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PubMed1996

[Serologic studies of domestic cats for potential human pathogenic virus infections from wild rodents].

For several viral infections a reservoir in wild rodents has been demonstrated. Some of the agents are known or suspected to be pathogenic for humans. Because improvements in hygiene have reduced direct human contact with rodents, domestic cats could be acting as active transmitters of these viruses from rodents to man. We selected 4 such pathogens--ortho- and parapox-, hanta- and encephalomyocarditis viruses--which, in different ways, may lead to serious human illness: Ortho- and parapoxvirus infections may cause localized pox lesions following direct skin contact. In general, the lesions heal without complications; in immunosuppressed or -deficient individuals, however, infection may generalize and take a dramatic course. Hantaviruses exist in various serotypes with different pathogenicity for human beings, varying from asymptomatic infection to highly fatal disease. In central and northern Europe the Puumala serotype is predominant causing influenza-like symptoms and renal dysfunction. Human infections arise from inhalation of aerosolized excreta of persistently infected rodents. Infections of man associated with encephalomyocarditis virus were demonstrated sporadically in cases of encephalitis and meningitis. In the present study, we investigated in 200 feline serum samples the prevalence of antibodies to ortho- and parapox-, hanta- and encephalomyocarditis virus. All serum samples were from cats that had been allowed to roam outside and to hunt. They were submitted from all parts of Austria for routine diagnosis in 1993. Four per cent of cats showed antibodies to orthopoxviruses with haemagglutination inhibition (HI) titres of 16-512; because of extensive cross-reactivity, positive samples reacted with all investigated orthopoxviruses (a feline orthopoxvirus recently isolated in Vienna, the reference strain of cowpox virus, Brighton, and vaccinia virus, strain IHD), only varying in titre. The specificity of the results was confirmed by virus neutralisation (VN) test, in which the same sera showed titres of 4-32. These data imply that, at least in Austria, unrecognized or subclinical orthopoxvirus infection in cats is more common than previously thought. In contrast to orthopoxviruses, all serum samples proved negative to parapoxvirus (parapoxvirus bovis 1) in VN test. In the same 200 samples, a seroprevalence of 5% was found to hantavirus (immunofluorescence antibody assay), indicating that domestic cats are susceptible to this virus and that infection is not uncommon in cat populations. Because higher titres were obtained against the Puumala serotype compared to the more pathogenic serotype Hantaan, it is most likely that the cats had experienced Puumala infections. Using HI test, antibodies to encephalomyocarditis virus (EMCV) were demonstrated in only 1.5% of the feline serum samples; although the antibody titres were low (16 and 32, respectively) we consider them specific, because these sera proved positive in VN test as well. Nevertheless, EMCV infection in domestic cats seems to be of low importance. The serological results presented in this paper, together with virological and epidemiological data, indicate that the domestic cat plays an important role only in the transmission of orthopoxviruses to human beings, but not in the case of parapox-, hanta-, and encephalomyocarditis virus.

Animals↗
PubMed1998

Bacterial flora in the duodenum of healthy cats, and effect of dietary supplementation with fructo-oligosaccharides.

