Catalase transgenic mice: characterization and sensitivity to oxidative stress

The role of catalase in the antioxidant defense system was studied using transgenic mice [Tg(CAT)] harboring a human genomic clone containing the entire human CAT gene. Catalase activity was 2-fold higher in the tissues of hemizygous [Tg(CAT)(+/o)] mice and 3- to 4-fold higher in the tissues of homozygous [Tg(CAT)(+/+)] mice compared to wild type mice. The human CAT transgene was expressed in a tissue-specific pattern that was similar to the endogenous catalase gene. The levels of other major antioxidant enzymes were not altered in the tissues of the transgenic mice. Hepatocytes and fibroblasts from the Tg(CAT)(+/+) mice were more resistant to hydrogen peroxide-induced cell death but were more sensitive to paraquat and TNFalpha toxicity. Fibroblasts from the Tg(CAT)(+/+) mice showed reduced growth rate in culture without treatment and reduced colony-forming capability after gamma-irradiation compared to fibroblasts from wild type mice. In addition, the Tg(CAT)(+/+) animals were more sensitive to gamma-irradiation.     

Albumin recovery with centrifugal adsorption technology (CAT)

Centrifugal adsorption technology (CAT) is a new compact, countercurrent technology for efficient adsorption from large liquid streams by using adsorbent particles in the micrometer range. CAT seems particularly suited for the recovery of macromolecules at low concentrations, because the small particle dimensions lead to fast mass transfer rates. In this work, the potential of CAT for protein recovery is studied by model and experiment. A predictive model for the separation performance of CAT is presented, incorporating mass transfer resistance and axial dispersion transport in the liquid and the adsorbent phases. The model calculations were compared to experimental data for the adsorption of bovine serum albumin (BSA) on a standard commercial anion-exchange resin with particle diameter d(p) = 50 microm in a pilot-scale CAT apparatus. The model calculations accurately predicted the separation efficiency of CAT. The experimental set-up is shown to be mass transfer limited for the conducted experiments, which agrees with the model predictions. The model was also used to estimate the dimensions and performance of a CAT apparatus for the large-scale recovery of human serum albumin (HSA) from fermentation broth at the scale of 40 tons per year. The resulting equipment dimensions proved to be very small indeed, making CAT a potentially very attractive technology.     

Translational control of synthesis of simian virus 40 late proteins from polycistronic 19S late mRNA.

The simian virus 40 (SV40) 19S late mRNA is polycistronic, encoding multiple late proteins: agnoprotein, VP2, and VP3. We constructed a chloramphenicol acetyltransferase (CAT) transient expression vector in which the SV40 sequences between nucleotides 5171 and 1046 (via the SV40 origin of replication and including the late promoter) were inserted 5' to the cat gene; therefore, the AUG for CAT expression occurs after the AUGs for agnoprotein, VP2, and VP3. CAT enzyme activity assayed after transfection of these constructions indicates the level of CAT AUG utilization and, therefore, can be used as a measure of the ability of prior AUGs to intercept scanning ribosomes. Specifically, deletions and point mutations of the viral AUGs resulted in increased CAT enzyme activity owing to increased utilization of the downstream CAT AUG. To compare a variety of mutants, we used the levels of increase to calculate the translational efficiency of the viral AUGs. Some of our data agree with predictions of the modified scanning model (MSM). Little variation in downstream CAT AUG utilization was noted regardless of whether the VP2 AUG (in a weak MSM sequence context) was intact or removed. Hence, a scanning ribosome may easily bypass it. Similar analysis of the VP3 AUG (in a favorable MSM sequence context) demonstrated that it could efficiently intercept ribosomes prior to the downstream AUG. Overall, these data indicate that the structure of the 19S late mRNA and the relative efficiency of translational start codon utilization can account for the VP3/VP2 ratio found in infected cells. The agnoprotein reading frame, depending on how the mRNA precursor is spliced, is either not contained in the mRNA or is terminated near the VP2 AUG. Under these conditions, the ability of the agnoprotein AUG to block downstream CAT AUG utilization was found to be minimal in our assay. However, we directly tested the blocking ability of the agnoprotein AUG under conditions in which the reading frame terminated well after the CAT AUG. Although the agnoprotein AUG lies in a very good sequence context, this direct analysis showed that it interfered minimally with utilization of the CAT AUG when under the control of the SV40 late promoter. However, expected high levels of interference were regained when the late promoter was replaced with the Rous sarcoma virus long terminal repeat.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antioxidant defense in mitochondria during diapause and postdiapause development of European corn borer (Ostrinia nubilalis, Hubn.)

