Uterine infarctions in cynomolgus monkeys (Macaca fascicularis).

Uterine infarctions have not been reported in domestic animals, and there are few reports in the human medical literature. In a retrospective study, uterine infarctions were identified in 9 of 323 (2.8%) female cynomolgus monkeys (Macaca fascicularis) necropsied over a 13-year period. The infarctions were grossly visible, after fixation, on the serosal surface of the uterus in 2 monkeys; the remainder were first recognized in histologic sections. Histologically, the lesions consisted of well-demarcated regions of endometrial and myometrial necrosis and of hemorrhage. All affected monkeys had histologic evidence of a previous pregnancy, which included enlarged myometrial vessels with an expanded perivascular matrix. In all monkeys with uterine infarctions, there was clinical evidence of severe systemic illness, which included trauma, diarrhea, hypovolemia, or septicemia. The major pathologic findings in affected monkeys included cutaneous or skeletal muscle necrosis (n = 5), enterocolitis (n = 4), pulmonary edema or diffuse alveolar damage (n = 3), and intestinal amyloidosis (n = 1). Histopathologic evidence of intravascular fibrin thrombi in multiple organs of 5 monkeys was consistent with a diagnosis of disseminated intravascular coagulopathy (DIC). Based on these findings, it appears that uterine infarction is an uncommon finding in cynomolgus monkeys and may occur secondary to a severe systemic illness, predisposing to DIC.

Recognition of a novel naturally processed, A2 restricted, HCV-NS4 epitope triggers IFN-gamma release in absence of detectable cytopathicity.

Using short term CTL lines derived from HLA A2/Kb transgenic mice and IFN-gamma release assays we demonstrate that the NS4.1769 epitope, is generated from natural processing of the NS4 antigen, and presented in the context of the A2/Kb molecules. Interestingly, T cell recognition of the naturally processed form of the NS4. 1769 epitope was associated with significant IFN-gamma release, but no direct cytolytic activity. Epitopes of this phenotype might be of interest, in terms of therapy of chronic HCV infection by associating the benefit of localized lymphokine release with low or absent direct cytopathicity.

Gastric infarction in cynomolgus monkeys (Macaca fascicularis).

Five cases of gastric infarction were observed in adolescent or adult cynomolgus monkeys (Macaca fascicularis) over a 20-month period. Gastric infarcts were encountered as striking and unexpected findings at necropsy. Gross and microscopic findings included gastric necrosis, hemorrhage, and edema that involved large areas of the fundus and pylorus. A consistent finding was the presence of thrombi in the gastric microvasculature, particularly in the venous system. All animals had acute clinical episodes with substantial tissue damage resulting from a variety of causes, including trauma, pancreatitis, necrotizing cystitis, and intestinal intussusception. In addition, three animals had microvascular thrombosis in nongastric tissues. Our findings suggest that cynomolgus monkeys may be predisposed to developing gastric infarction under conditions of severe systemic insult that predispose to disseminated intravascular coagulation.

Localized retroperitoneal fibromatosis causing intestinal obstruction in a cynomolgus monkey (Macaca fascicularis).

Gross examination of a 24-month-old, male cynomolgus monkey (Macaca fascicularis) revealed obstruction of the ileum by a mass that entrapped and compressed the ileocecal junction. The mass was well circumscribed, firm, and white on cut surface. Histologically, the mass consisted of spindle-shaped cells arranged in interweaving bundles or as narrow cords and individual cells widely separated by dense collagen. A diagnosis of localized retroperitoneal fibromatosis was made based on the characteristic gross and microscopic findings and isolation of type D simian retrovirus, serotype-2, from spleen and mesenteric lymph node. Monkeys with localized retroperitoneal fibromatosis generally exhibit signs only of a palpable mass at the ileocecal junction and/or nonspecific diarrhea. This case represents an unusual presentation of localized retroperitoneal fibromatosis in which the lesion produced intestinal obstruction and death.

Identification of a novel simian parvovirus in cynomolgus monkeys with severe anemia. A paradigm of human B19 parvovirus infection.

Although human B19 parvovirus infection has been clearly associated with a number of distinct syndromes (including severe anemia, abortion, and arthritis), detailed knowledge of its pathogenesis has been hindered by the lack of a suitable animal model. We have identified a novel simian parvovirus in cynomolgus monkeys with severe anemia. Sequencing of a 723-bp fragment of cloned viral DNA extracted from serum revealed that the simian parvovirus has 65% homology at the DNA level with the human B19 parvovirus but little homology with other known parvoviruses. Light microscopic examination of bone marrow from infected animals showed intranuclear inclusion bodies, and ultrastructural studies showed viral arrays characteristic of parvoviruses. Another striking feature was the presence of marked dyserythropoiesis in cells of the erythroid lineage, raising the possibility that B19 parvovirus infection may underlie related dyserythropoietic syndromes in human beings. Affected animals had concurrent infection with the immunosuppressive type D simian retrovirus, analogous to HIV patients who develop severe anemia because of infection with B19 parvovirus. The remarkable similarities between the simian and B19 parvoviruses suggest that experimentally infected cynomolgus monkeys may serve as a useful animal model of human B19 infection.