OBJECTIVE: To investigate changes in the duodenal flora of healthy cats over time, and evaluate the effect of dietary supplementation with fructo-oligosaccharides (FOS). ANIMALS: 12 healthy, barrier-maintained, specific-pathogen-free cats. PROCEDURE: Duodenal juice for bacteriologic examination was collected via oral endoscopy 5 times from each cat over a 32-week period. Cats were allotted randomly to 2 groups, and a crossover design study, during which they were fed either a replete dry (basal) diet or, for 12 consecutive weeks, basal diet supplemented with 0.75% FOS, was done. Samples (3 from cats fed the basal and 2 from cats fed the FOS diet) were collected for a minimum of 6 weeks after commencement of feeding, and a minimum of 6 weeks apart. RESULTS: Mean aerobic, anaerobic, and total bacterial counts did not differ significantly among sample collection times. After pooling of the results, mean (+/- SD) log10 colony-forming units (CFU) of aerobic, anaerobic, and total bacteria/ml were 5.5 +/- 1.1, 4.8 +/- 1.0 and 5.6 +/- 1.1, respectively. However, individual cats had considerable variation in counts: mean (range) intraindividual coefficients of variation were: 19.0 (6.1 to 34.2), 19.9 (4.8 to 35.5), and 18.1 (5.5 to 32.6)%, respectively. In 1 cat, total bacterial count varied between < 3.0 and 6.3 CFU/ml. Bacterial flora varied qualitatively: only Enterococcus faecalis, Clostridium perfringens, Bacteroides, Pasteurella, and Streptococcus spp, and unidentified gram-negative (aerobic) rods were present in > 50% of the samples. CONCLUSIONS: Wide quantitative and qualitative variation in the duodenal flora of healthy cats was observed over time, which was not affected by dietary supplementation with FOS.

Animal Feed↗
PubMed1998

Assessment of intestinal function in cats with chronic diarrhea after infection with feline immunodeficiency virus.

OBJECTIVE: To identify consistent relevant mechanisms of small intestinal dysfunction in cats with experimentally induced feline immunodeficiency virus infection (FIV) that developed chronic diarrhea during the time they were being used in studies of pathogenicity and transmission of FIV. ANIMALS: 10 cats. PROCEDURE: The following investigative tests and techniques were performed on each of the cats: routine hematologic and serum biochemical analyses; urinalysis; fecal parasitologic and microbiologic examinations; breath hydrogen lactulose (BH2LT) and xylose (BH2XT) tests; intestinal permeability test; endoscopic examination of the intestinal mucosa; bacteriologic culture of endoscopically collected small intestinal juice; and histologic examination of endoscopically obtained intestinal biopsy specimens. RESULTS: Neutrophilia was evident in 3 cats, and lymphopenia was detected in 2 cats. Serum biochemical abnormalities were not observed. Urinalysis results were unremarkable. Fecal bacteriologic and parasitologic results were normal, except for isolation of Campylobacter sp from 1 cat. Abnormal BH2XT values suggestive of D-xylose malabsorption were identified in 2 cats, and BH2LT values indicated evidence of small intestinal bacterial overgrowth in 1 cat. Finally, permeability test results, quantitation of bacterial flora from the proximal part of the small intestine and histologic examination of biopsy specimens did not reveal any abnormalities. CONCLUSIONS: Enteric pathogens did not account for the development of diarrhea in cats with experimentally induced FIV infection, and consistent relevant mechanisms of small intestinal dysfunction were not identified.

Animals↗
PubMed2000

Prevalence of Bartonella henselae, Bartonella clarridgeiae and the 16S rRNA gene types of Bartonella henselae among pet cats in Japan.

The authors investigated bacteriologically the prevalence of Bartonella infection among 690 pet cats derived from 10 private animal hospitals in six cities (Sapporo, Hokkaido Prefecture; Sendai, Miyagi Prefecture; Joetsu, Niigata Prefecture; Fujisawa, Kanagawa Prefecutre; Kyoto, Kyoto Prefecture; Sanda, Hyogo Prefecutre) and 4 counties (Mishima, Osaka Prefecture; Hikawa, Shimane Prefecture; Aira, Kagoshima Prefecture; Shimajiri, Okinawa Prefecture) located from the north to the south of Japan. Bartonella species were isolated from 7.2% (50/690) of all the cats examined. No Bartonella species were isolated from the cats in Sapporo or Sendai. The isolation rate varied from 2% in Joetsu and Sanda to 20% in Shimajiri. Bartonella clarridgeiae was isolated from two of 50 cats in Kyoto, three of 50 in Mishima and one of 50 in Shimajiri, but not in cats from the other cities or counties. Though the cats of Joetsu, Fujisawa, Kyoto, Sanda, Aira and Shimajiri were infected with either B. henselae or B. clarridgeiae, one of eight infected cats in Mishima was harboring both Bartonella species. Type I of 16S rRNA gene was the predominant type among the isolates of B. henselae, but only one isolate derived from Shimajiri was found to be of type II. Prevalence of B. clarridgeiae and the 16S rRNA gene type of B. henselae among cats in Japan was demonstrated for the first time in this investigation.