Antioxidant enzymes (CAT, catalase; GPx, selenium nondependent glutathione peroxidase; GST, glutathione-S-transferase; GR, glutathione reductase; DHAR, dehydroascorbate reductase) were determined in the mitochondria of diapausing and non-diapausing larvae and pupae of both diapausing and non-diapausing larvae of the European corn borer (Ostrinia nubilalis, Hubn., Lepidoptera: Pyralidae). CAT, GST, and DHAR activity in mitochondria of diapausing larvae were reduced compared to non-diapausing larvae. Pupae of diapaused-larvae possessed lower GST, but higher DHAR activities compared to pupae of non-diapaused individuals. Comparison between larvae and pupae revealed lower GPx activity in the mitochondria of pupae. CAT activity in the mitochondria of pupae was higher compared to diapausing larvae, but lower than in non-diapausing ones. Correlation and canonical discriminant analyses revealed different antioxidant enzyme compositions for a particular stage and developmental pattern. Our results show that antioxidant enzymes have a similar role in the regulation of energetics in mitochondria as that in diapause and metamorphosis.     

Requirement of a polypyrimidine tract for trans-splicing in trypanosomes: discriminating the PARP promoter from the immediately adjacent 3'' splice acceptor site.

We studied sequence requirements for trans-splicing at the 3' splice acceptor site of a procyclic acidic repetitive protein (PARP) coding gene in trypanosomes. In transient CAT transfection assays with linker scanning (LS) mutants in a PARP promoter--3' splice acceptor site--CAT construct, minor differences in the sequence composition of the polypyrimidine tract (nt -36 to -5 with respect to the 3' splice acceptor site) severely affected the CAT activity. Analysis of steady-state CAT RNA in stably transformed trypanosomes revealed that the LS mutations had indeed affected the pre-mRNA splicing efficiency. The data indicate that mini-exon addition is not required simply for maturation of polycistronic pre-mRNA but is also essential for the generation of functional mRNA from monocistronic genes, since unspliced monocistronic pre-mRNA did not accumulate or allow synthesis of CAT. We postulate that mini-exon addition at polycistronically transcribed genes, which can have drastically different polypyrimidine tracts at each of their 3' splice acceptor sites, can occur with different efficiencies for each gene of the array thus affecting mRNA abundance.     

Ornithine Decarboxylase Activity during Sporulation of Bacillus subtilis

A simple method for overproduction of a target protein by genetic engineering techniques has been established. This method involves rearranging the target gene, which contains a ribosome binding sequence for expression, in plurally repeated form, and inserting it in a 3′ lower part of promoters.

The chloramphenicol acetyltransferase (CAT) structural gene was used to demonstrate the validity of this method. E. coli harboring a CAT expression plasmid, pUS(CAT)₁, which had one inserted CAT gene, was able to produce CAT at the level of only 4% of the total cellular protein according to densitometric scanning on Coomassie-blue-stained SDS-polyacrylamide gel and had the CAT activity of 3.9 × 10³ units/mg protein. However, E. coli harboring a CAT expression plasmid, pUS(CAT)₄, which had inserted four directly repeated copies of the CAT gene, could synthesize CAT up to 16% of the total cellular protein and had the CAT activity of 2.8 × 10⁴ units/mg protein. This suggests that this method should be useful for overproducing many important peptides or proteins in bacteria.     

