gunma Prefecture, Japan, by using a commercial latex agglutination test (LAT). Antibodies (LAT, 1:64 or higher) to T. gondii were found in 6.3% of wild boars and 1.9% of sika deer. This is the first record of T. gondii infection in wild deer in Japan, and deer and wild boar meat should be cooked well before human consumption.