Microheterogeneity of molecular forms of arginase in mammalian tissues

Two isoforms of arginase, A1 and A2, were found in rat liver, submaxillary gland and kidney as well as beef kidney. In beef liver, however, A2 was the only detectable form. Two additional forms, A3 and A4, found only in rat kidney were probably artifactitious. A1 and A2 exhibited chromatographic and immunological microheterogeneity. While A1 in rat liver and submaxillary gland was excluded by DEAE-cellulose (pH 8.3) and retained on CM-cellulose (pH 7.5), that (A'1) in beef and rat kidneys was excluded by both ion-exchangers. A2 in all tissues was retained on DEAE-cellulose, but not on CM-cellulose. Both A1 and A2 in rat liver and beef kidney, A1 from rat submaxillary gland and A2 from beef liver were precipitated by antibodies to rat and beef liver arginases. None of the forms in rat kidney (A1, A2, A3 and A4) showed any cross-reactivity to either antibody. Rat submaxillary gland A2 was precipitated by anti-rat liver arginase, but activated by anti-beef liver arginase. While the major molecular forms were A1 in rat liver and submaxillary gland and A2 in beef liver and rat kidney, the two forms occurred in equal proportions in beef kidney. It appears that different isoforms might function as components of the urea cycle in the liver of different mammals and of the arginine catabolic pathway in different extrahepatic tissues.     

Red cell aging results in a change of cell surface carbohydrate epitopes allowing for recognition by galactose-specific receptors of rat liver macrophages

Binding and uptake studies of in vitro aged or senescent rat erythrocytes by isolated rat liver macrophages suggest recognition by galactose-specific receptors on the cell surface of the macrophages. We analyzed carbohydrates exposed on old erythrocytes by plant lectins in an agglutination assay in comparison with freshly isolated untreated erythrocytes. Rat erythrocytes aged in vitro by storage are agglutinated by a panel of lectins that do not react with freshly isolated erythrocytes. Specificity of agglutination was shown by inhibition with monosaccharides. Antibodies eluted from senescent rat erythrocytes agglutinate in vitro aged as well as senescent rat erythrocytes, but not freshly isolated cells nor human erythrocytes. Galactose-specific lectins isolated from rat liver give similar results; they also agglutinate normal human erythrocytes. Agglutination by the liver lectin is inhibitable by galactose and N-acetylgalactosamine but not by N-acetylglucosamine or mannose. Furthermore, rat liver macrophages devoid of galactose-specific receptors show markedly reduced binding of senescent rat erythrocytes. We conclude that recognition of old rat erythrocytes is mediated by two systems: old erythrocytes expose different terminal sugar residues or a different arrangement of glycans when compared to young erythrocytes, rendering them recognizable by liver lectins and by autoantibodies.     

Multicolor spectral karyotyping of rat chromosomes

Rat and mouse have become important animal models to study various human diseases such as cancer. Cytogenetic analysis of the respective karyotypes is frequently required to investigate the causative genetic defects and especially neoplastic cells often show complex chromosome aberrations and many different marker chromosomes. However, structural homogeneity of the chromosomes in these species as well as less pronounced differences in banding patterns make it difficult to assign genetic abnormalities to certain chromosomes by conventional banding techniques. Here we report for the first time the successful application of multicolor spectral karyotyping (SKY) to rat chromosomes, which allows unequivocal identification of all rat chromosomes with the exception of chromosomes 13 and 14 in different colors, thus enabling the elucidation of even complex rearrangements in the rat karyotype. Flow-sorted chromosome specific painting probes for all 22 rat chromosomes (20 autosomes, X, and Y) were combinatorially labeled by a set of five different fluorochromes and hybridized in situ to metaphase spreads of a healthy rat, to diakineses from testicular material, and to cells from a rat FAO hepatoma cell line. Measuring the complete spectrum at each image point by using the SpectraCube((R)) spectral imaging system and respective computer software allowed identification of the individual rat chromosomes by their specific emission spectra. Classification algorithms in the analysis software can then display the rat chromosomes in specific pseudo-colors and automatically order them in a karyotype table. After its successful application to human and mouse chromosomes, spectral karyotyping of rat chromosomes now also allows cytogenetic screening of the complete rat genome by a single hybridization.     

gamma-Glutamyl transpeptidase: a single copy gene in the rat and a multigene family in the human genome

gamma-Glutamyl transpeptidase (GGT) genomic sequences were isolated from rat and human libraries using a rat GGT cDNA as a cross-species hybridization probe. Characterization of the human GGT clones by restriction mapping clearly establishes that at least four different GGT genes or pseudogenes are present in the human genome. All the rat genomic clones cover a 12.5-kilobase sequence and exhibit a unique restriction pattern. A precise quantitation of the rat GGT gene copy number by Southern blot analysis demonstrates that this sequence is present as a single copy/rat haploid genome. Therefore, the GGT gene organization is different between rat and human species; this raises the possibility of different regulatory mechanisms in the two species.     