IDENTIFICATION AND CHARACTERIZATION OF THE CATALASE GENE PyCAT FROM THE RED ALGA PYROPIA YEZOENSIS (BANGIALES,RHODOPHYTA)1

Catalase is an antioxidant enzyme that plays a significant role in protection against oxidative stress by reducing hydrogen peroxide. The full‐length catalase cDNA sequence as isolated from expressed sequence tags (ESTs) of Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi (PyCAT) through rapid amplification of cDNA ends (RACE) was identified and characterized. It encoded a polypeptide of 529 amino acids, which shared 36%–44% similarity with other known catalase proteins. Phylogenetic analysis revealed that PyCAT was closer to the catalases from plants than from other organisms. The PyCAT mRNA expression was investigated using real‐time PCR to determine life‐cycle‐specific expression and the expression pattern during desiccation. The mRNA expression level in gametophytes was significantly higher than in sporophytes, and the mRNA expression level of PyCAT was significantly up‐regulated during the desiccation process. The recombinant PyCAT protein was purified and analyzed biochemically. The recombinant PyCAT protein exhibited high enzymatic activity (28,000 U·mg^?1) with high thermal stability and a broad pH range. All these results indicate that the PyCAT is a typical member of the plant and algal catalase family and may play a significant role in minimizing the effect of oxidative damage in P. yezoensis during desiccation.     

Antioxidant levels from different Antarctic fish caught around South Georgia Island and Shag Rocks

Antarctic fish have been isolated for over several million years in an environment with a very low and constant temperature and high oxygen concentration. In such conditions the oxidative stress might be an important factor affecting their metabolic adaptive strategies. Activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), vitamin E levels and total antioxidant capacity (TRAP) were measured in liver, gill, heart and muscle homogenates of red-blooded (Nototheniidae) and white-blooded (Channichthyidae) Antarctic fish. SOD activity was also measured in blood samples. Gill SOD activity was threefold higher in channichthyids than in nototheniids while CAT and GPx were significantly higher in the gills of channichthyids. The increased SOD activity of channichthyids probably reflects the large PO₂ gradient across their gills. The H₂O₂ produced seems to be preferentially eliminated by diffusion, according to the low levels of CAT and GPx found in the gills of these species. In contrast, blood SOD was about fivefold higher in the latter group, which possesses erythrocytes and thus a much higher oxygen-carrying capacity. CAT activity was always higher in nototheniids except in muscle. However, vitamin E did not show clear differences between families except for the pattern observed in muscle. The higher content of vitamin E in this tissue shown in channichthyids is related to the higher volume density of mitochondria reported for this group, since vitamin E is responsible for preventing membrane lipid peroxidation. Accordingly, TRAP (representative of hydrosoluble antioxidant capacity) was also higher in muscle of channichthyids. This is probably related to the role of ascorbic (a hydrosoluble compound) acid in regenerating vitamin E. Accepted: 4 September 1999     

Contribution of the hormone-response elements of the proximal ApoA-I promoter, ApoCIII enhancer, and C/EBP binding site of the proximal ApoA-I promoter to the hepatic and intestinal expression of the ApoA-I and ApoCIII genes in transgenic mice

We have generated and studied the pattern of expression of transgenic mouse lines carrying the human apoA-I and apoCIII gene cluster mutated at different sites. In two lines, we have either mutated the hormone-response element (HRE) of element G of the apoCIII enhancer or the C/EBP binding site of the proximal apoA-I promoter. In a third line, we have mutated the two HREs of the apoA-I promoter and the HRE of the apoCIII enhancer. Mutations in the HRE of element G reduced the hepatic and intestinal expressions of the reporter chloramphenicol acetyltransferase (CAT) gene (which substituted the apoCIII gene) to 4 and 13% of the wild-type (WT) control, whereas the hepatic and intestinal expressions of the apoA-I gene were reduced to 92 and 25% of the WT control, respectively. A mutation in the C/EBP site increased the hepatic and intestinal expressions of the apoA-I gene approximately 1.25- and 1.6-fold, respectively, and did not affect the expression of the CAT gene. The mutation in the three HNF-4 binding sites of the apoA-I promoter/apoCIII enhancer nearly abolished the expression of apoA-I and the reporter CAT gene in all tissues. These findings establish the importance of the HREs for the hepatic and intestinal expressions of the apoA-I and apoCIII genes and suggest that C/EBP does not play a central role in the expression of the apoA-I gene.     

