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.

     

Light Activation of NADP-Malate Dehydrogenase in Guard Cell Protoplasts from Vicia faba L

Light-induced swelling of guard cell protoplasts (GCP) from Vicia faba was accompanied by increases in content of K⁺ and malate. DCMU inhibited the increase of K⁺ and malate, and consequently swelling.

Effect of light on the activity of selected enzymes that take part in malate formation was studied. When isolated GCP were illuminated, NADP-malate dehydrogenase (NADP-MDH) was activated, and the activity reached a maximum within 5 minutes. The enzyme activity underwent 5- to 6-fold increase in the light. Upon turning off the light, the enzyme was inactivated in 5 minutes NAD-MDH and phosphoenolpyruvate carboxylase (PEPC) were not influenced by light. The rapid light activation of NADP-MDH was inhibited by DCMU, suggesting that the enzyme was activated by reductants from the linear electron transport in chloroplasts. An enzyme localization study by differential centrifugation indicates that NADP-MDH is located in the chloroplasts, NAD-MDH in the cytosol and mitochondria, and PEPC in the cytosol. After light activation, the activity of NADP-MDH in guard cells was 10 times that in mesophyll cells on a chlorophyll basis. The physiological significance of light-dependent activation of NADP-MDH in guard cells is discussed in relation to stomatal movement.

     

The heterogeneity of human fibroblasts as determined from the effects of hydrocortisone on cell growth and specific dexamethasone binding

To elucidate the heterogeneity of human fibroblasts from lung and skin, the effects of hydrocortisone on cell proliferation and the specific dexamethasone binding to cells were studied. Hydrocortisone at physiological concentrations stimulated the proliferation in three strains of human fetal lung fibroblasts and inhibited it in two strains. There are two kinds of fibroblasts in the human fetal lung in addition to the human skin fibroblasts reported previously. Dexamethasone-binding experiments showed that human fibroblasts may be classified into two groups with respect to the dissociation constant (Kd) of the binding reaction. The heterogeneity of human fibroblasts shown by Kd could not be correlated to classification on the basis of the effects of hydrocortisone on cell proliferation. The differences in Kd for the binding reactions suggest differences in donor tissues from which human fibroblasts are derived.     

Studies on the metabolism of unsaturated fatty acids. XVI. Purification and general properties of 2,4-dienoyl-CoA reductase from Candida lipolytica

2,4-Dienoyl-CoA reductase has been purified to homogeneity from Candida lipolytica cultivated in the presence of linoleic acid. The native enzyme had a molecular weight close to 360,000 as estimated by gel filtration on Sepharose CL-4B, whereas the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 33,000. The purified 2,4-dienoyl-CoA reductase from C. lipolytica gave a single precipitin line with antibodies raised against the purified enzyme from C. lipolytica. The general properties of the 2,4-dienyl-CoA reductase from C. lipolytica were examined. The enzyme had optimal pH at 6.5 and was inactivated by heat treatment at 50 degrees C for 10 min. trans-2,trans-4-Octadienoyl-CoA was the most active substrate of the dienoyl-CoA esters examined.     

Aspartate aminotransferase isozymes from rabbit liver. Purification and properties

Cytosolic and mitochondrial isozymes of aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase [EC 2.6.1.1] ) were purified to homogeneity from rabbit liver. The rabbit liver isozymes were closely similar to the corresponding isozymes from other sources, including human heart, pig heart, chicken heart, and rat liver, in their molecular weights, absorption spectra, amino acid compositions, isoelectric points, and Michaelis constants for the substrates. The NH2-terminal amino acid sequences of rabbit liver isozymes were identified up to 30 residues, and showed some differences from those of the corresponding isozymes obtained from other animals so far studied.     

Amino acid sequences of the two kinds of regulatory light chains of adductor smooth muscle myosin from Patinopecten yessoensis

Smooth muscle myosin from scallop (Patinopecten yessoensis) adductor muscle contains two kinds of regulatory light chains (regulatory light chains a and b), and myosin having regulatory light chain a is suggested to be suitable for inducing "catch contraction" rather than myosin having regulatory light chain b (Kondo, S. & Morita, F. (1981) J. Biochem. 90, 673-681). The amino acid sequences of these two light chains were determined and compared. Regulatory light chain a consists of 161 amino acid residues, while regulatory light chain b consist of 156 amino acid residues. Amino acid substitutions and insertions were found only in the N-terminal regions of the sequences. The structural difference between the two light chains may contribute to the functional difference between myosins having regulatory light chains a and b.     