Expression of Neuronal Kinesin Heavy Chain Is Developmentally Regulated in the Central Nervous System of the Rat

Abstract: The kinesin family of motor proteins comprises at least two isoforms of conventional kinesin encoded by different genes: ubiquitous kinesin, expressed in all cells and tissues, and neuronal kinesin, expressed exclusively in neuronal cells. In the present study, we have analyzed the expression of the two kinesin isoforms by immunochemistry at different stages of development of the rat CNS. We have found that the level of expression of neuronal kinesin is five to eight times higher in developing than in adult rat brains, whereas that of ubiquitous kinesin is only ∼2.5 times higher in maturing versus adult brains. Moreover, we have studied the distribution of neuronal kinesin by light microscopic immunocytochemistry in the rat brain at different postnatal ages and have found this protein not only to be more highly expressed in juvenile than in adult rat brains but also to show a different pattern of distribution. In particular, tracts of axonal fibers were clearly stained at early postnatal stages of development but were markedly unlabeled in adult rat brains. Our results indicate that the expression of at least one isoform of conventional neuron-specific kinesin is up-regulated in the developing rat CNS and suggest that this protein might play an important role in microtubule-based transport during the maturation of neuronal cells in vivo.     

大鼠FMR1同源基因cDNA的克隆、测序与选择剪接的初步分析

应用ＲＴ－ＰＣＲ方法,从新生大鼠脑组织总ＲＮＡ扩增大鼠ＦＭＲ１同源基因的ｃＤＮＡ片段,克降至ｐＵＣ１８质粒中进行序列分析．获得从终止密码子起共１６８１ｂｐ的编码序列,尚缺少约２００ｂｐ的５′序列．所克隆的这部分大鼠ＦＭＲ１ｃＤＮＡ,不含有对应于人ＦＭＲ１基因的外显子１２及外显子１７第一和第三剪接受点之间的序列,提示大鼠ＦＭＲ１基因也有选择剪接表达．同源性分析显示,大鼠ＦＭＲ１与小鼠ＦＭＲ１基因的同源性为９７．７％,与人ＦＭＲ１基因的同源性为９４．９％;与小鼠ＦＭＲＰ（ＦＭＲ１蛋白）的氨基酸序列同源性为９８．４％,与人ＦＭＲＰ的氨基酸序列同源性为９７．９％．以大鼠ＦＭＲ１ｃＤＮＡ片段为探针检测到大鼠不同组织中ＦＭＲ１基因的选择剪接表达．上述结果为以大鼠为动物模型深入研究ＦＭＲ１基因功能奠定了基础．     

Uncoupling of muscle-specific protein expression in myocyte X myoblast heterokaryons

The inducibility of several rat skeletal muscle proteins was examined in heterokaryons formed by fusing differentiated chick myocytes to undifferentiated rat myoblasts. Chicken and rat proteins were distinguished using species-specific antibodies or by their different migrations in polyacrylamide or agarose gels. Both rat skeletal myosin light chain 1 and rat α-tropomyosin were induced in the heterokaryons. In contrast, neither rat acetylcholine receptors nor creatine kinase could be detected. These results suggest that chick myocytes may contain quantities of regulatory factors that are sufficient for the activation of some but not all of these rat muscle-specific proteins within the cellular context of the heterokaryon.     

Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies

Antibodies against a peptide (SP19) corresponding to a highly conserved, predicted intracellular region of the sodium channel alpha subunit bind rat brain sodium channels with a similar affinity as the peptide antigen, indicating that the corresponding segment of the alpha subunit is fully accessible in the intact channel structure. These antibodies recognize sodium channel alpha subunits from rat or eel brain, rat skeletal muscle, rat heart, eel electroplax, and locust nervous system. alpha subunits from all these tissues except rat skeletal muscle are substrates for phosphorylation by cAMP-dependent protein kinase. Disulfide linkage of alpha and beta 2 subunits was observed for both the RI and RII subtypes of rat brain sodium channels and for sodium channels from eel brain but not for sodium channels from rat heart, eel electroplax, or locust nerve cord. Treatment with neuraminidase reduced the apparent molecular weight of sodium channel alpha subunits from rat and eel brain and eel electroplax by 22,000-58,000, those from heart by 8000, and those from locust nerve cord by less than 4000. Our results provide the first identification of sodium channel alpha subunits from rat heart and locust brain and nerve cord and show that sodium channel alpha subunits are expressed with different subunit associations and posttranslational modifications in different excitable tissues.     

