Direct Observation of Reversible and Irreversible Stomatal Responses of Attached Sunflower Leaves to SO(2)

The effects of SO₂ on stomatal aperture of attached sunflower leaves were observed with a remote-control light microscope system that permitted continuous observation of stomatal responses over periods of several hours. The relationship between actual stomatal aperture and stomatal conductance, measured with a porometer, also was examined on leaves before and after exposure to SO₂.

A distinction between uninjured and injured regions was clearly visible on leaves after exposure to 1.5 microliters per liter SO₂ for less than an hour. During the exposure, the mean value of apertures for many stomata, which indicates stomatal conductance and transpiration rate, tended to decrease simultaneously in the uninjured and injured regions. However, the rate of decrease in the injured region was slower than that in the uninjured region because of a transient opening induced by water-soaking in the injured region. The transient opening was less common in stomata near veins and veinlets.

There was a good correlation between pore width and stomatal conductance measured with a porometer before exposure to SO₂. This correlation continued in leaves exposed to SO₂ until visible, irreversible injury occurred, but then it disappeared.

The results of these experiments indicate the necessity of continuous observation of individual stomata under the microscope to understand the effects of air pollutants such as SO₂ on stomatal behavior.

     

Distribution of vasopressin and oxytocin in rat brain

Arginine-vasopressin and oxytocin in various portions of rat brain were determined by radioimmunoassays. The hormones were extracted from tissue samples into 0.1 N HCl and then purified partially with acetone-petroleum ether extraction. The non-equilibration method was used for the assays. In this method recovery rates of arginine-vasopressin and oxytocin were 73.0 +/- 4.4% and 75.0 +/- 3.8%, respectively. Sensitivities of the assays were 1 pg of arginine-vasopressin and 0.75 pg (0.3 microU) of oxytocin per assay tube. The higher concentrations of arginine-vasopressin and oxytocin were confirmed in the hypothalamo-neurohypophyseal system, where these hormones are synthesized, transported and stored. Relatively high concentrations of these hormones, especially oxytocin, were detected in spinal cord. Amygdala, hippocampus, limbic forebrain and pineal body contained a certain amount of arginine-vasopressin (2-20 pg/mg protein). Oxytocin (1-7 pg/mg protein) was also detected in amygdala, pons and medulla oblongata, pineal body and midbrain. The low concentrations of these hormones were also found in cerebral cortex and cerebellum.     

Induction of peroxisomal beta-oxidation enzymes in primary cultured rat hepatocytes by clofibric acid

Rat hepatocytes were cultured for 72 h with or without the addition of 0.5 mM clofibric acid. The activities of individual enzymes of the peroxisomal beta-oxidation pathway (acyl-CoA oxidase, enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and 3-ketoacyl-CoA thiolase) decreased in the control culture, but markedly increased synchronously in the clofibric acid-treated culture. The levels of mRNAs coding for these enzymes and the rates of synthesis of the enzymes were also elevated in the clofibric acid-treated culture, although no proportional relationship was observed between the time-dependent changes of these parameters. The increase in mRNAs was much higher than the increase in the rate of synthesis of the enzymes. The activity of catalase, its mRNA level and the rate of its synthesis were slightly affected. The effects of clofibric acid on the peroxisomal beta-oxidation enzymes and catalase in primary cultured hepatocytes were very similar to those observed in vivo. These results, therefore, suggest that primary culture of hepatocytes should provide a useful means for investigating the mechanism of induction of peroxisomal enzymes and the mechanism of action of peroxisome proliferators.     

Effect of free Mg2+ on liver phosphorylase kinase activity

When pig liver phosphorylase kinase was assayed at various concentrations of Mg2+, about 2-fold stimulation was observed around 2-3 mM Mg2+ (Mg2+/ATP ratio, 20-30) compared with the activity at 0.3 mM Mg2+ (Mg2+/ATP ratio, 3). This stimulation was specific for Mg2+ among the divalent cations tested and the process was reversible. Km values for ATP and phosphorylase b were decreased 3.6- and 9.5-fold, respectively, at 3 mM Mg2+ compared with those obtained at 0.3 mM Mg2+. These results indicate that the activity of liver phosphorylase kinase is influenced by free Mg2+.     

