Human smooth muscle alpha-actin gene is a transcriptional target of the p53 tumor suppressor protein.

Smooth muscle (sm) alpha-actin is expressed in vascular smooth muscle cells and fibroblast cells. Its expression is regulated by cell proliferation and repressed during oncogenic transformation. In this study, we demonstrate that p53 activation is associated with a dramatic increase in organized microfilament bundles and an increase in sm alpha-actin mRNA level. Wild-type p53, but not mutant p53, strongly stimulated human sm alpha-actin promoter activity in p53 null cell lines. The sequences homologous to the p53 consensus sequence and to the p53 binding sequence from the muscle creatine kinase, were found within a specific region of the sm alpha-actin promoter. This sequence was sufficient to confer p53-dependent activation to a heterologous promoter and p53 was capable of binding to this sequence as assessed by gel shift analysis. Ionizing irradiation of colorectal tumor cells caused an increase in alpha-actin mRNA level in a p53-dependent manner. Taken together, these results demonstrate that human sm alpha-actin gene is a transcriptional target for p53 tumor suppressor protein and represents the first example of a cytoskeletal gene with a functionally defined p53 response element.

Human dehydroepiandrosterone sulfotransferase. Purification, molecular cloning, and characterization.

Human tissues possess at least four distinct forms of cytosolic ST, three of which are involved in the sulfation of steroids. DHEA-ST is responsible for the majority of hydroxysteroid and bile acid sulfation in human tissues and abundant levels of the enzyme are present in human liver and adrenal tissues. In the adult human adrenal, DHEA-ST has been localized immunologically to the zona reticularis of the adrenal cortex. No age- or gender-related differences in the expression of DHEA-ST activity in adult human liver cytosols have been reported. The cDNA encoding DHEA-ST has been isolated from a human liver cDNA library and expressed in both mammalian COS cells and E. coli. Purification and molecular characterization studies suggest a single form of DHEA-ST in human tissues. The properties of DHEA-ST expressed in either mammalian or bacterial cells are very similar to those of the native enzyme. DHEA-ST can also bioactivate a number of procarcinogens to reactive electrophilic forms. Hydroxymethyl PAHs are sulfated and bioactivated at a relatively rapid rate by DHEA-ST, whereas 1'-hydroxysafrole and N-hydroxy-2-acetylaminofluorene are bioactivated to a lesser extent.

Comparison of the Abbott Cell Dyn 3000 SL and the Coulter STKS hematology analyzers.

A side by side comparison of the Cell Dyn 3000 SL and Coulter STKS using identical samples was performed over a four month period in the Clinical Hematology Laboratory at the University of Cincinnati Medical Center. A total of 444 samples comprised of 114 sophomore medical students and 330 randomly selected clinical patients were compared in 20 hemogram parameters, and the ability of each instrument to generate differential suspect flags was analyzed. Correlation coefficients for leukocytes, erythrocytes, hemoglobin, hematocrit, and platelets were 0.99. Correlation coefficients for the Cell Dyn and STKS compared with a 600 cell manual differential were 0.98 and 0.89 for neutrophils, 0.96 and 0.87 for lymphocytes, and 0.72 and 0.48 for monocytes, respectively. Both instruments demonstrated high precision and accuracy by internal and external quality control standards. Each analyzer exhibited strength as a screening instrument for abnormal cell populations. The Coulter STKS had overall sensitivity of 75%, specificity of 94%, 25% false negatives, and 6% false positives. Sensitivity, specificity, false negative and positive rates for the Cell Dyn 3000 SL were 68%, 92%, 32%, and 8%, respectively. Based upon this extensive side by side comparison using an identical sample population, it has been concluded that although statistically significant systematic bias (p < 0.05) exists between the two instruments, both analyzers can adequately support the needs of the clinical hematology laboratory.

Canine leishmaniasis caused by Leishmania leishmania infantum in two Labrador retrievers.

Canine leishmaniasis, a generally fatal parasitic disease, was diagnosed in 2 dogs with a medical history of foreign travel, lymphadenopathy, emaciation, anorexia, intermittent fever, and cutaneous lesions. Clinically, hyperproteinemia, proteinuria, azotemia, and glomerulopathy were evident. Isolation of Leishmania species was done using Schneider's Drosophila medium. Syrian hamsters were used for infectivity studies. Clear taxonomic identification was done biochemically by isoenzyme analysis and comparison of zymogram banding patterns with 6 World Health Organization reference strains. Based on the geographic origin of affected dogs, clinicopathologic presentation, visceralization with hepatosplenomegaly in hamsters, and isoenzyme analysis, a diagnosis of Leishmania leishmania infantum was made. This study, representing the first taxonomic identification of an isolate from canine leishmaniasis, demonstrates the zoonotic and epidemiologic implications of this disease.

Carpet deodorizer as a contact allergen in a dog.

Allergic contact dermatitis was diagnosed in a 7-year-old dog. Clinical signs and history were consistent with a contact allergen being the cause of pruritic skin disease. Patch testing confirmed that carpet deodorizer was the allergen.

