Immunocytochemical localization of ornithine transcarbamylase in rat intestinal mucosa. Light and electron microscopic study

We investigated light and electron microscopic localization of ornithine transcarbamylase (OTC) in rat intestinal mucosa. In the immunoblotting assay of OTC-related protein, a single protein band with a molecular weight of about 36,500 is observed in extracts of liver and small intestinal mucosa but is not observed in those of stomach and large intestine. For light microscopy, tissue slices of the digestive system were embedded in Epon and stained by using anti-bovine OTC rabbit IgG and the immunoenzyme technique. For electron microscopy, slices of these and the liver tissues were embedded in Lowicryl K4M and stained by the protein A-gold technique. By light microscopy, the absorptive epithelial cells of duodenum, jejunum, and ileum stained positively for OTC, but stomach, large intestine, rectum, and propria mucosa of small intestine were not stained. Electron microscopy showed that gold particles representing the antigenic sites for OTC were confined to the mitochondrial matrix of hepatocytes and small intestinal epithelial cells. However, the enzyme was detected in mitochondria of neither liver endothelial cells, submucosal cells of small intestine, nor large intestinal epithelial cells. Labeling density of mitochondria in the absorptive epithelial cells of duodenum, jejunum, and ileum was about half of that in liver cells.     

CO(2) Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities

The growth of the halotolerant cyanobacterium Aphanothece halophytica, previously adapted to 0.5 molar NaCl, was optimal when NaCl concentration in culture medium was in the range 0.5 to 1.0 molar. The growth was delayed at either too low or too high salinities with lag time of ca. 0.5 day in 0.25 molar NaCl and ca. 2 days in 2 molar NaCl under the experimental conditions. However, the growth rates at the logarithmic phase were similar in the culture media containing NaCl in the range 0.25 to 2.0 molar. The capacity of photosynthetic CO₂ fixation increased 3.7-fold in the cells at the logarithmic phase as NaCl concentration in the culture medium increased from 0.25 to 2.0 molar. The protein level of ribulose 1,5-bisphosphate carboxylase/oxygenase was also found to increase with increasing salinity using both an immunoblotting method and protein A-gold immunoelectron microscopy. These results indicate that high photosynthetic capacity and high ribulose 1,5-bisphosphate carboxylase/oxygenase content may entail an important role in betaine synthesis and adaptation of the A. halophytica cells to high NaCl level.     

Altering the position of the first horizontal cleavage furrow of the amphibian (Xenopus) egg reduces embryonic survival.

The animal/vegetal cleavage ratio (AVCR), defined as the ratio of the height of the animal blastomere to the height of the Xenopus embryo at the 8 cell stage, can be shifted by placing embryos in novel gravitational fields: clinostating (microgravity simulation) increases AVCR, and centrifugation (hypergravity simulation) reduces AVCR. This report contributes to an understanding of the subcellular mechanism responsible for the furrow relocation and assesses its significance. Embryo inversion and D2O immersion were found to increase AVCR, and cold shock was found to reduce AVCR. Based on the additive or antagonistic effects of combined treatments, it is postulated that the primary cause of AVCR changes is an alteration in the distribution of yolk platelets and the rearrangement of microtubule arrays. Embryos with a decreased AVCR exhibited reduced survival in early developmental stages, indicating serious difficulties in cleavage, blastulation and/or gastrulation. Cold-shocked embryos with a reduced AVCR could be rescued by D2O pretreatment or clinostating, an observation which supports the notion that changes accompanying AVCR modifications represent the primary cause of the reduction in percent survival.     