Molecular cloning of cDNA for rat acyl-CoA oxidase

Poly(A+) RNA was prepared from hepatic free polysomes of rats which had been fed di(2-ethylhexyl) phthalate for the induction of peroxisomal beta-oxidation enzymes. This preparation was enriched for the mRNAs of these enzymes by sucrose density gradient centrifugation, and used for the synthesis of double-stranded cDNA. Recombinant plasmids were constructed from the cDNA and pBR322 by dG X dC-tailing method and used for the transformation of an Escherichia coli strain, chi 1776. By differential colony hybridization using [32P]cDNA of partially purified liver poly(A+) RNA from induced and noninduced rats as probes, and then by hybridization-selected translation, we obtained two clones with cDNA inserts which specifically selected acyl-CoA oxidase mRNA. On Northern blotting, both cDNA inserts hybridized to 3.8-kilobase RNA which was increased about 10-fold by di(2-ethylhexyl) phthalate treatment of the rats. The cleavage maps of the cDNA inserts showed they overlap with each other. We conclude that the above two recombinant plasmid clones contain cDNA sequences for rat acyl-CoA oxidase.     

Kinetics of formation of maltose and isomaltose through condensation of glucose by glucoamylase

A kinetic model was devised for the hydrolysis and synthesis of maltose and isomaltose by two glucoamylases from Rhizopus niveus and Aspergillus niger, and the validity of the model was verified experimentally at 313 K and pH 5.0. For both enzymes, the formations of maltose and isomaltose from glucose were parallel reversible reactions, and glucosyl transfer between maltose and isomaltose was not observed. The enzymes catalyzed rapid hydrolysis and synthesis of maltose. Isomaltose was hydrolyzed and synthesized more slowly, but the level produced from glucose was much higher than that of maltose. These hydrolysis and condensation reactions were expressed well by the model.     

A new type of glycogen storage disease caused by deficiency of cardiac phosphorylase kinase

A five-month-old Japanese boy was found to have marked glycogen accumulation only in the heart. A survey of enzymes revealed normal activities of phosphorylase, cyclic AMP-dependent protein kinase, acid maltase and amylo-1,6-glucosidase. However, the heart had capacity of activating neither rabbit muscle phosphorylase b nor endogenous phosphorylase b, which was converted to active form only when supplemented rabbit muscle phosphorylase kinase. In contrast to the heart, activities of phosphorylase kinase were found within normal levels in other organ tissues so far tested. These findings indicate that the present case of the cardiac glycogenosis is caused by deficiency of cardiac phosphorylase kinase.     

Molecular cloning of cDNA for rat liver catalase

For the studies on the induction of peroxisomal enzymes by hypolipidemic agents, we have tried to isolate a cDNA clone for rat liver catalase. A recombinant clone, pMJ501, was isolated, of which cDNA insert specifically hybridized to catalase mRNA in hybridization-selected translation. On RNA blot hybridization, it hybridized to 2.4-kilobases RNA which was increased about 1.5-fold by the administration of di-(2-ethylhexyl)phthalate to the rats. The nucleotide sequence of the cDNA contains a reading frame for 109 amino acid residues which match the reported amino acid sequence of bovine liver catalase at the carboxyl end with 82% homology. It is concluded that pMJ501 contains a cDNA sequence for rat liver catalase.     

Triterpenes from Periandra dulcis roots

Three oleanane triterpenes were isolated from the roots of Periandra dulcis,and identified as 3β-hydroxy-25-al-olean-18-en-30-oic acid (periandric acid I), 3β-hydroxy-25-al-olean-12-en-30-oic acid (periandric acid II) and 3-oxo-25-hydroxy-olean-12-en-30-oic acid. The former two compounds (periandric acids I and II) were identical with the aglycones obtained by hydrolysis of periandrin I and II, respectively and the latter one was a new triterpene.