The role of the C-terminal region of rat brain natriuretic peptide in receptor selectivity.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have different C-terminal tail structures compared with the rather conservative ring structures which consist of 17 amino acid residues. To examine the different effects of the tail structures of ANP and BNP on their interaction with receptors, we synthesized several peptide analogs and measured their biological actions in three different assay systems. Deletion of the C-terminal tail from rat BNP did not effect the vasorelaxation activity against rat aorta, but it promoted cGMP production in cultured rat aortic smooth muscle cells (RASMC). Deletion of the C-terminal tail from rat ANP diminished both vasorelaxant and cGMP producing activities. In a binding competition assay with RASMC and [125I]rat ANP-(1-28), the competition activities of both ANP and BNP were greatly reduced by C-terminal deletion. In addition, we obtained agonists with novel receptor selectivity.     

Responsibility of tRNA(Ile) for spermine stimulation of rat liver Ile-tRNA formation

To determine whether tRNA or aminoacyl-tRNA synthetase is responsible for spermine stimulation of rat liver Ile-tRNA formation, homologous and heterologous Ile-tRNA formations were carried out with Escherichia coli and rat liver tRNA(Ile) and their respective purified Ile-tRNA synthetases. Spermine stimulation was observed only when tRNA from the rat liver was used. Spermine bound to rat liver tRNA(Ile) but not to the purified aminoacyl-tRNA synthetase complex. Kinetic analysis of Ile-tRNA formation revealed that spermine increased the Vmax and Km values for rat liver tRNA(Ile). The Km value for ATP and isoleucine did not change significantly in the presence of spermine. Furthermore, higher concentrations of rat liver tRNA(Ile) tended to inhibit Ile-tRNA formation if spermine was absent. Spermine restored isoleucine-dependent PPi-ATP exchange in the presence of rat liver tRNA(Ile), an inhibitor of this exchange. The nucleotide sequence of rat liver tRNA(Ile) was determined and compared with that of E. coli tRNA(Ile). Differences in nucleotide sequences of the two tRNAs(Ile) were observed mainly in the acceptor and anticodon stems. Limited ribonuclease V1 digestion of the 3'-32P-labeled rat liver tRNA(Ile) showed that both the anticodon and acceptor stems were structurally changed by spermine, and that the structural change by spermine was different from that by Mg2+. The influence of spermine on the ribonuclease V1 digestion of E. coli tRNA(Ile) was different from that of rat liver tRNA(Ile). The results suggest that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation.     

The distribution of phosphofructokinase isoenzymes in the liver of camel, rat and rabbit

1. The distribution of phosphofructokinase isoenzymes have been compared among camel, rat and rabbit livers. 2. Only a single phosphofructokinase isoenzyme is present in the camel liver which has shown different physical and regulatory properties from the isoenzymes of rat and rabbit liver. 3. The ammonium sulphate precipitation curves of the camel and rabbit enzymes were monophasic, whereas the rat enzyme was biphasic. 4. Rabbit liver phosphofructokinase was slightly more anodic than the rat enzyme, whereas the camel enzyme was the least anodic as shown by the techniques of DEAE-cellulose chromatography and cellulose acetate electrophoresis. 5. Partially purified camel liver phosphofructokinase showed different regulatory properties from the rabbit and rat isoenzymes as the apparent Km values were 0.58, 0.45 and 0.82 mM respectively.     

Isolation and identification of a cDNA clone of rat placental lactogen II

The developing rat placenta expresses two placental lactogens at different stages of pregnancy: rat placental lactogen I from Days 11 to 13 of pregnancy and rat placental lactogen II (rPLII) from Day 12 to term. In this paper, we describe cDNA clones for rPLII, which have been isolated from a Day 18 rat placental cDNA library. The rPLII clones hybrid-select a mRNA which translates in vitro to a protein of 25,000 daltons. This protein is processed by dog pancreatic microsomes to a 22,000-dalton form, identical in size to rPLII isolated from pregnant rat serum. Both forms are precipitated by an anti-rPLII antiserum and an anti-ovine prolactin antiserum. The mRNA for rPLII is first expressed in Day 12 placenta and reaches a maximum at about Day 18 of pregnancy, in parallel with the appearance of the hormone in serum. Sequencing of the cDNA shows that, unlike human placental lactogen which is 85% homologous to human growth hormone at the amino acid level, rPLII is much more closely related to the prolactins. Thus, rPLII is 52% homologous to rat prolactin at the amino acid level, but only 34% related to rat growth hormone. This is the second placental lactogen to be fully characterized, and in the rat this hormone appears to have evolved by a route quite different from that which produced placental lactogen in humans.     

