CAT is coming--CAT is coming--CAT is coming.
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Expression of human tyrosine hydroxylase-chloramphenicol acetyltransferase (CAT) fusion gene in the brains of transgenic mice as examined by CAT immunocytochemistry.
We have produced transgenic (Tg) mice carrying 5.0-kb fragment from the 5'-flanking region of the human tyrosine hydroxylase (hTH) gene fused to a reporter gene, chloramphenicol acetyltransferase (CAT) [Sasaoka et al. (1992) Mol Brain Res 16: 274-286]. In the brain of the Tg mice, CAT expression has been observed in catecholaminergic (CAnergic) neurons and also in non-CAnergic neurons. The aim of the present study is to examine in detail the cell-type specific expression of the hTH-CAT fusion gene in the brain of the Tg mice, by use of immunohistochemistry for CAT, TH, and aromatic L-amino acid decarboxylase (AADC). CAT-immunoreactive cells were found in CAnergic brain regions which contained TH-positive cells, and also in non-CAnergic brain regions which contained no TH-labeled cells. The non-CAnergic brain regions that represented CAT-stained cells were further divided into two groups: (i) regions containing AADC-labeled cells, for example, bed nucleus of the stria terminalis, nucleus suprachiasmaticus, mammillary body, nucleus raphe dorsalis, inferior colliculus, and nucleus parabrachialis, and (ii) regions containing no AADC-positive cells, for example, main olfactory bulb (except A16), accessory olfactory bulb, nucleus olfactorius anterior, caudoputamen, septum, nucleus accumbens, hippocampus, medial nucleus of the amygdala, entorhinal cortex, nucleus supraopticus, and parasubiculum. The results indicate that the 5.0-kb DNA fragment flanking the 5' end of the hTH gene may contain the element(s) specific for neuron-specific TH expression but which may be insufficient to attenuate ectopic expression.
Animals↗
Application of the two-phase partition assay for chloramphenicol acetyl transferase (CAT) to transfections with simian virus 40-CAT plasmids.
The two-phase partition assay for chloramphenicol acetyl transferase (CAT) was tested for its applicability in transfection experiments involving CAT-expression plasmids containing simian virus 40 (SV40) transcriptional control elements. The assay was shown to be simple, sensitive, reproducible, and, with the modification described, cost-effective. However, CAT transfection experiments themselves gave highly variable results. Yields of CAT per microgram of protein in pSV2-CAT transfections of CV-1 cells varied by 60%. This variability could not be explained by variation in the quality of DNAs used, differences in growth states of the cells at transfection, the culture medium employed, or the time at which transfected cells were extracted for CAT assay.
Chloramphenicol O-Acetyltransferase↗
The low level expression of chloramphenicol acetyltransferase (CAT) mRNA in Escherichia coli is not dependent on either Shine-Dalgarno or the downstream boxes in the CAT gene.
Recent studies have shown that the canonical Shine-Dalgarno (SD)-anti-SD interaction is dispensable for the initiation of translation of certain mRNAs in Escherichia coli. Alternative non-SD sequences (located upstream from the initiation codon) and also downstream sequences ("downstream boxes") complementary to 16S rRNA were found to be involved in the initiation of translation of mRNAs devoid of either SD or any leader sequences. In this study the chloramphenicol acetyltransferase (CAT) gene was modified to remove the 5' terminal non-translated region and/or the two potential downstream boxes in the CAT gene. Thus a series of ten CAT gene constructs was created and expressed in E. coli under a strong constitutive promoter. The results showed that CAT mRNAs devoid of both leader sequence nucleotides and the two downstream boxes in the CAT gene remained active in vivo and produced CAT protein in sufficient amounts for survival of the transformed cells at chloramphenicol concentrations up to 20-30 micrograms/ml.
5' Untranslated Regions↗
Nuclear transfer of synchronized african wild cat somatic cells into enucleated domestic cat oocytes.