Results of urinalysis and bacterial culture of canine urine obtained by antepubic cystocentesis, catheterization, and the midstream voided methods.

Results of urinalysis and bacterial culture of urine specimens obtained during midstream voiding, by catheterization, and by cystocentesis were studied in 50 clinically normal dogs (25 females and 25 males). All of the specimens obtained by cystocentesis were bacteriologically sterile. Bacterial growth occurred in 26% of the catheterized specimens and in 85% of the voided specimens. The proportion of positive urine cultures from catheterized and voided specimens was higher for females than for males. Results or urinalysis did not correlate well with results of bacterial culture.

Canine hyperadrenocorticism: pretreatment clinical and laboratory evaluation of 117 cases.

The physical findings, clinical signs, age, breed and sex distributions, and laboratory data of 117 dogs with untreated Cushing's syndrome were reviewed. Poodles, Dachshunds, and Boxers of all ages were found to be at increased risk, as were dogs of all breeds greater than or equal to 6 years old. Polydipsia, polyuria, progressive bilaterally symmetric alopecia, and abdominal distention were the most frequently observed clinical signs and physical findings. Lymphopenia, eosinopenia, above normal values of serum alkaline phosphatase, serum cholesterol, and sulfobromphthalein dye retention, and below normal urine specific gravity were the most frequent abnormalities found in the laboratory data. About 50% of the dogs had urinary tract infections. Final diagnosis was established on the basis of abnormally high plasma corticosteroid values in response to an intramuscular injection of adrenocorticotropic hormone.

Comparison of procedures for assessing adequacy of dog foods.

Dog foods with similar claims for nutritional adequacy were tested by chemical analysis and the American Association of Feed Control Officials' growth trial. All foods tested were similar chemically, however, dogs given one regionally marketed food had lower growth rate and food efficiency as well as suboptimal PCV and hemoglobin values during the growth trial. Pups fed this diet also had clinical signs typical of zinc and copper deficiencies. We concluded that American Association of Feed Control Officials' approved feeding tests provide valid assessment of pet food quality, and procedures involving only chemical analysis or calculated values may not.

Rocky Mountain spotted fever.

Rocky Mountain spotted fever is an endemic tickborne disease found throughout the United States and other regions of the world. Exposure may result in a spectrum of disease from subclinical infection to severe or fatal multiorgan collapse. The disease is maintained in nature in Ixodid tick vectors and their hosts. The most important ticks in the United States are Dermacentor variabilis and Dermacentor andersoni. Small mammals are the natural reservoirs in the wild. Dogs become infected when a tick harboring Rickettsia rickettsii feeds on the dog. Dogs do not develop sufficient rickettsemia to act as a reservoir in the transmission of Rickettsia rickettsii. Thus, although dogs act as sentinels to the presence of the disease, they cannot directly transmit infection. Signs in early stages of disease often are nonspecific. The most characteristic laboratory abnormality is thrombocytopenia, but serologic testing is necessary for confirmation of infection. Tetracycline and chloramphenicol are effective antibiotics to treat infection. Treatment should continue for 14 to 21 days to allow host immune defenses to develop and eradicate the organism. Prevention requires avoidance of tick-infested areas and rapid removal of ticks should exposure occur.

Soft x-ray virtual diagnostics for tokamak simulations.

The numerical toolset, FAR-TECH Virtual Diagnostic Utility, for generating virtual experimental data based on theoretical models and comparing it with experimental data, has been developed for soft x-ray diagnostics on DIII-D. The virtual (or synthetic) soft x-ray signals for a sample DIII-D discharge are compared with the experimental data. The plasma density and temperature radial profiles needed in the soft x-ray signal modeling are obtained from experimental data, i.e., from Thomson scattering and electron cyclotron emission. The virtual soft x-ray diagnostics for the equilibriums have a good agreement with the experimental data. The virtual diagnostics based on an ideal linear instability also agree reasonably well with the experimental data. The agreements are good enough to justify the methodology presented here for utilizing virtual diagnostics for routine comparison of experimental data. The agreements also motivate further detailed simulations with improved physical models such as the nonideal magnetohydrodynamics contributions (resistivity, viscosity, nonaxisymmetric error fields, etc.) and other nonlinear effects, which can be tested by virtual diagnostics with various stability modeling.

Immunological characterization of dehydroepiandrosterone sulfotransferase from human liver and adrenal.