Expression and functional significance of alternatively spliced CS1 fibronectin in rheumatoid arthritis microvasculature.

Expression of fibronectin (FN) isoforms containing CS1, a 25-amino acid sequence present within the alternatively spliced IIICS region of FN, has been analyzed in rheumatoid arthritis (RA) synovium. Unexpectedly, CS1-containing FN variants were exclusively found on endothelium but not extracellular matrix (ECM) of RA synovium. Lumenal expression of CS1 on RA endothelial cells, as observed by electron microscopy, correlated with inflammation in RA, since normal synovium expressed little CS1 without appreciable decrease in ECM FN. CS1 expression on human endothelial cells was further shown by FN mRNA analyses. In adhesion assays on frozen RA synovial sections, T lymphoblastoid cells expressing functionally activated alpha 4 beta 1 integrin specifically attached to the intravascular surface of RA endothelium. Binding was abrogated by both anti-alpha 4 integrin and CS1 peptides. Our observations suggest direct involvement of CS1-containing FN in recruitment of alpha 4 beta 1-expressing mononuclear leukocytes in synovitis, and provide basis for therapeutic intervention in RA.

Insensitivity of the chicken embryo to the ototoxicity of aminoglycoside antibiotics and a loop diuretic.

Guinea pigs are routinely used in the histological evaluation of the cochlea as a method of testing for ototoxicity, but the procedures are very time-consuming. Because the avian cochlea is easier to examine and newly hatched chicks are sensitive to the ototoxic effects of gentamicin, birds may be useful in testing for ototoxicity. The use of chicken embryos would be even better for testing, but whether or not chicken embryos are sensitive to ototoxicants is unknown. In an attempt to determine whether or not chicken embryos may be used instead of guinea pigs in screening tests for ototoxicity, aminoglycoside antibiotics and a loop diuretic, ethacrynic acid, were administered to chicken embryos. A maximum-tolerated dose of gentamicin, kanamycin, streptomycin, ethacrynic acid, or a combination of gentamicin and ethacrynic acid was administered to fertile eggs of White Leghorn chickens on incubation days 10-17. To compare the effect of route of exposure on ototoxicity, gentamicin was administered by injection into the allantoic space, yolk sac, and air cell as well as by submerging the egg in gentamicin solution. With the preferred air cell route the effects of the ototoxic drugs kanamycin, streptomycin, ethacrynic acid, and a combination of ethacrynic acid and gentamicin were compared. On incubation day 18, cochleas were removed from the chicken embryos. Serial sections of these avian cochleas were examined and hair cells were counted. No significant difference was seen between the number of hair cells in cochleas of control chicken embryos and those from chicken embryos treated with drugs. Therefore, the chicken embryo appears to be insensitive to the ototoxicity of aminoglycoside antibiotics and a loop diuretic.

Distribution of gentamicin to the cochlea of the chicken embryo.

Aminoglycoside antibiotics are ototoxic in mammals and birds, including recently hatched chicks, but chicken embryos are insensitive to the ototoxicity of gentamicin, kanamycin, and streptomycin. To determine whether or not the insensitivity is due to a lack of antibiotic distribution to the avian cochlea, the distribution of gentamicin to the cochlea of the White Leghorn chicken embryo was compared to the distribution to the cochlea of the recently hatched White Leghorn chick. Fertile eggs were injected with a maximally tolerated dose of gentamicin sulfate (0.1 mg/egg/day) on incubation days 10-18, and the chicks were injected subcutaneously with either 5 mg (non-ototoxic) or 100 mg (ototoxic) gentamicin sulfate/kg body weight on days 1-9 after hatching. Gentamicin sulfate was histochemically detected within the basilar papilla (the avian equivalent of the organ of Corti) in all treated chicken embryos and chicks by 1 day after the first injection, and the staining was intense after 3 days of treatment. By ultrastructural immunocytochemistry, mild, diffuse labeling for gentamicin sulfate was detected within the endoplasmic reticulum of short and tall hair cells of chicken embryos by incubation day 17. Moderate labeling of gentamicin sulfate was detected in the infracuticular region of lysosomes of hair cells in chicks receiving 5 treatments of gentamicin sulfate at 5.0 mg/kg body weight and after 1 treatment of gentamicin sulfate at 100 mg/kg body weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in yeast HAP2/HAP3 define a hybrid CCAAT box binding domain.

We describe a detailed genetic analysis of the DNA-binding regions in the HAP2/HAP3 CCAAT-binding heteromeric complex. The DNA-binding domain of HAP2 is shown to be a 21 residue region containing three critical histidines and three critical arginines. Mutation of an arginine at position 199 to leucine alters the DNA-binding specificity of the complex to favor CCAAC over CCAAT. Residues in HAP3 that are critical for DNA-binding comprise a short, seven amino acid region. Three different mutations in the HAP2 DNA-binding domain are suppressed by a mutation in the HAP3 DNA-binding domain. This HAP3 mutation also suppresses mutations in a different region of HAP2 which promotes subunit assembly of the complex. These findings suggest that short regions of HAP2 and HAP3 comprise a hybrid DNA-binding domain and that this domain can help hold the two subunits together in the CCAAT-binding complex.