Molecular cloning and nucleotide sequence of the cDNA for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme

For the studies on the mechanism of induction of peroxisomal beta-oxidation enzymes and biogenesis of the organelle, we have isolated cDNA clones for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. On blotting experiments with liver RNA, the cDNAs hybridized to a 3.0-kilobase RNA which was increased 5-7-fold by the administration of di-(2-ethylhexyl)phthalate to rats. Nucleotide sequencing was carried out for four cloned cDNAs and one obtained by a primer extension method. By overlapping these sequences with each other, we identified 20 nucleotides of 5'-noncoding, 2,166 nucleotides of coding, and 910 nucleotides of 3'-noncoding regions. The deduced amino acid sequence of the enzyme is composed of 722 residues, and the composition agrees with that of the protein data. The sequence was confirmed by the amino acid compositions and sequence analyses of some of the tryptic peptides. The molecular weight of the mature enzyme is calculated to be 78,511 from the predicted amino acid sequence. The enzyme has no terminal peptide extension as a signal for translocation into peroxisomes.     

X-ray diffraction photographs of chicken gizzard G-actin.DNase I complex crystals taken with synchrotron radiation

X-ray diffraction photographs of a chicken gizzard G-actin.DNase I complex crystal have been recorded using the synchrotron radiation beam emitted by the Synchrotron Radiation Source at Daresbury and the Photon Factory at Tsukuba. The resolution limit was extended to 2.4 A and the exposure time was reduced approximately by a factor of 10, when data recorded at the Photon Factory, were compared with those recorded with a conventional rotating-anode source. Using a newly designed Weissenberg camera equipped with a multi-layer line screen, the diffraction data in a 36 degrees oscillation range were recorded on a single film up to 3.5 A resolution.     

Cloning and characterization of the alkA gene of Escherichia coli that encodes 3-methyladenine DNA glycosylase II

By in vitro recombination we have constructed hybrid plasmids which can suppress the increased methylmethane sulfonate sensitivity caused by the alkA1 mutation in Escherichia coli. Since the cloned DNA fragment was mapped at 44 to 45 min of the E. coli K12 genetic map, an area where the alkA gene is located, we conclude that the cloned DNA fragment contains the alkA gene itself but not other gene(s) that suppresses the alkA mutation. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the alkA codes for a polypeptide whose molecular weight is about 30,000. When cells harboring the alkA+ plasmids were grown in the presence of low doses of a simple alkylating agent (adapted condition), the activity of 3-methyladenine DNA glycosylase II was increased. The enzyme activity was copurified with the Mr 30,000 polypeptide. These results indicate that the alkA gene codes for 3-methyladenine DNA glycosylase II. Taking advantage of overproduction of the alkA protein in adapted cells that harbor multicopy plasmids carrying the alkA+ gene, 3-methyladenine DNA glycosylase II has been purified to apparent physical homogeneity.     

Reexamination of the reality or artifacts of the microtrabeculae

The polyethylene glycol (PEG) method revealed that model systems such as erythrocytes and protein solutions, which are supposed to lack structured components, exhibit lattice structures not unlike the microtrabeculae. The compactness of the lattice was dependent on the concentration of proteins. The gelated state of gelatin exhibited lattices more compact than those of the solated state at any given concentration. Comparison of images by PEG and rapid-freezing, deep-etching replica methods showed no basic differences in the ultrastructure of the intestinal epithelial cell. This indicates that the PEG method, including chemical fixation, produces little, if any, disorganization of the cytoskeleton. All of the present findings suggest that cytoplasmic protein, nonstructure-bound or structure-forming, might be present in intact cells which could form microtrabecular structures when specimens are fixed by chemical fixatives without any extractions. Therefore, the microtrabeculae should generally be regarded as a simple marker for the presence of proteinaceous macromolecules. It is also suggested that the microtrabecular lattice, as a whole, might represent a gelated state in a given compartment when another, looser lattice is simultaneously present in the same compartment, i.e., within a single cell.