Freeze-fracture images of exocytosis and endocytosis in anterior pituitary cells of rabbits and mice

Summary Freeze-fracture images of exocytosis and endocytosis were studied in various kinds of secretory cells of the anterior pituitary of mice and rabbits. Exocytotic figures are frequently observed in thin section of the anterior pituitary cells. In freeze-fracture images, small elevated membrane areas without membrane particles are often seen on the PF of the plasma membrane of the secretory cells. There is a secretory granule in the cytoplasm just beneath the particle-free membrane area, and limiting membrane of the granule is also devoid of the membrane particles at the part facing the plasma membrane. The fusion of membranes for exocytosis may occur at this particle-free area.The limiting membrane of the granule which is continuous with the plasma membrane is almost always coated after release of the granule core. This invagination of coated membrane may be an initiation site for the membrane retrieval after exocytosis. In freeze-fracture images, this depressed region with an accumulation of the membrane particles is observed on the PF of the plasma membrane. This particle-rich depressed region is thought to correspond to the coated area of the plasma membrane observed in thin section. It is thought that the membrane retrieval by pinocytosis initiates at the particle-rich depressed region of the plasma membrane.This study was supported by a grant from the Japan Ministry of Education     

Pyrone derivatives: effective inhibitors of photosynthetic electron flow system

The inhibitory effects of the pyrone derivatives, 6-()-decenyl-2,3-dimethyl--pyrone(DDP) and 6-farnesyl-2,3-dimethyI--pyrone (FDP), on the photosyntheticelectron flow system was investigated using the blue-green algaAnabaena variabilis and the green alga Chlorella pyrenoidosa. Both reagents inhibited photosynthesis in intact cells; 50%inhibition occurred at 2.7 x 10^–5 M with DDP and at 4.3 lO^–6 M with FDP in Anabaena photosynthesis. The reagentssuppressed the photosystem II reaction [water to 2,6-dichlorophenolindophenol (DCIP)] of Anabaena membrane fragments, but werefar less inhibitory on the photo-system I reaction (DCIPH₂ tomethyl viologen). The kinetics of the fluorescence inductionindicated that the reagents do not block Q-reduction, but dosuppress the oxidation of reduced Q indirectly. Oxygen evolutionunder repetitive flashes at a low repetition rate (5 Hz) wasinsensitive to the reagents even at concentrations which inducedmore than 50% inhibition. These results are evidence that DDPand FDP inhibit the plastoquinone reaction by slowing down itsturnover rate. The advantages of pyrone derivatives are that they are inactivein the oxidation-reduction reaction and do not quench the fluorescenceof chlorophyll in vivo. (Received April 14, 1980; )     

Methane production by anaerobic digestion of animal manures

Anaerobic digestion of swine manure was performed at mesophilic and thermophilic temperatures. In addition, the possibility of enhancement of biogas production by the co-digestion of manures with cellulosic waste residues (e.g., corn stover), was investigated. In the latter studies, the effect of particle size on the gasification efficiency was assessed.Methane productivity, G(m³ methane/m³ slurry volume.day), in the digesters operating at a stationary state could be correlated with the volatile solids loading, L (kg/m³.day), Retention time, (days), and pH as follows: G = L/(1 + 10^pK-pH) + [k/(1 + k)] where is dependent primarily on the nature of the utilizable carbohydrate fraction. The constant, , is found to be a function of the organic nitrogenous fraction of the manure. The constants pK and k (days^-1) are dependent on the temperature only.     

Comparison of some minor activities accompanying a preparation of sodium-plus-potassium ion-stimulated adenosine triphosphatase from pig brain

1. An ATPase (adenosine triphosphatase) preparation obtained from pig brain microsomes by treatment with sodium iodide showed four apparently different ouabain-sensitive activities under various conditions. They were (a) ouabain-sensitive Mg(2+)-stimulated ATPase, (b) K(+)-stimulated ATPase, (c) (Na(+),K(+))-stimulated ATPase and (d) Na(+)-stimulated ATPase activities. 2. These activities showed the same substrate specificity, ATP being preferentially hydrolysed and CTP slightly. AMP was not hydrolysed. 3. These activities were inhibited by low concentration of ouabain. The concentration producing 50% inhibition was 0.1mum for ouabain-sensitive Mg(2+)-stimulated ATPase, 0.2mum for K(+)-stimulated ATPase, 0.1mum for (Na(+),K(+))-stimulated ATPase and 0.003mum for Na(+)-stimulated ATPase activity. 4. The ouabain-sensitive ATPase activities were inactivated by N-ethylmaleimide but the insensitive ATPase activity was not. 5. The three ouabain-sensitive ATPase activities were inhibited about 50% by 1mm-Ca(2+), whereas the ouabain-sensitive Mg(2+)-stimulated ATPase activity was activated by the same concentration of Ca(2+). The preparation was treated with ultrasonics at 20kcyc./sec. The 2min. ultrasonic treatment inactivated the ATPase activities by 50%. 7. The temperature coefficient Q(10) was 6.6 for K(+)-stimulated ATPase activity, 3.7 for (Na(+),K(+))-stimulated ATPase and 2.6 for Na(+)-stimulated ATPase. 8. Organic solvents inactivated the ATPase activities, to which treatment the K(+)-stimulated ATPase was the most resistant. 9. The phosphorylation of the enzyme preparation became less dependent on Na(+) with decreasing pH. This Na(+)-independent phosphorylation at low pH was sensitive to K(+) and hydroxylamine as well as the Na(+)-dependent phosphorylation at neutral pH.     