Animals↗
PubMed1997

Development of Cat-301 immunoreactivity in auditory brainstem nuclei of the gerbil.

The developing brainstem auditory system has been studied in detail by using anatomical and physiological techniques. However, it is not known whether immature auditory neurons exhibit different molecular characteristics than those of physiologically mature neurons. To address this issue, we examined the distribution of Cat-301 immunoreactivity in the developing auditory brainstem of gerbils. Cat-301 is a monoclonal antibody that recognizes a 680-kD chondroitin sulfate proteoglycan similar to aggrecan, a high-molecular-weight chondroitin sulfate proteoglycan found in cartilage. In the central nervous system, Cat-301 immunoreactivity is localized to the extrasynaptic surface of neurons. It has been hypothesized by Hockfield and co-workers (Hockfield et al. [1990a]Cold Spring Harbor Symp. Quart. Biol. 55:504-514) that the Cat-301 proteoglycan is a molecular marker indicating that a neuron has acquired mature neuronal properties. In the current study, Cat-301 staining is first seen at 7 days after birth in the anterior ventral cochlear nucleus (AVCN), the posterior VCN (PVCN), and the medial nucleus of the trapezoid body (MNTB) shortly before the onset of sound-evoked activity. By 21 days after birth, neurons in the AVCN, the PVCN, and the lateral and medial superior olive have attained adult-like distributions of Cat-301 staining concomitant with the physiological maturation of these neurons. Neurons in MNTB attain adult-like distributions of Cat-301 immunoreactivity at 1 year. The maturation of Cat-301 immunoreactivity parallels the physiological maturation of gerbil auditory neurons, and the Cat-301 proteoglycan may play a role in the formation and/or stabilization of auditory synapses.

Animals↗
PubMed1996

Molecular characterization of a plasmid-borne (pTC82) chloramphenicol resistance determinant (cat-TC) from Lactobacillus reuteri G4.

Lactobacillus reuteri G4 contains a 7.0-kb plasmid (pTC82) encoding resistance to chloramphenicol (Cm). Determination of the nucleotide sequence of the genetic determinant (cat-TC) encoding resistance to Cm on pTC82 revealed an open reading frame for a 238-amino-acid Cm acetyltransferase (CAT) monomer. This structural cat gene, 714 bp in length, was highly related (ca. 95% nucleotide and ca. 81% amino acid identity) to the 648-bp cat gene from Staphylococcus aureus plasmid pC194. A total of 6 bp transversions and 4 bp deletions was observed along the whole DNA sequence of cat-TC compared to that of cat-pC194. To determine the activity of the putative cat-TC gene, recombinant plasmid pUC8217 containing the cat determinant from pTC82 was subjected to a maxicell analysis. The observed molecular mass of the synthesized protein, based on electrophoretic mobility, was in reasonable agreement with the 27.3 kDa predicted from the DNA sequence. This is the first reported nucleotide sequence of a Cm-resistance determinant from L. reuteri and also the first evidence of adding Lactobacillus to the list of versatile bacterial genera which naturally acquire the cat-pC194 gene in the microbial ecological system.

Amino Acid Sequence↗
PubMed1991

Expression of luteinising hormone-beta subunit chloramphenicol acetyltransferase (LH-beta-CAT) fusion gene in rat pituitary cells: induction by cyclic 3'-adenosine monophosphate (cAMP).