The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei were synchronized by serum starvation (83.0%) than after roscovitine (80.0%) or contact-inhibition (80.0%). The fusion efficiency of in vivo and in vitro matured oocytes used as recipient cytoplasts of AWC donor nuclei (86.6% vs. 85.2%) was similar to the rates obtained with DSH donor nuclei, 83.7% vs. 73.0%, respectively. The only significant effect of source of donor nucleus (AWC vs. DSH) was on the rate of blastocyst formation in vitro. A higher percentage of the embryos derived from AWC nuclei developed to the blastocyst stage than did embryos produced from DSH nuclei, 24.2% vs. 3.3%, respectively (P < 0.05). In experiment 4, the effect of calcium in the fusion medium on induction of oocyte activation and development of AWC-DSH-cloned embryos was determined. The presence of calcium in the fusion medium induced a high incidence of cleavage of DSH oocytes (54.3%), while oocyte cleavage frequency was much lower in the absence of calcium (16.6%). The presence or absence of calcium in the fusion medium did not affect the fusion, cleavage, and blastocyst development of AWC-DSH-cloned embryos. In experiment 5, AWC-DSH-cloned embryos were transferred to the uteri of 11 synchronized domestic cat recipients on Day 6 or 7 after oocyte aspiration. Recipients were assessed by ultrasonography on Day 21 postovulation, but no pregnancies were observed. In the present study, after NT, AWC donor nuclei were able to dedifferentiate in DSH cytoplasts and support high rates of blastocyst development in vitro. Incomplete reprogramming of the differentiated nucleus may be a major constraint to the in vivo developmental potential of the embryos.
Animals↗
Study of heterogeneity of chloramphenicol acetyltransferase (CAT) genes in streptococci and enterococci by polymerase chain reaction: characterization of a new CAT determinant.
An assay based on the utilization of degenerate primers that enable enzymatic amplification of an internal fragment of cat genes known to be present in gram-positive cocci was developed to identify the genes encoding chloramphenicol resistance in streptococci and enterococci. The functionality of this system was illustrated by the detection of cat genes belonging to four different hydridization classes represented by the staphylococcal genes catpC221, catpC194, catpSCS7, and the clostridial gene catP, and by the characterization of a new streptococcal cat gene designated catS. A sequence related to the clostridial catQ gene, which was present in one streptococcal strain, was not detected by this assay. These results reveal that these six cat genes account for chromosomal-borne chloramphenicol resistance in 12 group A, B, and G streptococci tested. By contrast, only three of these six cat genes (catpC221, catpC194, and catpSCS7) were detected on the 10 enterococcal plasmids studied here that encode resistance to chloramphenicol.
Amino Acid Sequence↗
Detection of feline coronaviruses by culture and reverse transcriptase-polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis.
A reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of the feline coronavirus (FCoV) genome and a co-cultivation method for the isolation of field strains of FCoV are described. Using the RT-PCR assay to assess blood samples from cats with feline infectious peritonitis (FIP) (n = 47) and healthy cats from households with endemic FCoV (n = 69) it was shown that approximately 80% of the cats were viraemic, irrespective of their health status. It was also shown that, over a 12-month period, a similar percentage of healthy cats remained viraemic, and that the presence of viraemia did not appear to predispose the cats to the development of FIP. The co-cultivation system proved to be a suitable method for the culture of field strains of FCoV from blood samples, so long as the cultures were maintained for at least 4 weeks. Using this system, followed by the RT-PCR, viraemia was detected as frequently as by RT-PCR on RNA extracted directly from peripheral blood mononuclear cells.
Animals↗
Brugia pahangi in cats: the passive transfer of anti-microfilarial immunity from immune to non-immune cats.
Serum from cats (Felis catus) that were repeatedly infected with Brugia pahangi and had become amicrofilaraemic (mf-ve) was injected intravenously into microfilaraemic (mf+ve) cats. If more than 1 microliter of immune serum per 1000 mf was injected, microfilarial counts fell dramatically within minutes and, in some cats, mf completely disappeared. In most cases mf reappeared 21-44 days later. However, in two experiments mf never reappeared and circulating antigen (indicative of the presence of living adults) could not be detected. At autopsy no adult worms were found, but in one cat 6 mf/ml were detected by filtration of cardiac blood. Passive transfer of single Ig isotypes showed that IgG is the immunoglobulin responsible for the mf killing effect of immune serum, and that IgG1 is probably the most active isotype. Mf killing and destruction, occurred in the lungs in an antibody dependent cell mediated reaction involving neutrophils, eosinophils and mononuclear cells. Three of the 20 recipient cats died from what appeared to be anaphylactic shock while under anaesthesia probably due to the sudden release of inflammatory mediators in the lung.
Animals↗
Post-exposure treatment of cats with mouse-cat chimeric antibodies against feline herpesvirus type 1 and feline calicivirus.