Germination ecology of coastal plants in relation to salt environment

Responses of seed germination to salinity were examined using 37 species collected from salt marshes, cliffs, and fore (unstable) and hind (stable) sand dunes along Japanese coasts. For comparison, seed germination of nine inland species was also examined. The soil salinities in salt marshes ranged from 150 to 300 mmol/L NaCl, whereas those in fore and hind dunes ranged from 0 to 150 mmol/L NaCl, with a few exceptions. Cliff soils showed relatively high salinities up to 300 mmol/L NaCl. Ciff and foredune soils that encountered a typhoon and storm showed high salinities >300 mmol/L NaCl. Salt tolerance in seed germination of coastal plants was ordered by comparing the responses of percentage and rate of germination to salinity conditions up to 200 mmol/L NaCl, being in the order of salt marsh>cliff>foredune≅hind dune≅inland. Thse results indicate that salt tolerance in seed germination of coastal plants is closely related to the salinity conditions of their habitats. Germination experiments under favorable conditions showed that a high percentage of the seeds of salt marsh species germinate rapidly, those of diff species germinate slowly and those of foredune species exhibit a low percentage and low rate of germination. It seems that these germination characteristics contribute to the success of germination at the ‘safe site’ and the subsequent survivorship of emerged plants in their natural habitats.     

Use of mammalian cell expression cloning systems to identify genes for cytokines, receptors, and regulatory proteins.

Mammalian cell expression cloning has become a standard technique for the isolation of mammalian genes or cDNAs. Its advantage is that the biological functions of the gene of interest are used for cloning. Therefore, the identified cDNAs or genes should be functional in vivo, and there is no need for physical or chemical information about the gene products, so that protein purification in sufficient quantity to raise antibodies or to obtain amino acid sequences is not necessary. Here, we summarize recent progress in mammalian cell cloning systems, and discuss the possible directions in which this technique will lead.     

In vitro and in vivo transferrable beta-lactam resistance due to a new plasmid-mediated oxyiminocephalosporinase from a clinical isolate of Proteus mirabilis

A new plasmid-mediated beta-lactamase (FPM-1) with an isoelectric point of 7.2 and a molecular weight of 26,000 was found in a cefuroxime-resistant clinical isolate of Proteus mirabilis strain 6003. FPM-1 can be classified as a type I oxyimino-cephalosporinase on the basis of its substrate specificity and inhibition pattern by clavulanic acid etc., and its conferred resistance on both the strain and transconjugants against most oxyme-type cephalosporins as well as the older ones but not against cefamycins and a few exceptional oxyme-type cephalosporins such as ceftizoxime, ceftazidime and cefixime. In a murine systemic infection model, only these FPM-1-stable drugs exhibited protective activity against the FPM-1-producing P. mirabilis 6003 similar to that against a nonproducing derivative strain. The FPM-1-mediated cefuroxime resistance in P. mirabilis 6003 was transferred to co-infected Escherichia coli 7004 at frequencies between 3.8 x 10(-3) and 4.0 x 10(-2) in a murine ascending urinary tract infection model. In the same infection model due to the FPM-1-producing E. coli transconjugant, FPM-1-stable cefixime was significantly more effective than FPM-1-labile cefteram pivoxil, although both drugs had similar therapeutic effect against its FPM-1-nonproducing counterpart strain.     

Immunocytochemical localization of 2,4-dienoyl-CoA reductase in the liver of normal and di-(2-ethylhexyl)phthalate-fed rats

Summary Localization of 2,4-dienoyl-CoA reductase (DCR) in rat liver was studied using immunoenzyme and immunogold techniques. The animals were fed on a laboratory diet with or without 2% di-(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, for two weeks. For light microscopy (LM), semithin Epon sections were stained by immunoenzyme technique after removal of the epoxy resin. For electron microscopy (EM), ultrathin Lowicryl K4M sections were stained by the protein A-gold technique. By LM, in untreated rats reaction deposits showing the antigenic sites for DCR were present in the cytoplasmic granules. Hepatocytes, epithelial cells of interlobular bile duct, and sinus-lining cells contained these granules. After administration of DEHP, the cytoplasmic granules stained similarly. The staining intensity of the heaptocytes increased markedly, but that of the other cells decreased. The sinus-lining cells became mostly negative. By EM, gold particles indicating the antigenic sites for DCR were present in both the mitochondria and peroxisomes of hepatocytes of untreated rats. In the other cells, the gold label was confined to the mitochondria. After administration of DEHP, labelling intensity of the hepatocyte mitochondria increased markedly, but that of the peroxisomes conversely decreased. Quantitative analysis of labelling density showed that the mitochondrial DCR increased to about three times that in the untreated rat, but the peroxisomal DCR decreased to 1/6. The results show that in the rat liver, DCR exists in both, mitochondria and peroxisomes. DEHP can induce mitochondrial DCR, but not peroxisomal DCR.     