In this study we determined the activity of the rat luteinising hormone-beta gene promoter in a heterologous rat pituitary cell line (GH3 cells). 1.7 kb of LH-beta 5' flanking sequence and the first 5 bp of the 5' untranslated region were ligated to the chloramphenicol acetyltransferase (CAT) receptor gene (LH-beta-CAT) and transiently transfected by calcium phosphate precipitation into subconfluent cultures of GH3 cells. Basal low-level CAT activity was only detected in GH3 cells, being absent in two non-pituitary cell lines (BeWo and HeLa) RNase analysis revealed that mRNA from transfected GH3 cells protected a fragment of labelled antisense probe of correct size for transcription initiation from the LH-beta CAP site, confirming that promoter activity reflected correctly initiated LH-beta-CAT fusion gene transcripts. CAT activity was consistently induced by an average of 3-5-fold from the full-length 1.7 kb promoter, in a dose- and time-dependent manner, by forskolin, dibutyryl cAMP, and 8-bromo cAMP implying presence of a cAMP-responsive cis-acting domain in the LH-beta promoter region. Transfection of deletion mutants delta-615-CAT, delta-385-CAT and delta-250-CAT each reduced forskolin inducibility to 1.7-fold but did not abolish induction completely suggesting a domain between -1.7 and -0.6 kb contained a cAMP-responsive element(s) (CRE). Further deletion of LH-beta 5' flanking sequences to delta-85-CAT restored forskolin induction to wild-type levels (3-5-fold), suggesting the presence of a weak inhibitory element between -600 and -85 kb, and a cAMP-responsive domain in the proximal promoter region. The LH-beta promoter does not contain perfect tandem repeat palindromic CRE DNA sequences, though there are several octanucleotide sequences differing by only 1 bp from AP-2 binding sites, the consensus CRE, and the vasointestinal peptide gene CRE. Although these data suggest that the LH-beta gene is cAMP responsive this is likely mediated by several and complex protein interactions with multiple DNA sequences in the proximal and distal LH-beta promoter enhancer.

Adenylyl Cyclases↗
PubMed1985

Isolation and expression of a constitutive variant of the chloramphenicol-inducible plasmid gene cat-86 under control of the Bacillus subtilis 168 amylase promoter.

The amyR1 region controls the regulated expression of the Bacillus subtilis 168 amylase gene amyE. When cloned into the B. subtilis promoter-cloning plasmid pPL603, amyR1 has been shown to activate expression of the promoter-indicator gene cat-86. In this chimeric plasmid, p5' alpha B10, cat-86 expression was maximal in stationary phase B. subtilis cells and cat-86 expression was repressible by glucose. Both these properties are similar to the regulated expression of the B. subtilis amyE gene. In addition, cat-86 expression in p5' alpha B10 was inducible with chloramphenicol (Cm). The inducibility phenotype of cat-86 has been shown to be independent of the promoter that is used to activate the gene, and inducibility has been suggested to result from the presence of a pair of inverted-repeat sequences that span the ribosome-binding site (RBS) for cat-86. A spontaneous deletion mutant of p5' alpha B10 was isolated, p5' alpha B10 delta 1, in which cat-86 expression was constitutive with respect to Cm, but the basic pattern of amyR1-directed regulation of cat-86 was intact. The rightward deletion endpoint was within the upstream member of the pair of inverted repeats that immediately precede cat-86. This result is therefore consistent with the role proposed for the inverted repeats in Cm inducibility. The leftward endpoint of the deletion is within the amyR1 region and thus allows a more precise determination of the functional domain of amyR1.

Acetyltransferases↗
PubMed1984

Selective expression of a plasmid cat gene at a late stage of Bacillus subtilis sporulation.

