Adenosine 3′,5′-Cyclic Monophosphate-Deficient Mutants of Vibrio cholerae

Two adenosine 3',5'-cyclic monophosphate (AMP)-deficient mutants of Vibrio cholerae (biotype El Tor) were successfully isolated by nitrosoguanidine treatment followed by pencillin screening for pleiotropic sugar-negative clones. Exogenous cyclic AMP is required for the fermentation of sucrose, trehalose, fructose, maltose, and mannose but not of glucose, as well as for the formation of normal flagella and specific somatic antigens. A striking characteristic of the mutants is their growth behavior at higher temperatures. They cannot grow on TCBS selective plates at 37 C or higher unless they are provided with a supply of exogenous cyclic AMP, although they are capable of producing colonies on the same medium, even without cyclic AMP, at temperatures lower than 30 C. Since the mutants are converted to spheroplasts, spindle forms, and spiral filaments in cyclic AMP-free media at 37 C, and this phenomenon is stopped by the addition of cyclic AMP or a combination of 20% sucrose and 0.2% magnesium chloride, it is assumed that cyclic AMP is essential for the synthesis of the cell wall of V. cholerae at higher temperatures.     

Electron microscopic demonstration of carbonic anhydrase activity in mouse liver cells

Summary Carbonic anhydrase (CAH) activity was demonstrated ultracytochemically in the mouse liver cells fixed with 1% glutaraldehyde buffered to pH 7.2 with 0.1 M cacodylate buffer containing 0.1 M sucrose and other aldehyde fixatives. After the fixed 25–40 section were incubated in Hansson's incubation medium containing 0.2 M sucrose, the cobalt phosphate formed by the action of CAH was converted to lead phosphate by immersing the incubated sections into 0.1% lead nitrate aqueous solution.The lead phosphate precipitate was observed very well on the plasma membrane of hepatocytes in Disse space and of endothelial cells or erythrocytes, and very slightly on the external coat of microvilli in bili canaliculi.In the tissues fixed with 4% formaldehyde, the deposits were found very barely on the microvilli in the space of Disse and the plasma membrane of the endothelial cells or the erythrocytes.As the -hydroxyadipaldehyde-fixed tissues showed the highest the CAH activity but had not a good preservation of morphology, this fixative is not suitable for the electron microscopic histochemistry of CAH.The tissue incubated in medium containing Diamox exhibited non-specific deposits in all over the cell, which were lost when the tissue was treated in Diamox solution before incubation.     

Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes

We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid freezing and the freeze-substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at -80 degrees C. Some of the freeze-substituted tissues were embedded in Lowicryl K4M at -20 degrees C; the others were returned to room temperature and embedded in Epok 812 at 60 degrees C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepatocytes was very well preserved compared with that of conventionally processed tissues. The labeling for catalase was confined to peroxisomes. When the labeling density was compared among freeze-substituted tissues and conventionally processed tissues, that of freeze-substituted and Lowicryl K4M-embedded tissues was the most intense. These results show the usefulness of freeze-substituted tissues for immunohistochemical analysis of cell organelles.     

Secretory carcinoma of the endometrium

A case of secretory carcinoma of the endometrium in a 21-year-old woman is reported. Endometrial smears were interpreted as showing a differentiated adenocarcinoma. Smears of ascitic fluid obtained during subsequent surgery showed similar findings; periodic acid-Schiff staining of the smears revealed abundant positive material in the cytoplasm. These findings were interpreted as evidence of secretory carcinoma, which was confirmed by histopathologic study of the biopsy and surgical specimens.     

Purification and characterization of the active serine: pyruvate aminotransferase of rat liver mitochondria expressed in Escherichia coli

In the previous study (Oda, T., et al. (1985) Eur. J. Biochem. 150, 415-421), we isolated a cDNA clone which expressed in Escherichia coli a specific size of product having the activity of rat liver serine:pyruvate aminotransferase (SPTm). This specific product (SPT10) was purified to homogeneity through three different column chromatographies. The amino acid composition and N-terminal amino acid sequence of the purified enzyme agreed with those predicted from the nucleotide sequence of cDNA and showed that SPT10 consists of the whole amino acid sequence of mature SPTm and several extra amino acid residues at the N-terminus. The catalytic and physical properties of SPT10, such as substrate specificity, Km for alpha-keto acids, electric charge, and quaternary structure, were all very similar to those of SPTm. Using several cDNA clones which lack a 5'-terminal sequence corresponding to a portion of the N-terminal amino acid sequence of SPTm, we examined the expression profile of the specific product in bacteria transformed with each cDNA clone. The products encoded by these cDNAs were segregated into inclusion bodies and were neither catalytically active nor easily solubilized by sonication. In contrast, the inclusion bodies were not formed in the bacteria transformed with the cDNA clone for SPT10.     