Diagnostic perspectives of mobile radiographic computerized tomography]

The authors assess mobile methods of x-ray computer-aided tomography (CAT) and suggest an organization and methodological scheme of its application. Their program of the first and up to now the only one in this country mobile CAT device is based on the new principles of mobile CAT application. It is realized in special hospitals of large regions, where the patients with the optimal indications for CAT are assembled. Over 15,000 examinations were carried out with the use of the suggested CAT program over 4 years, that resulted in detection of 1295 brain tumors, 804 cases with neoplastic involvement of the abdominal cavity and the retroperitoneal space. The authors claim that wide application of mobile CAT devices according to the program they suggest will help decide the problem of unavailability of such examinations, for it will rule out the principal cause of this unavailability--economic problems arising because of high price of this equipment. One mobile device may replace 3 permanent CAT devices, if used according to the program suggested by the authors.     

Computed tomography in the diagnosis of stomach cancer and the assessment of its spread]

CAT was carried out in 110 patients with gastric carcinoma and its findings compared with the intraoperative findings. CAT involved a polypositional scanning (depending on the tumor site) against the background of oral contrast label of the stomach and intestine. CAT potentialities in the diagnosis of gastric carcinoma, assessment of the tumor size and involvement of the adjacent organs and structures, detection of the metastases were studied. Use of CAT in complex with other methods will help choose the optimal treatment strategy and determine the volume of supposed surgical intervention.     

Activities of enzymes that detoxify superoxide anion and related toxic oxyradicals in Trichoplusia ni

In third-, fourth-, and fifth-instar larvae of the cabbage looper moth, Trichoplusia ni, the activities of the antioxidant enzymes, superoxide dismutase (SOD*), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR) were examined using 850 g supernatants of whole-body homogenates. The enzyme activities, expressed as units mg⁻¹ protein min⁻¹ at 25°C ranged as follows: SOD, 0.67-2.13 units; CAT, 180.5-307.5 units; GPOX, none detectable; and GR, 0.40-1.19 units. There was a similar pattern of changes for SOD and CAT activities with larval ontogeny, but not for GR. The cabbage looper apparently uses SOD and CAT to form a “defensive team” effective against endogenously produced superoxide anion (O₂⪸). Glutathione may serve as an antioxidant for the destruction of any organic/lipid peroxides formed, and GSH oxidized to glutathione disulfide would be recycled by GR. Bioassays against pro-oxidant compounds exogenous sources of (O₂⪸) show high sensitivity of mid-fifth instars to the linear furanocoumarin, 8-methoxypsoralen (xanthotoxin) primarily from photoactivation (320-380 nm), and auto-oxidation of the flavonoid, quercetin. The LC₅₀s are 0.0004 and 0.0045% (w/w) concentration of xanthotoxin and quercetin, respectively. Both pro-oxidants have multiple target sites for lethal action and, in this context, the role of antioxidant enzymes is discussed.     

渗透胁迫对黑麦幼苗活性氧和抗氧化酶活性的影响

用20%聚乙二醇(PEG 6000)研究了渗透胁迫对黑麦(Secale cereale L.)幼苗活性氧(reactive oxygen species, ROS)和主要抗氧化酶—— 超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)、抗坏血酸过氧化物酶(ascorbate peroxidase, APX)和谷胱甘肽还原酶(glutathione reductase, GR)活性的影响。结果表明, 与对照相比, PEG处理明显提高了叶子和根中丙二醛(malondialdehyde, MDA)的含量、ROS的水平和以上4种抗氧化酶的活性。渗透胁迫下,叶子和根中MDA和ROS水平变化的规律基本相似, 但抗氧化酶活性在2种器官中表现不完全相同, 叶子中CAT的活性在对照和处理中无显著差异, 但在根中差异明显, 表明叶子中SOD、APX和GR在植物应答渗透胁迫中起重要作用, 而根中这4种抗氧化酶都参与植物对胁迫的反应。GR活性随PEG处理变化幅度显著高于其它抗氧化酶, 表明GR在黑麦应答渗透胁迫中所起作用可能强于其它抗氧化酶。     