The cat-86 gene in plasmid pPL603 specifies chloramphenicol acetyltransferase (CAT) and is selectively expressed in Bacillus subtilis at a stage in sporulation in which internal spores are first observed (approximately T8). The gene is unexpressed in vegetatively growing cells. cat-86 expression and spore formation are both blocked when cells are grown in excess glucose. cat-86 expression at T8 is due to selective transcription of the gene, since cat-86 mRNA is undetectable in vegetatively growing cells but is readily demonstrated in sporulating cells. The transcription start site for cat-86 mRNA from sporulating cells is within a 203-base-pair restriction fragment designated P1, which is located upstream from the cat coding region on pPL603 . Deletion of P1 from pPL603 eliminates the sporulation -associated expression of cat-86. Host sporulation genes, whose function is absolutely required for cat-86 expression at T8, include six early sporulation, spo0 , genes and spoIIE . Therefore, pPL603 provides a novel system in which the in vivo expression of a known, plasmid-linked gene is dependent on sporulation-specific changes in B. subtilis.

Acetyltransferases↗
PubMed1989

Change in desensitization of cat muscle acetylcholine receptor caused by coexpression of Torpedo acetylcholine receptor subunits in Xenopus oocytes.

Cat muscle acetylcholine receptors (AcChoR) expressed in Xenopus oocytes desensitized more slowly than Torpedo electric organ AcChoRs, also expressed in oocytes. To examine the bases for the different degrees of desensitization, cat-Torpedo AcChoR hybrids were formed by injecting oocytes with cat denervated muscle mRNA mixed with a large excess of cloned Torpedo AcChoR subunit mRNAs. Hybrid AcChoRs formed by coinjection of cat muscle mRNA with the Torpedo beta or delta subunit mRNAs desensitized as slowly as cat AcChoR. In contrast, the hybrid AcChoRs expressed by coinjection with the Torpedo gamma subunit mRNA desensitized much more rapidly than cat AcChoR. The AcChoRs expressed in oocytes injected with cat muscle mRNA together with the Torpedo beta, gamma, and delta subunit mRNAs desensitized as rapidly as Torpedo AcChoR, indicating that the cat alpha subunit does not play an important role in determining the slow rate of desensitization. It is concluded that the difference in the rates of desensitization of cat and Torpedo AcChoRs is determined mainly by differences in their respective gamma subunits.

Animals↗
PubMed2002

Translation mediated by the internal ribosome entry site of the cat-1 mRNA is regulated by glucose availability in a PERK kinase-dependent manner.

The cationic amino acid transporter, Cat-1, is a high affinity transporter of the essential amino acids, arginine and lysine. Expression of the cat-1 gene is known to be regulated by amino acid availability. It is shown here that cat-1 gene expression is also induced by Glc limitation, which causes a 7-fold increase in cat-1 mRNA, a 30-fold induction of Cat-1 protein levels, and a 4-fold stimulation of arginine uptake. Glc limitation is known to induce the unfolded protein response (UPR) by altering protein glycosylation in the endoplasmic reticulum (ER). The studies here demonstrate that synthesis of Cat-1 occurs during the UPR when global protein synthesis is inhibited. The 5'-UTR of the cat-1 mRNA contains an internal ribosomal entry site (IRES) that is activated by amino acid starvation by a mechanism that involves phosphorylation of the translation initiation factor, eukaryotic initiation factor 2alpha, by the GCN2 kinase. It is shown here that translation from the cat-1/IRES is also induced by Glc deprivation in a manner dependent upon phosphorylation of eukaryotic initiation factor 2alpha by the transmembrane ER kinase, PERK. Because PERK is a key constituent of the UPR, it is concluded that induction of cat-1 gene expression is part of the adaptive response of cells to ER stress. These results also demonstrate that regulation of IRES activity in cellular mRNAs is part of the mechanism by which the UPR protects cells from unfolded proteins in the ER.

3T3 Cells↗
PubMed1995

Chlamydia infection in cats in New Zealand.