Expression and regulation of human and rat phosphodiesterase type IV isogenes

Type IV phosphodiesterases (PDE IV) specifically hydrolyze cAMP and are inhibited by rolipram. RT-PCR was applied to analyze the expression patterns of mRNAs for four cloned human and rat phosphodiesterase type IV isogenes (PDE IV-A, -B, -C and -D). Although these patterns were mostly coincident for the human and rat PDE IV genes, some differences were found between the two species. PDE IV-A expression was detectable in human blood but not in rat blood, suggesting a species-specific difference in the expression of this PDE IV isogene. PDE IV-C was neither detected in human or rat blood nor in different cell populations of the human immune system. It is further demonstrated that the PDE IV isogene expression is differentially regulated by cAMP in different cell types.     

Determination of the functional molecular size of vasopressin isoreceptors

The molecular size of vasopressin receptors in the intact membrane-bound state was determined by radiation inactivation (target size analysis). For the V1 receptor in rat liver a molecular size of (76 +/- 8) kDa was determined. For the V2 receptor in rat kidney and bovine kidney molecular sizes of (95 +/- 4) and (108 +/- 11) kDa were found. Statistical analysis gave evidence for size differences between rat liver and rat kidney receptors or differences between rat liver and bovine kidney receptors, but not between kidney receptors from different species. The results suggest that V1 and V2 receptors can be distinguished by functional properties as well as by their size.     

Diversity and complexity of uterine mRNA from rats of differing hormonal states.

The diversity and complexity of the uterine mRNA population has been compared at two different stages of uterine growth and development in the rat. Analysis by cDNA hybridization to homologous mRNA indicates that there are 8000 different sequences expressed in the immature rat uterus responding to just 4 h of oestradiol-17 beta-induced growth while the fully developed uterus expresses 36 000 sequences. As a check on our results, the complexity is re-analysed by hybridization of mercurated mRNA to total unique DNA. Analysis by this method indicates that the hormone-stimulated immature rat uterus contains 12 000 poly(A)-containing mRNA sequences while the fully differentiated adult tissue contains 53 000 diverse sequences.     

Immunological characterization of rat kininogens with monoclonal antibodies to T-kininogen. Distinction between the different domains of T-kininogen and the multiple rat kininogens.

A panel of 16 monoclonal antibodies (mAb) were produced against rat T-kininogen to characterize this family of proteins. These mAbs bound 125I-T-kininogen by radioimmunoassay as well as reacting strongly with immobilized T-kininogen in an enzyme-linked immunosorbent assay (ELISA). The reactivity of these antibodies with proteolytic fragments of T-kininogen demonstrated the recognition of several different epitopes. One antibody was specific for the domain 1 of the heavy chain and/or the light chain, twelve antibodies were specific for domain 2 and three antibodies were specific for domain 3. All monoclonal antibodies recognized the two forms of T-kininogen encoded by the two different T-kininogen genes, TI and TII kininogen, except antibody TK 16-3.1 which uniquely reacted with TII kininogen. Two antibodies recognizing domain 2 cross-reacted with the high-molecular-mass kininogen (H-kininogen), whereas all the other monoclonal antibodies were specific to T-kininogen and did not recognize the heavy chain of H-kininogen. None of the antibodies tested altered the thiol protease inhibitory activity of T-kininogen, its partial proteolysis by rat mast cell chymase or the hydrolysis of H-kininogen by rat urinary kallikrein. The use of these antibodies in the development of sensitive ELISA to measure T-kininogen levels in plasma, urine, liver microsomes and hepatocytes is described. Two different forms of T-kininogen were distinguished by these monoclonal antibodies in Western blotting using rat plasma. The localization of T-kininogen was defined using these monoclonal antibodies by immunohistochemistry in rat liver hepatocytes and rat kidney.     

Target size of 5'-nucleotidase in smooth muscle

Target size of the 5'-nucleotidase in six different smooth muscles was determined by radiation inactivation. The enzyme in the soluble fraction of rat myometrium and vas deferens gave a target size of approximately 80,000 daltons. The plasma membrane bound 5'-nucleotidase however, gave target size of 80,000 to 110,000 daltons in rat gastric fundus and vas deferens and dog stomach and ileum, 135,000 daltons in rat mesenteric artery and 210,000 daltons in rat myometrium.