Antioxidative effects of docosahexaenoic acid in the cerebrum versus cerebellum and brainstem of aged hypercholesterolemic rats

Female Wistar rats (100 weeks old) were divided into two groups; one group was fed a high-cholesterol diet (HC) and the other a high-cholesterol diet plus docosahexaenoic acid (HC-fed DHA rats). Fatty acid concentrations in brain tissues were analyzed by gas chromatography. In the HC-fed DHA rats, brain catalase (CAT), GSH, and glutathione peroxidase (GPx) increased in the cerebrum but not in the brainstem or cerebellum. The rate of increase was 23.0% for CAT, 24.5% for GSH, and 26.3% for GPx compared with that in the HC animals (p < 0.05). In the cerebrum of the HC-fed DHA rats, CAT and GPx increased, with an increase in the ratio of DHA to arachidonic acid. The cerebrum, unlike the other areas of the brain, seems to be more sensitive to DHA in stimulating CAT and GPx. We suggest that DHA plays an important role in inducing an antioxidative defense against active oxygen by enhancing the cerebral activities of CAT, GPx, and GSH.     

Sustained nitric oxide production in macrophages requires the arginine transporter CAT2

The aberrant production of nitric oxide (NO) contributes to the pathogenesis of diseases as diverse as cancer and arthritis. Sustained NO production via the inducible enzyme, nitric-oxide synthase 2 (NOS2), requires extracellular arginine uptake. Three closely related cationic amino acid transporter genes (Cat1-3) encode the transporters that mediate most arginine uptake in mammalian cells. Because CAT2 is induced coordinately with NOS2 in numerous cell types, we investigated a possible role for CAT2-mediated arginine transport in regulating NO production. The complexity of arginine transport systems and their biochemically similar transport properties called for a genetic approach to determine the role of CAT2. CAT2-deficient mice were generated and found to be healthy and fertile in contrast to Cat1(-/-) animals. Analysis of cytokine-activated macrophages from Cat2(-/-) mice revealed a 92% reduction in NO production and a 95% reduction in l-Arg uptake. The reduction in NO production was not due to differences in NOS2 protein expression, NOS2 activity, or intracellular l-arginine content. In conclusion, our results show that sustained abundant NO synthesis by macrophages requires arginine transport via the CAT2 transporter.     

Micro topography of Tyrosine Hydroxylase, Glutamic Acid Decarboxylase, and Choline Acetyltransferase in the Substantia Nigra and Ventral Tegmental Area of Control and Parkinsonian Brains

Tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD), and choline acetyl transferase (CAT) were used as markers for catecholamine, gamma-aminobutyric acid, and acetylcholine containing neurons in human mesencephalon. Their rostrocaudal, mediolateral, and dorsoventral distribution was investigated within the substantia nigra pars compacta (SNC) and pars reticulata (SNR) and in the ventral tegmental area (VTA). TH activity was highest in the caudal, medial, and ventral SNC and in the middle of VTA medio-ventrally. The enzyme activity in SNR was low and uniformly distributed. In SNC as well as SNR, GAD activity was high and greater laterally and in the middle of the rostro-caudal extent. No particular pattern of distribution was observed in VTA. an area with low GAD content. In the substantia nigra, CAT activity was low. A characteristic medio-ventral distribution with a peak of high enzyme activity in the middle of the rostrocaudal extent was observed. In VTA, enzyme levels were high and also concentrated medio-ventrally and in the middle of the area. In parkinsonian brains, the distribution of TH was uniformly affected throughout the rostro-caudal extent. In VTA the enzyme activity was not as reduced as in SNC and SNR; the CAT pattern was only disrupted in a very localized part of SNC but not in SNR and VTA. In all three areas, GAD activity was reduced to a uniformly low distribution.