Metachromatic Reaction of Pancreatic B Cells to Toluidine Blue; Influence of pH on Staining

The granules of islet B cells show an intense β metachromasia when paraffin sections of pancreas fixed in Bouin's fluid or formalin are dipped for 1 min in a 0.1% aqueous solution of toluidine blue O₂ buffered to pH 6.0 with acetate or phosphate. This reaction provides a quick method for surveying the condition of B cells in experimental work. A weak staining is observable at pH 4.5 and becomes distinct at pH 5.5-6.0. Oxidation of sections (0.25% KMnO₄ in 0.5% H₂SO₄, for 1 min, recommended) prior to staining intensifies the metachomatic reaction conspicuously. The metachromatic substance could not be demonstrated after fixation in either ethanol or acetone. It corresponds to the aldehyde fuchsin-positive and pseudoisocyanin-metachromatic substance in its occurrence and distribution in the B cells, as shown by different physiological states of various animals, including fasted and glucose-administered guinea pigs. It is thought to be topographically coincident but not necessarily identical to insulin.     

A numerical solution of theVolterra equation for the growth of an animal population accompanying an autotoxic effect

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Contribution from the Department of Fisheries, Faculty of Agriculture, Kyoto University.     

Identification of Escherichia coli proteins cross-reacting with antibodies against region 2.2 peptide of RNA polymerase sigma subunit.

Antisera against a synthetic tetradecameric peptide with the sequence DLIQEGNIGLMKAV, which is present in region 2.2 of both sigma 70 and sigma 32 subunits of Escherichia coli RNA polymerase, cross-reacted with more than 10 E. coli proteins including these two sigma subunits. Four major species of these cross-reacting proteins (SCRPs) were purified. N-Terminal amino acid sequence analysis revealed that one of them (SCRP-27A) was an as yet unidentified protein while the other three (SCRP-34, SCRP-27B and SCRP-23) were thioredoxin reductase, ribosomal protein S2, and alkyl hydroperoxide reductase, respectively. Immunological competition experiments with various fragments of this sigma region 2.2 peptide indicated that the anti-sigma peptide serum contained at least three different species of antibodies. All the four SCRPs analyzed here reacted with an antibody against a C-terminus-proximal epitope.     

Isolation of cDNA clone encoding rat senescence marker protein-30 (SMP30) and its tissue distribution.

We have isolated and characterized two cDNA clones encoding senescence marker protein-30 (SMP30), the amounts of which are known to decrease androgen-independently with aging in the livers of rats. Of these cDNA clones, one consisted of 1588 bp nucleotides and the other of 1195 bp nucleotides generated by alternative polyadenylation. These two cDNA clones shared the same open reading frame, but the larger species had 393 bp nucleotides of 3' untranslated region in addition to the first polyadenylation site of smaller species. Northern hybridization analysis showed that two species of mRNA (1.7 kb and 1.4 kb) located in the liver and kidney were consistent with these short and long forms of cDNA. The open reading frame, 897 bp could encode 299 amino acids. The estimated molecular weight and pI of the deduced polypeptide were 33,387 and 5.1, respectively. Furthermore, immunohistochemical analysis confirmed that SMP30 was preferentially localized in the hepatocytes and renal proximal tubular epithelium. Genomic Southern hybridization analysis demonstrated that SMP30 was widely conserved among higher animals. A computer-assisted homology analysis of nucleic acid and protein databases revealed no remarkable homology with other known proteins. Therefore, SMP30 seems to be a novel protein. In addition, the existence of putative A-U rich mRNA degradation signals and protein degradation signals (PEST sequence) in the structure of SMP30 may suggest important regulatory function of this unique protein manifested by changes in its concentrations.