Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases

A metagenomic cosmid library was prepared in Escherichia coli from DNA extracted from the contents of rabbit cecum and screened for cellulase activities. Eleven independent clones expressing cellulase activities (four endo-β-1,4-glucanases and seven β-glucosidases) were isolated. Subcloning and sequencing analysis of these clones identified 11 cellulase genes; the encoded products of which shared less than 50% identities and 70% similarities to cellulases in the databases. All four endo-β-1,4-glucanases and all seven β-glucosidases, respectively, belonged to glycosyl hydrolase family 5 (GHF 5) and family 3 (GHF 3) and formed two separate branches in the phylogenetic tree. Ten of the 11 cloned cellulases exhibited highest activities at pH 5.5 ∼ 7.0 and 40 ∼ 55°C, a condition similar to that in the rabbit cecum. All the four endo-β-1,4-glucanases could hydrolyze a wide range of β-1,4-, β-1,4/β-1,3- or β-1,3/β-1,6-linked polysaccharides. One endo-β-1, 4-glucanase gene, umcel5G, was overexpressed in E. coli, and the purified recombinant enzyme was characterized in detail. The enzymes cloned in this work represented at least some of the cellulases operating efficiently in the rabbit cecum. This work provides the first snapshot on the cellulases produced by bacteria in rabbit cecum.     

Aminopeptidase N- and human blood group A-antigenicity along the digestive tract and associated glands in the rabbit

Summary The cellular localization of an aminopeptidase N homologous to the brush-border intestinal enzyme and that of human blood group A-substances were investigated using the immunofluorescence technique on thin frozen sections (200 nm) of the digestive tract and associated glands of A⁺ and A^– rabbits. Aminopeptidase N was found to be a common specific marker of both the apical region of plasma membrane of acinar cells in submaxillary and parotid glands and pancreas and the brush border of jejunum and colon absorbing cells. In hepatocytes, the enzyme was localized in the sinusoidal domains. Soluble A-substances were present in mucus secretory granules of intestinal goblet cells and those of stomach and gall bladder mucous cells. In contrast, the mucous acini of sublingual and submaxillary glands were devoid of A-antigenicity. The columnar cells of striated ducts of these glands exhibited A-antigenicity. Soluble A-substances were also found in zymogen granules of parotid and pancreas acinar cells and those of stomach chief cells. Moreover, in all cells secreting A-substances, and in the non-secreting absorbing intestinal cells, the glycoproteins of the plasma membrane bore A-determinants. Aminopeptidase N was one of the membrane-bound glycoproteins that bore A-determinants in cells that expressed A-antigenicity.     

Parthenogenesis of rabbit oocytes activated by different stimuli

Oocyte activation is one of the essential elements determining the success of nuclear transfer and the subsequent development of cloned embryos both in vitro and in vivo. Experiments were conducted to optimize the protocol for oocyte activation in a regular nuclear transfer study. In vivo derived oocytes were collected at 14–15 h from New Zealand white rabbits after ovulation treatment and were activated +18 h post-ovulation treatment. Single activation agents including calcium ionophore (A23187, 5 μM, 5 min), ethanol (Eth, 7%, 7 min), and thimerosal (200 μM, 10 min) were tested. Cleavage rates were highest in the ethanol-treated group (37%) compared to other treatments (19–25%). Very low blastocyst rates (2–3%) resulted which were not significantly different among treatments (P>0.05). Combined single agent treatment (calcium stimulators) with protein kinase inhibitor, 6-DMAP were used to achieve a full oocyte activation. Both pronuclear and blastocyst formation rates were significantly higher (P<0.05) in the Eth+6-DMAP treatment group (38 and 27%) than in the other groups (16–21 and 7–9%, respectively, P<0.05). Low (0.2 mM) and high (2.5 mM) concentrations of 6-DMAP treatments with different treatment lengths (1.5 and 3.5 h) in the combined groups were also compared. Blastocyst formation and cleavage rates were greater in the high concentration with less treatment time groups (36% versus 4–20%, P<0.05). In conclusion, single activation agents, either Ca²⁺ stimulators or protein kinase inhibitors, could not fully activate mature rabbit oocytes. The best activation procedure obtained in this study was the Eth+6-DMAP combined treatment, which may be incorporated into regular nuclear transfer or cloning protocols.     