Dehydroepiandrosterone sulfotransferase (DHEA-ST), a steroid sulfotransferase (ST), has recently been purified from human liver cytosol and partially characterized. DHEA-ST has a subunit molecular mass of 35 kDa and is responsible for the majority of the sulfation of steroids and bile acids in the liver. For these studies, polyclonal antibodies to human liver DHEA-ST were raised in rabbits. The anti-human liver DHEA-ST antibodies were used to characterize the immunoreactivity of DHEA-ST in human liver and to study the relationship of human adrenal DHEA-ST to the liver form of the enzyme. Immunoblot analysis of several different human liver cytosol samples with the rabbit anti-human liver DHEA-ST antiserum detected only a single 35-kDa protein in each liver. Anti-human liver DHEA-ST antibodies also did not react with either form of phenol sulfotransferase (PST), P-PST or M-PST, present in human liver cytosol. DHEA-ST activity was purified from the 100,000 x g supernatant fraction of human adrenal tissue by DEAE-Sepharose CL-6B chromatography and 3',5'-diphosphoadenosine-agarose affinity chromatography. Human adrenal DHEA-ST was shown to have a molecular mass of 35 kDa, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis of human adrenal cytosol revealed that the anti-human liver DHEA-ST antibodies reacted specifically with the 35-kDa subunit of DHEA-ST. The apparent Km values for DHEA and 3'-phosphodenosine-5'-phosphosulfate obtained with human adrenal DHEA-ST were 1.0 microM and 1.6 microM, respectively. Adrenal DHEA-ST demonstrated the same pattern of reactivity towards different steroid substrates as did human liver DHEA-ST, and neither form of DHEA-ST was found to sulfate cortisol. The results of this study suggest that DHEA-ST is the major steroid ST present in human liver and adrenal tissue and that the physical, biochemical, and kinetic properties of adrenal DHEA-ST are similar if not identical to those of the liver form of the enzyme.

Cloning and expression of human liver dehydroepiandrosterone sulphotransferase.

Dehydroepiandrosterone sulphotransferase (DHEA-ST) catalyses the 3'-phosphoadenosine 5'-phosphosulphate-dependent sulphation of a wide variety of steroids in human liver and adrenal tissue and is responsible for most, if not all, of the sulphation of bile acids in human liver. This report describes the isolation, characterization and expression of a cDNA which encodes human liver DHEA-ST. The DHEA-ST cDNA, designated DHEA-ST8, was isolated from a Uni-Zap XR human liver cDNA library and is composed of 1060 bp and contains an open reading frame encoding a 285-amino-acid protein with a molecular mass of approx. 33765 Da. Translation of DHEA-ST8 in vitro generated a protein identical in molecular size with that of DHEA-ST. Expression of DHEA-ST8 in COS-7 cells produces an active DHEA-ST protein which is capable of sulphating DHEA, has the same molecular mass as human liver DHEA-ST and is recognized by rabbit anti-(human liver DHEA-ST) antibodies. Northern-blot analysis of human liver RNA detects the presence of three different size transcripts; however, Southern-blot analysis of human DNA suggests that only one gene may be present in the genome. These results describe the cloning of a human ST which has an important role in the sulphation of steroids and bile acids in human liver and adrenals.

A unique alpha chain in hemoglobin of "Skive" Danish Mus musculus.

The primary structures of the alpha chains in hemoglobins from three stocks of mice with the Hbaw2, Hbaw3, and Hbaw4 haplotypes were determined to establish whether the tentative alpha-chain assignments based on the results of isoelectric focusing patterns were correct. These Hba haplotypes were identified in laboratory descendants of feral mice captured in different parts of the world. Hemoglobin from "Centreville", Maryland, Mus musculus domesticus (Hbaw2) contains equal amounts of alpha chains 1 and 3. Hemoglobin from "Czech" Mus musculus musculus (Hbaw4) contains equal amounts of alpha chains 3 and 4. Amino acid analysis of the alpha-globins of "Skive" Danish Mus musculus musculus (Hbaw3) establishes that its hemoglobin is comprised of about one-third alpha chain 2 as expected plus a greater amount of a unique alpha chain that has not been described previously. This unique alpha chain has glycine at position 25, isoleucine at position 62, and serine at position 68; it is called chain 7. It may represent an intermediate in the evolution of genes that code for chain 2 (which has glycine, valine, and serine at positions 25, 62, and 68, respectively) and chain 4 (which has valine, isoleucine, and serine at positions 25, 62, and 62, respectively).

Human liver steroid sulphotransferase sulphates bile acids.

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.

Sequence analysis and expression of the cDNA for the phenol-sulfating form of human liver phenol sulfotransferase.

A cDNA encoding the human liver phenol-sulfating form of phenol sulfotransferase (P-PST) has been isolated and characterized from a lambda Uni-Zap XR human liver cDNA library. P-PST is the major form of phenol sulfotransferase involved in drug and xenobiotic metabolism in human liver. P-PST is also responsible for the sulfation and activation of minoxidil to its therapeutically active sulfate ester. The full length cDNA, P-PST-1, is 1206 base pairs in length and encodes a 295-amino acid protein with a molecular mass of 34,097 Da. The translation sequence of P-PST-1 is 96% similar to the amino acid sequences of five peptides derived from the purified protein. In vitro transcription and translation of P-PST-1 generated a protein that comigrates with immunoreactive P-PST from human liver. Significant increases in sulfotransferase activity toward two P-PST-specific substrates, minoxidil and 4-nitrophenol, were detected in cytosol prepared from COS-7 cells transfected with P-PST-1 in the expression vector p-SV-SPORT-1. Northern blot analysis of human liver RNA detected a transcript of approximately 1300 nucleotides in length. Characterization of P-PST at the molecular level provides insight into the structure and heterogeneity of this major class of drug-metabolizing enzymes.