Conjunctival swabs collected in 1991-92 from 333 pedigree and non-pedigree cats were tested for the presence of Chlamydia spp. antigen using an ELISA antigen kit. Forty (18.4%) of the 217 samples from cats with conjunctivitis were positive. Seven (6%) of 116 samples from cats which were in contact with cats with conjunctivitis but which showed no clinical signs at the time of sample collection were positive. Positive-testing cats were frequently from multi-cat households. Chlamydia spp. is present and associated with conjunctivitis in cats in New Zealand. Infection may occur concurrently with viral diseases. Feline calicivirus was recovered from 27 (21 with conjunctivitis) of 37 cats tested in five catteries. Four cats (with conjunctivitis) were FIV-positive.

Journal Article↗
PubMed1994

Red cell morphologic alterations in cats with hepatic disease.

Medical records from 39 cats with hepatic disease, examined at the Veterinary Medical Teaching Hospital, University of Florida, between 1987 and 1992 were retrospectively evaluated for alterations in red blood cell (RBC) morphology. Diagnoses included: hepatic lipidosis, neoplasia, cholangiohepatitis, hepatitis/hepatopathy, systemic histoplasmosis, and portocaval shunt. A total of 56 laboratory data sets were studied which included complete blood counts and serum chemistry results. Stained blood smears were evaluated from 51 of the data sets. Twenty-two cats (56%) were determined to have poikilocytosis on the basis of blood smear evaluation. Eleven (28%) cats had moderate to marked poikilocytosis (2+ to 4+). Acanthocytes accounted for 62.6 -/+ 22.1% of morphologically abnormal RBC and were observed in blood smears from 100% of cats with poikilocytosis. Elliptocytes (ovalocytes) comprised 19.5 -/+ 15.8% of poikilocytes and were found in smears from 82% of cats with poikilocytosis. Keratocytes (7.0 -/+ 6.8%), schistocytes (3.6 -/+ 4.4%), and blister cells (2.6 -/+ 6.4%) were present in lower numbers and in fewer cats. Serum total cholesterol values were significantly greater (p < 0.05) in cats with moderate to marked alterations in RBC morphology. Cats with hepatic lipidosis were significantly (p < 0.04) more likely to have poikilocytosis than cats with other types of hepatic disease.

Journal Article↗
PubMed2003

Asexual development is increased in Neurospora crassa cat-3-null mutant strains.

We use asexual development of Neurospora crassa as a model system with which to determine the causes of cell differentiation. Air exposure of a mycelial mat induces hyphal adhesion, and adherent hyphae grow aerial hyphae that, in turn, form conidia. Previous work indicated the development of a hyperoxidant state at the start of these morphogenetic transitions and a large increase in catalase activity during conidiation. Catalase 3 (CAT-3) increases at the end of exponential growth and is induced by different stress conditions. Here we analyzed the effects of cat-3-null strains on growth and asexual development. The lack of CAT-3 was not compensated by other catalases, even under oxidative stress conditions, and cat-3(RIP) colonies were sensitive to H(2)O(2), indicating that wild-type (Wt) resistance to external H(2)O(2) was due to CAT-3. cat-3(RIP) colonies grown in the dark produced high levels of carotenes as a consequence of oxidative stress. Light exacerbated oxidative stress and further increased carotene synthesis. In the cat-3(RIP) mutant strain, increased aeration in liquid cultures led to increased hyphal adhesion and protein oxidation. Compared to the Wt, the cat-3(RIP) mutant strain produced six times more aerial hyphae and conidia in air-exposed mycelial mats, as a result of longer and more densely packed aerial hyphae. Protein oxidation in colonies was threefold higher and showed more aerial hyphae and conidia in mutant strains than did the Wt. Results indicate that oxidative stress due to lack of CAT-3 induces carotene synthesis, hyphal adhesion, and more aerial hyphae and conidia.

Animals↗
PubMed1984

Regulatory regions that control expression of two chloramphenicol-inducible cat genes cloned in Bacillus subtilis.