In vitro loss of hydrophobicity of trehalase from the brush border membrane of rabbit kidney cortex

Trehalase solubilized with 0.5% Triton X-100 and 0.5% deoxycholate from the brush border membrane of rabbit kidney cortex was all adsorbed on phenyl-Sepharose equilibrated with elution buffer containing no detergents, and all the adsorbed enzyme was eluted in one peak on the addition of 0.5% Triton X-100 to the elution buffer, in contrast to the results reported by Nakano and Sacktor (J. Biochem. 97, 1329-1335 (1985], who separated two forms of trehalase differing in hydrophobicity from rabbit kidney. On concentration of detergent-solubilized extracts, followed by incubation at 37 degrees C, however, there appeared trehalase nonadsorbable on phenyl-Sepharose, i.e. a hydrophilic trehalase. Various protease inhibitors added to the concentrated extracts did not inhibit this conversion at all. The concentration-incubation treatment also increased the proportion of trehalase that interacts with Con A-Sepharose. These results indicate that kidney trehalase that interacts with Con A-Sepharose. These results indicate that kidney trehalase is susceptible to some lytic action of a factor(s) intrinsic to the brush border membrane (limited autolysis), as seen with rabbit intestinal trehalase (Yokota et al., (1986) Biochim. Biophys. Acta 881, 405-414). Therefore, in studies of the molecular form of trehalase (and other proteins) in the brush border membrane of the kidney and intestine where a lot of hydrolases exist, it is very important to take account of limited autolysis which results in some chemical modifications without affecting enzymatic activity.     

A noncleavable signal for mitochondrial import of 3-oxoacyl-CoA thiolase

Rat 3-oxoacyl-CoA thiolase, an enzyme of the fatty acid beta-oxidation cycle, is located in the mitochondrial matrix. Unlike most mitochondrial matrix proteins, the thiolase is synthesized with no transient presequence and possesses information for mitochondrial targeting and import in the mature protein of 397 amino acid residues. cDNA sequences encoding various portions of the thiolase were fused in frame to the cDNA encoding the mature portion of rat ornithine transcarbamylase (lacking its own presequence). The fusion genes were transfected into COS cells, and subcellular localization of the fusion proteins was analyzed by cell fractionation with digitonin. When the mature portion of ornithine transcarbamylase was expressed, it was recovered in the soluble fraction. On the other hand, the fusion proteins containing the NH2-terminal 392, 161, or 61 amino acid residues of the thiolase were recovered in the particulate fraction, whereas the fusion protein containing the COOH-terminal 331 residues (residues 62-392) was recovered in the soluble fraction. Enzyme immunocytochemical and immunoelectron microscopic analyses using an anti-ornithine transcarbamylase antibody showed mitochondrial localization of the fusion proteins containing the NH2-terminal portions of the thiolase. These results indicate that the NH2-terminal 61 amino acids of rat 3-oxoacyl-CoA thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. Pulse-chase experiments showed that the ornithine transcarbamylase precursor and the thiolase traveled from the cytosol to the mitochondria with half-lives of less than 5 min, whereas the three fusion proteins traveled with half-lives of 10-15 min. Interestingly, in the cells expressing the fusion proteins, the mitochondria showed abnormal shapes and were filled with immunogold-positive crystalloid structures.     

Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.