In order to confirm the in vivo effectiveness of anti- feline herpesvirus type 1 (FHV-1) mouse-cat chimeric antibody (FJH2), and anti-feline calicivirus (FCV) mouse-cat chimeric antibody (F1D7), cats that had been experimentally infected with FHV-1 or FCV were administered intravenously with the chimeric antibodies, and observed for clinical manifestations. The symptoms due to FHV-1 or FCV infection in the cats administered FJH2 or F1D7 were obviously decreased when compared with those of the non-administered control cats. From these results, it was confirmed that both FJH2 and F1D7 were effective at reducing the appearance of symptoms due to FHV-1 and FCV infection, respectively.
Animals↗
Multiple myeloma in cats: variable presentation with different immunoglobulin isotypes in two cats.
Multiple myeloma was diagnosed in two cats with monoclonal hyperglobulinemia, proteinuria, and plasma cell proliferations in bone marrow. An immunoglobulin G-producing myeloma occurred in the vertebral bone marrow of one cat, and twice responded to surgical reduction followed by a combination of local irradiation and chemotherapy. The cat's survival time was approximately 2 years. The other myeloma in a cat that presented with hypercalcemia and renal insufficiency involved visceral organs and produced a biclonal peak due to immunoglobulin A dimer formation on serum electrophoresis. This cat's tumor did not respond to chemotherapy.
Animals↗
Infection and seroconversion of cats exposed to cat fleas (Ctenocephalides felis Bouché) infected with Rickettsia felis.
Cats with no prior exposure to cat fleas were exposed to fleas infected with Rickettsia felis and monitored monthly for seroconversion via an indirect fluorescent assay (IFA). Each of 12 cats continually infested with fleas, seroconverted by four months post-exposure. Three of six cats fed on by 50 fleas for 15 minutes once per week also seroconverted by month four. Rickettsia felis DNA was detected by PCR in the blood of five of the 16 cats.
Animals↗
[A comparison of corneal sensitivity between healthy cats and cats with corneal sequestra].
In order to establish reference values for corneal sensitivity in ophthalmologically healthy persians (n = 40) and domestic short hair cats (n = 60) a prospective study was conducted. Furthermore corneal sensitivity in 48 cats with a corneal sequestrum was measured. Corneal sensitivity was recorded with the help of the aesthesiometer according to Cochet and Bonnet in five different corneal locations (central, nasal, dorsal, temporal, and ventral). The sensitivity for the central corneal region was recorded as amounting to 3.58 +/- 0.56 cm in ophthalmologically healthy domestic short hair cats and to 2.97 +/- 0.58 cm in healthy persian cats. The sensitivity of the central corneal area of a cat with a corneal sequester only amounts to 2.03 +/- 0.53 cm. Between the diseased and the healthy eyes no statistical difference could be demonstrated for any of the measured corneal locations. The sensitivity of the peripheral corneal locations is significantly lower than that of the central corneal region in all three groups examined.
Animals↗
Cryptosporidium infection in cats: prevalence of infection in domestic and feral cats in the Glasgow area.
A clinical and post mortem survey of domestic and feral cats in the Glasgow area revealed that 19 of 235 (8.1 per cent) were infected with Cryptosporidium species. More kittens than adults were infected (P less than 0.01), and of 51 of the cats which had diarrhoea, four also had cryptosporidium infection. Of seven domestic cats with cryptosporidium infection, two were also positive for feline immunodeficiency virus. There was no significant difference between the prevalence of cryptosporidium infection in domestic and feral cats. Cryptosporidium oocysts were detected in faecal and mucosal impression smears stained with auramine-phenol and modified Ziehl-Nielsen techniques. Endogenous developmental stages of cryptosporidium were found in the microvillus region of enterocytes of eight of 19 positive cats in sections stained with haematoxylin and eosin. The results suggest that cryptosporidium infection is common among young and newborn kittens, and that the disease is usually asymptomatic.
Age Factors↗
Primary structure of cat preproendothelin-2 and cat renal mRNA expression of preproendothelin-1 and preproendothelin-2 in naturally occurring renal failure.