Plasmid pPL603 is a promoter cloning vector for Bacillus subtilis and consists of a 1.1-kilobase fragment of Bacillus pumilus DNA inserted between the EcoRI and BamHI sites of pUB110. The gene cat-86, specifying chloramphenicol-inducible chloramphenicol acetyltransferase, is located on the 1.1-kilobase cloned DNA. When pPL603 is present in B. subtilis, cat-86 is unexpressed during vegetative growth but expressed during sporulation. The regulation of cat-86 in pPL603 is due to sequences within two restriction fragments, designated P1 and R1, that precede the main coding portion of the gene. The P1 fragment promotes transcription of cat-86 only during sporulation, whereas the adjacent R1 fragment lacks promoter function but contains sequences essential to chloramphenicol inducibility. A second B. pumilus gene, cat-66, was cloned in B. subtilis and is expressed throughout the vegetative growth and sporulation cycle. The cat-66 coding region is preceded by two adjacent restriction fragments designated as P2 and R2. P1 and P2 are identical in size and share 95% conservation of base sequence. R1 and R2 are also identical in size and share 91% conservation of base sequence. Fragment substitution experiments demonstrate that R2 can functionally replace R1. The substitution of P2 for P1 promotes cat-86 expression throughout vegetative growth and sporulation. Analysis of a derivative of pPL603 in which P2 has replaced P1 demonstrates that P2 promotes transcription of cat-86 during vegetative growth and that P2 contains the start site for transcription of cat-86. Thus, P1 and P2 differ strikingly in vegetative promoter function, yet they differ by single-base substitutions at only 11 positions of 203.

Bacillus subtilis↗
PubMed1987

Fixation of the unmethylated or the 5'-CCGG-3' methylated adenovirus late E2A promoter-cat gene construct in the genome of hamster cells: gene expression and stability of methylation patterns.

The late E2A promoter of adenovirus type 2 (Ad2) DNA can be inactivated by in vitro methylation of three 5'-CCGG-3' sequences at positions +23, +5, and -215 relative to the cap site in this promoter. This inactivation has been documented in transient expression experiments both in Xenopus laevis oocytes and in mammalian cells (K.-D. Langner, L. Vardimon, D. Renz, and W. Doerfler, Proc. Natl. Acad. Sci. USA 81:2950-2954, 1984; K.-D. Langner, U. Weyer, and W. Doerfler, Proc. Natl. Acad. Sci. USA 83:1598-1602, 1986). In the present study, in vitro-methylated or unmethylated promoter-gene assemblies were permanently fixed by integration in the hamster genome. In individually established cell lines, the degree of promoter methylation was correlated to gene activity. The pAd2E2AL-CAT construct, in which the late E2A promoter controls expression of the procaryotic chloramphenicol acetyltransferase (cat) gene, was fixed in BHK21 hamster cells by cotransfection with and selection for the pSV2-neo construct (P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1:327-341, 1982) in which the early simian virus 40 promoter controls the gene for neomycin phosphotransferase. The pAd2E2AL-CAT construct was transfected in the unmethylated or in the 5'-CCGG-3' methylated form. The pSV2-neo plasmid was cotransfected in the unmethylated form. The stability of in vitro-imposed methylation patterns and cat gene expression were followed and correlated in a number of established cell lines which contained the constructs integrated in a non-rearranged configuration. The foreign DNA did not persist in the episomal state but was integrated, frequently in multiple tandems of the plasmid DNA. Among 19 cell lines established after transfecting the unmethylated pAd2E2AL-CAT construct, the late E2A promoter remained unmethylated (examined in 10 cell lines), and the cat gene was expressed in 18 cell lines. On the other hand, among 14 cell lines which were generated by transfection with the methylated construct, 7 cell lines did not express the cat gene, and the three 5'-CCGG-3' sequences in the late E2A promoter remained almost completely methylated. In five cell lines, the E2A promoter sequences were partly demethylated and the cat gene was expressed at low levels. Last, in two cell lines, demethylations were found to be extensive and strong cat expression was observed. It remained a question of considerable interest what factors determined the stability of methylation patterns that had been preimposed by in vitro methylation on specific sequences in a promoter, after this promoter was fixed by integration in the mammalian genome.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyltransferases↗
PubMed2000

Nuclear factor-kappaB mediates simultaneous induction of inducible nitric-oxide synthase and Up-regulation of the cationic amino acid transporter CAT-2B in rat alveolar macrophages.