A study of the mechanism of action of pyridoxal isonicotinoyl hydrazone at the cellular level using reticulocytes loaded with non-heme 59Fe

Pyridoxal isonicotinoyl hydrazone (PIH) has recently been identified as a new iron chelating agent with a high degree of iron mobilizing activity in vitro and in vivo which makes this compound a candidate drug in the treatment of iron overload. This study was undertaken to elucidate the mechanism of action of the iron mobilizing activity of PIH at the cellular level. An in vitro system of rabbit reticulocytes with a high level of non-heme ⁵⁹Fe was used as a model of iron overload. The effects of various biochemical and physiological manoeuvers on the mobilization of ⁵⁹Fe by PIH from the cells were studied. The fate of [¹⁴C]-PIH in the in vitro system was also studied. Studies were also carried out using a crude mitochondrial fraction. The results indicate three phases of the iron mobilizing activity of PIH: (1) the entry of PIH into erythroid cells seems to be by passive diffusion; (2) chelation occurs mainly from mitochondria and may depend on the availability of iron in a low molecular weight, non-heme pool. Chelation seems to be enhanced by reduction of Fe (III) to Fe (II); (3) the exit of the PIH₂-Fe complex is an energy-dependent process. Iron mobilization by PIH is not dependent on (Na⁺ + K⁺)-ATPase activity, external ionic composition, or external hydrogen ion concentration. Membrane fluidity does not seem to play a role in PIH-Fe mobilization. The exit of the PIH₂-Fe complex is inhibited by anti-microtubule agents (vinca alkaloids but not colchicine)_suggesting that the PIH₂-Fe complex is actively extruded from the cell by a microtube-dependent event.     

Characterization of an endothelial protein in the developing rabbit kidney

A new protein (EnPo 1 antigen) abundant on endothelial cells and glomerular podocytes has been characterized by means of the mouse monoclonal antibody EnPo 1. Following electrophoretical separation of rabbit kidney homogenates EnPo 1 recognized a protein with a molecular weight of 110 kDa and an isoelectric point of 5.9 in Western blots. Using immunohistological techniques, the EnPo 1 antigen has been localized in high concentrations on glomerular podocytes of different developmental stages. Furthermore, the EnPo 1 antigen was expressed on endothelial cells of all adult rabbit organs tested so far. Detailed analysis of neonatal rabbit kidney revealed the abundance of EnPo 1 antigen on both differentiated vessels as well as on immature endothelial cells and endothelium of the microvasculature. Thus, for the first time a marker for in situ investigations of angiogenic processes within the mammalian kidney is available. Analysis of kidney cryosections by confocal laser scan microscopy revealed a direct connection between mature and differentiating vessels in the outer kidney cortex. Furthermore, two differentially organized cell populations discriminated by their EnPo 1 binding pattern were localized in the embryonic renal cortex. Morphologically, these cells were not distinguishable from other mesenchymal cells.     

Neuronal systems immunoreactive with antiserum to lamprey gonadotropin-releasing hormone in the brain of Petromyzon marinus

Summary The wall structure of arteriovenous anastomoses in the rabbit ear was investigated. (1) Clusters of epithelioid smooth muscle cells form 3–4 longitudinally oriented plicae. The channel shows a single, irregularly outlined lumen, and its wall is very thin between adjacent plicae. (2) Endothelial cells covering the plicae protrude into the lumen, thus suggesting active contraction or shortening of the plicae. (3) The tunica adventitia is composed of 4–6 sheaths of flat fibroblasts, which may serve as a barrier to prevent loss of neurotransmitters. Processes of some of the fibroblasts also extend into the tunica media. (4) The tunica media is composed of an outer circular layer of typical smooth muscle cells, and an inner longitudinally running plica of ramified smooth muscle cells. Wide intercellular spaces between these ramified cells are filled with collagen fibrils, microfibrils, amorphous intercellular substances, and fibroblasts. Fibroblasts form close membrane contacts with each other, and with the smooth muscle cells. (5) Fibroblasts and other connective tissue components may function as an elastic support during active motility of the anastomotic channel.     

Variations in (Ca2+ + Mg2+)-ATPase,its inhibitor protein and calmodulin of density (age) separated rabbit erythrocytes

Density (age) separated rabbit erythrocytes were examined for differences in the activities of calmodulin and the protein inhibitor of membrane (Ca²⁺ + Mg²⁺)-ATPase (Lee, K.S. and Au, K.S. (1983) Biochim. Biophys. Acta 742, 54–62) as well as response of the ATPase towards these protein modulators. It was found that activities of the cytosol protein-bound and free inhibitor as well as membrane-bound inhibitor were higher in top (young) cells as compared to bottom (old) cells. Though the activity of the divalent cation associated membrane calmodulin pool was also higher in young cells, calmodulin activity in the erythrosol remained constant in cells from both age groups. The pool of membrane-associated inhibitor was shown to have greater influence on the ATPase than the membrane-associated calmodulin pool. The influence was more pronounced with inhibitor derived from old than from young cell membranes. Response of the young cell ATPase towards the protein inhibitor was better than the old cell enzyme at low inhibitor concentration. At higher inhibitor concentration, however, response of the ATPase from both cell types was similar.     