Endothelin (ET)-1 is involved in the pathophysiology of various renal disorders, promoting renal cellular proliferation and extracellular matrix protein accumulation, and, thus, diminishing fundamental renal function, including filtration. To determine whether ET-1 and ET-2 play a role in feline chronic renal failure, we analyzed the messenger RNA (mRNA) expression of the prepro-ET (PPET )-1 and PPET-2 genes in affected cat kidney after molecular cloning of full-length PPET-2 complementary DNA (cDNA). Conceptual analysis of the primary structure of cat PPET-2 based on the cloned sequence demonstrated that the putative regions corresponding to a mature peptide and peptidase processing sites are present in cat PPET-2. Homology analysis showed that the similarity of the cat PPET-2 amino acid sequence with those from human, mouse, rat, rabbit, dog, ferret, cow, and horse was 73.0%, 68.6%, 69.1%, 76.4%, 81.2%, 83.1%, 76.3%, and 79.2%, respectively. Analysis of PPET-1 and PPET-2 mRNA in cat by reverse transcription polymerase chain reaction showed upregulated expression of both genes in kidneys affected by renal failure.
Amino Acid Sequence↗
Fecal proteolytic activity in clinically normal cats and in a cat with exocrine pancreatic insufficiency.
Fecal proteolytic activity determined in single samples collected on each of 3 consecutive days from each of 20 clinically normal cats ranged from 19 to 363 azocasein units (ACU)/g of feces when determined colorimetrically, using azocasein substrate, and ranged from undetectable (in 1 sample from 1 cat) to 21 mm of gel-clearing when determined using radial enzyme diffusion in agar gels containing a casein substrate. Corresponding mean 3-day values for each cat ranged from 29 to 207 ACU/g and from 5 to 16 mm, respectively. Studies of proteolytic activity remaining after treatment of fecal extracts with a specific trypsin inhibitor indicated that trypsin accounted for 0 to 77% of proteolytic activity. In a cat with exocrine pancreatic insufficiency, fecal proteolytic activity was 0, 0, and 3 ACU/g in a sample of feces collected from each of 3 consecutive days and was undetectable by use of radial enzyme diffusion. Assay of fecal proteolytic activity by use of either azocasein hydrolysis or radial enzyme diffusion allows evaluation of pancreatic function in cats, provided that several samples of feces are tested.
Animals↗
Platelet aggregation and ATP secretion in whole blood of normal cats and cats homozygous and heterozygous for Chediak-Higashi syndrome.
A rapid and simple technique using the Whole Blood Lumi-Aggregometer was used to study storage pool disease in Chediak-Higashi homozygote and heterozygote cats. Feline Chedlak-Higashi platelets aggregated after the addition of both ADP and collagen. During platelet aggregation, ATP secretion was assayed; the whole blood aggregometer is effective in detecting decreased levels of secretable ATP in homozygote cats. No storage pool deficiency was found in heterozygote cats. However, upon analysis of impedance tracings, a decreased platelet aggregation response was seen in both homozygote and heterozygote cats. These results suggest that prolonged bleeding times in Chediak-Higashi cats may involve a mechanism in addition to a dense granule deficiency.
Adenosine Triphosphate↗
[Glycosylated hemoglobin in the cat: affinity chromatography determination in healty, permanent diabetes mellitus and transient hyperglycemic cats].
The affinity method has proved to be the first technique that succeeded in measuring feline GHb as it is not affected by structural differences of haemoglobin as traditional proceedings are. By its help the reference interval for feline GHb was established from blood samples of 62 healthy cats. It ranged between 0.31 to 1.58% with a median of 0.92%. 21 newly diagnosed diabetic cats were reliably determined to be diabetic, because the GHb exceeded significantly the upper limit of the reference interval of 1.30%. The statistical difference between diabetic and healthy cats was highly significant (p < 0.00001). It was confirmed that the GHb value helps to distinguish diabetic from transient hyperglycemic cats. Newly presented hyperglycemic cats suffer from diabetes mellitus, if their GHb values are increased (p < 0.00001).
Animals↗
The cat hemoglobin polymorphism: Southern blot analysis of the beta-globin gene region from cats of various Hb A/Hb B phenotypes.
The molecular basis for the genetic control of variable proportions of the two cat hemoglobins Hb A(alpha 2 beta A2) and Hb B (alpha 2 beta B2) was investigated. Ratios of Hb A/Hb B vary between 50/50 and 90/10 among members of the mongrel cat population, with clusters around 50, 35, and 10% Hb B. Genomic DNA from cats of 50/50, 70/30, and 90/10 phenotypes were cut by restriction endonucleases HindIII, EcoRI, BamHI, BglII, and Pstl and hybridized to a fragment of the human beta-globin gene. The results of the Southern blots suggested a pattern of homozygote, heterozygote, homozygote for the respective cat phenotypes, 50/50, 70/30, and 90/10. Therefore, the cat hemoglobin polymorphism seems to result from the possible combinations of an allelic gene pair.
Animals↗