Immunogold Localization of Ribulose-1,5-Bisphosphate Carboxylase with Reference to Pyrenoid Morphology in Chloroplasts of Synchronized Euglena gracilis Cells

Euglena gracilis strain (Z) cells were synchronized under photoautotrophic conditions using a 14 hour light:10 hour dark regimen. The cells grew during the light period (growth phase) and divided during the following 10 hour period either in the dark or in the light (division phase). Changes in morphology of the pyrenoid and in the distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within the chloroplasts were followed by immunoelectron microscopy during the growth and division phases of Euglena cells. Epon-embedded sections were labeled with an antibody to the holoenzyme followed by protein A-gold. The immunoreactive proteins were concentrated in the pyrenoid, and less densely distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. Toward the end of the division phase, the pyrenoid began to form in the center of a chloroplast, and the immunoreactive proteins started to concentrate over that rudimentary pyrenoid. During the growth phase, small areas rich in gold particles, called `satellite pyrenoid,' were observed, in addition to the main pyrenoid. From a comparison of photosynthetic CO₂-fixation with the total carboxylase activity of Rubisco extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO₂-fixation in photosynthesis.     

Mechanism of Methane Transport from the Rhizosphere to the Atmosphere through Rice Plants

To clarify the mechanisms of methane transport from the rhizosphere into the atmosphere through rice plants (Oryza sativa L.), the methane emission rate was measured from a shoot whose roots had been kept in a culture solution with a high methane concentration or exposed to methane gas in the gas phase by using a cylindrical chamber. No clear correlation was observed between change in the transpiration rate and that in the methane emission rate. Methane was mostly released from the culm, which is an aggregation of leaf sheaths, but not from the leaf blade. Micropores which are different from stomata were newly found at the abaxial epidermis of the leaf sheath by scanning electron microscopy. The measured methane emission rate was much higher than the calculated methane emission rate that would result from transpiration and the methane concentration in the culture solution. Rice roots could absorb methane gas in the gas phase without water uptake. These results suggest that methane dissolved in the soil water surrounding the roots diffuses into the cell-wall water of the root cells, gasifies in the root cortex, and then is mostly released through the micropores in the leaf sheaths.     

Clonal dental pulp cells (RDP4-1, RPC-C2A) synthesize and secrete osteopontin (SPP1, 2ar).

Dental pulp cells play an important role in maintaining dental mineralized tissue throughout life. Supplementary mineralization such as reparative dentin and pulp stone frequently occurs after primary dentin formation. Dental pulp cells are thought to be closely associated with such mineralization. We found that clonal rat dental pulp cells, RDP4-1 and RPC-C2A, produce and secrete osteopontin, but do not synthesize phosphophoryn which is a major noncollagenous protein found in dentin. The dental pulp osteopontin was highly phosphorylated and identified by thrombin susceptibility and immunoprecipitation with osteopontin/2ar antibody. Osteopontin synthesis markedly increased by 12-O-tetradecanoylphorbol-13-acetate (TPA) as observed in many osteoblastic cells. This study indicates that these cells can produce osteopontin as a major phosphoprotein and suggests that the synthesis of osteopontin could be used as a characteristic marker of dental pulp cells.     

A restriction fragment length polymorphism (RFLP) locus of rat (Rattus norvegicus) linked to theCs-1 locus of rat linkage group XIII

A novel restriction fragment length polymorphism (RFLP) in inbred rats was revealed by Southern blot analysis with a clone arbitrarily chosen from a rat genomic library as a probe. A clone named alpha 403 showed interstrain variations in the length of the EcoRI and HindIII fragments. The EcoRI fragments were either 0.7 or 3 kb, those of HindIII were either 4.5 or 5 kb, and three types were identified as combinations of those fragments in 20 inbred rat strains. These types segregated in backcross progeny as codominant alleles. The locus for the RFLP was thus named A403. Analysis of linkage between the RFLP locus and 13 other loci reveal that the A403 locus was closely linked to the Cs-1 locus (15 +/- 5.2%), which belongs to rat linkage group XIII.