Evidence for two distinct lysophospholipase activities that degrade lysophosphatidylcholine and lysophosphatidic acid in neuronal nuclei of cerebral cortex

Neuronal nuclei were isolated from immature rabbit cerebral cortex and nuclear lysophospholipase activities studied using two different 1-acyl lysophospholipids: lysophosphatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA). Our interest in these two lysolipids arose from the observation that lysoPA could promote the acetylation of lysoPC by substantially inhibiting a very active nuclear lysoPC lysophospholipase activity, in a competitive manner (R.R. Baker, H.-y. Chang, Mol. Cell. Biochem. (1999) in press). As there was also evidence for nuclear lysoPA deacylation, it was of interest to see whether one activity could possibly utilize both lysolipid substrates. We now have evidence for two separate lysophospholipase activities in neuronal nuclei. The lysoPC lysophospholipase activity was the more active, more highly enriched in the neuronal nuclei, and showed optimal activity at pH 8.4–9, while the lysoPA lysophospholipase activity was maintained over a much broader pH range. The lysoPC activity was substantially inhibited by free fatty acid, and showed considerable stimulation by serum albumin, while the activity utilizing lysoPA was much less affected by these agents. When lysoPC was added to incubations containing radioactive lysoPA, there was no significant inhibition found in rates of release of radioactive fatty acid, indicating that the lysoPA lysophospholipase activity did not utilize the lysoPC substrate. In incubations with lysoPC, MgATP and CoA brought about a sizable formation of phosphatidylcholine whose radioactivity was equally distributed between the sn-1 and sn-2 positions suggesting labelling both directly from the lysoPC substrate and from fatty acid produced by the lysophospholipase activity. By comparison, with the radioactive lysoPA substrate, MgATP and CoA promoted relatively lower levels of phosphatidic acid formation whose principal labelling came directly from the radioactive lysoPA. Largely because of the high activity of the nuclear lysoPC lysophospholipase, there is considerable potential in the neuronal nucleus to limit the use of lysoPC in other reactions, such as the formation of acylPAF (1-acyl analogue of platelet activating factor). It is of interest that conditions associated with brain ischaemia such as increased free fatty acid levels, falling pH and declines in MgATP may allow a preservation of neuronal nuclear lysoPC levels for acetylation. The existence of a separate lysophospholipase activity for lysoPA allows an independent control of lysoPA which can serve as an important regulator of the nuclear lysoPC lysophospholipase.     

Scanning electron-microscopic study of Reissner's fiber in the caudal spinal cord of the cat and rabbit

Summary The caudal portion of Reissner's fiber was examined by scanning electron microscopy (SEM) in the spinal cord of the cat and rabbit. In some preparations of both species the fiber displayed in the sinus terminalis of the central canal either stump-like terminations or structural modifications such as knot-like swellings and convolutions. In the same area homogeneous material could also be found, which obviously originated from the disintegrating fiber.     

Regional Distribution of Substance P- and Thyrotrophin-Releasing Hormone-Like Immunoreactivity and Indoleamines in the Rabbit Spinal Cord

The distribution of thyrotrophin-releasing hormone (TRH), substance P, and the indoleamines [5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA)] has been examined in selected regions of the thoracic and lumbar spinal cord of the rabbit using sensitive radioimmunoassays for the first two and HPLC with electrochemical detection for the indoleamines. The levels of TRH- and substance P-like immunoreactivity (TRH-I and SP-I, respectively) were greatest in the ventral and dorsal grey matter, respectively. The level of TRH-I in most thoracic regions was greater than that in equivalent lumbar regions, but the only segmental difference in SP-I was in the ventral grey matter, where the lumbar segment contained more immunoreactivity. 5-HT and 5-HIAA were more evenly distributed than either peptide and showed no segmental variation in levels in equivalent regions, but the ventral grey matter contained significantly higher levels of 5-HT and had a greater 5-HT/5-HIAA ratio than all other regions. The absolute levels and the overall distribution of SP-I, TRH-I, and indoleamines in the thoracolumbar cord of the rabbit was very similar to that previously reported in both rats and humans, and the possible functional role of the peptides and indoleamines in spinal neurones is discussed.