The connection between the regulation of L-arginine transport and nitric oxide (NO) synthesis was studied in rat alveolar macrophages. Lipopolysaccharides (LPSs) and interferon-gamma stimulated in the same concentration- and time-dependent manner NO synthesis (measured by nitrite accumulation) and L-[(3)H]arginine uptake. This correlated with an increased mRNA expression for iNOS and the cationic amino acid transporter CAT-2B (analyzed by reverse transcription-polymerase chain reaction), with the same kinetics observed for the up-regulation of both mRNAs. Because nuclear factor-kappaB (NF-kappaB) is essential for induction of iNOS its role for the regulation of CAT-2B expression and L-arginine transport was investigated. The NF-kappaB inhibitors pyrrolidine dithiocarbamate and N(alpha)-p-tosyl-L-lysine chloromethyl ketone abrogated LPS- and interferon-gamma-induced increase of nitrite accumulation and L-[(3)H]arginine uptake as well as up-regulation of iNOS and CAT-2B mRNA. LPS-induced increase in iNOS and CAT-2B mRNA was also suppressed by specific NF-kappaB decoy oligodesoxynucleotides, confirming the essential role of NF-kappaB for iNOS and CAT-2B expression. Dexamethasone did not affect the initial (5 h) LPS-induced increase of iNOS and CAT-2B mRNA, but down-regulated both mRNAs after prolonged (20 h) exposure and this was accompanied by partial inhibition of LPS-stimulated nitrite accumulation and L-[(3)H]arginine uptake. These findings demonstrate parallel regulation of the expression of iNOS and CAT-2B, and of NO synthesis and L-arginine uptake in rat alveolar macrophages. NF-kappaB is an essential transcription factor not only for the induction of iNOS, but also for the up-regulation of CAT-2B. The simultaneous up-regulation of CAT-2B with iNOS is considered as a mechanism to ensure a high substrate supply for iNOS.

Amino Acid Transport Systems, Basic↗
PubMedDate not supplied

Effects of 2-chloro-4-acetotoluidine (CAT) toxicity on biochemical and morphological alterations in quail.

Morphological changes in response to 2-chloro-4-acetotoluidine (CAT) toxicity in the quail appeared in the form of progressive necrosis of the kidney, particularly the proximal tubular epithelial cells. Changes at 32 hr after the CAT administration included vacuolar degeneration, dilatation of distal tubules containing hyaline and granular casts, overt necrosis, and deposition of urate casts in collecting tubules. There were no striking histopathological changes in the liver at 24 hr. However, small focal necrotic lesions were seen 32 hr after the CAT administration. A 40% protection against the toxicity of CAT at the lower dose was seen in quail pretreated with phenobarbital. The protection offered by phenobarbital pretreatment was attributed to a quantitative shunting of CAT and/or its reactive metabolite along the microsomal-mediated metabolic pathway of the kidney responsible for their inactivation. Administration of reduced glutathione (GSH) to quail treated with CAT offered little protection against the toxicity. The quail treated with a toxic dose of CAT had an increased level of thiobarbituric acid (TBA) reacting products in the liver, with a concomitant decrease in GSH content. This suggests that lipid peroxidation may be involved in CAT-induced hepatic damage of quail. It was hypothesized that the depletion of protective GSH stores coincident with gradual shutdown of the protective peroxidase system of the kidney may occur in a more advanced stage of CAT toxicity in quail. This would then result in a severe disturbance in renal excretion of uric acid and in frank necrosis of renal tubules.

Aminopyrine N-Demethylase↗