Clinical, biochemical, electrophysiologic, and histologic assessment of chlorpyrifos induced delayed neuropathy in the cat.

The ability of a supralethal dose of chlorpyrifos to produce delayed neuropathy was examined using assessments of clinical signs, electromyography (EMG), motor nerve conduction velocity (MNCV), lymphocyte neuropathy target esterase activity (LNTE), and histologic changes in nervous system tissues. Cats were exposed to a single, im injection of corn oil (vehicle control), DFP (positive control) at 5.0 mg/kg, or chlorpyrifos at 300 mg/kg and observed for 60 days. Atropine and 2-PAM were administered to chlorpyrifos exposed cats one to two times a day for 14 to 24 days in response to the appearance of cholinergic signs. Anorectic cats during the acute toxicosis were force fed by hand and hydration was maintained by administering fluids sc. Onset of ataxia (mean +/- SD) for the positive control and chlorpyrifos exposed cats were 16.2 +/- 1.8 days (range of 14-19 days) and 19.0 +/- 1.4 days (range of 17-21 days), respectively. Functional deficits for both groups were confined to the hindlimbs and characterized by a crouched-waddling gait, hypermetria, and proprioceptive deficits. Maximal inhibition of LNTE activity was 96% at 24 hr postdosing in the positive control group and 46% at 7 days postdosing in the chlorpyrifos group. No EMG or MNCV abnormalities were detected in any of the treatment groups. Axonal degeneration was similar for the positive control and chlorpyrifos exposed cats. Ascending tracts of the cervical spinal cord and descending tracts of the thoracic and lumbar spinal cord were most severely affected and peripheral nerves were only mildly affected. The clinical and histologic effects produced indicate that chlorpyrifos can cause delayed neuropathy in the domestic cat. The moderate but prolonged inhibition of LNTE produced by chlorpyrifos is atypical of classic organophosphorus delayed neurotoxicants.

A cDNA encoding a human CCAAT-binding protein cloned by functional complementation in yeast.

We constructed a comprehensive cDNA library from HeLa cell mRNA in a vector that directs expression of the cDNA in Saccharomyces cerevisiae. We used this library to clone the human counterpart of the Sa. cerevisiae CCAAT-binding transcription factor, Hap2, by functional complementation of a hap2 mutation. The cDNA encoding the human Hap2 homolog encodes a protein of 257 amino acids that has a 62-amino acid carboxyl-terminal region 73% identical to the essential core region of Hap2. The amino terminus of the protein is highly enriched in glutamine residues, reminiscent of transcriptional activation domains of several other mammalian transcription factors. Analysis of human Hap2 expression reveals three major transcripts: a 4.1-kilobase species found in all cell types examined, a 7.0-kilobase species specific to B lymphocytes, and a 1.6-kilobase species that is expressed preferentially in HeLa cells and that likely corresponds to our cDNA clone. Thus, the human Hap2 homolog and related factors may play both a constitutive and cell type-specific role in gene expression. The general approach of cloning by complementation should allow the isolation of many human genes for which corresponding yeast mutations exist.

The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small essential core protein domain.

The fission yeast Schizosaccharomyces pombe is immensely diverged from budding yeast (Saccharomyces cerevisiae) on an evolutionary time scale. We have used a fission yeast library to clone a homolog of S. cerevisiae HAP2, which along with HAP3 and HAP4 forms a transcriptional activation complex that binds to the CCAAT box. The S. pombe homolog php2 (S. pombe HAP2) was obtained by functional complementation in an S. cerevisiae hap2 mutant and retains the ability to associate with HAP3 and HAP4. We have previously demonstrated that the HAP2 subunit of the CCAAT-binding transcriptional activation complex from S. cerevisiae contains a 65-amino-acid "essential core" structure that is divisible into subunit association and DNA recognition domains. Here we show that Php2 contains a 60-amino-acid block that is 82% identical to this core. The remainder of the 334-amino-acid protein is completely without homology to HAP2. The function of php2 in S. pombe was investigated by disrupting the gene. Strikingly, like HAP2 in S. cerevisiae, the S. pombe gene is specifically involved in mitochondrial function. This contrasts to the situation in mammals, in which the homologous CCAAT-binding complex is a global transcriptional activator.

Striking conservation of TFIID in Schizosaccharomyces pombe and Saccharomyces cerevisiae.

Eukaryotic promoters contain binding sites for basic transcription factors and gene-specific activator proteins. The transcription factors interact at the TATA box, which lies close to the position of transcription initiation. Activators typically bind to distant sites that can lie kilobases away from the initiation site. The factor TFIID binds specifically to the TATA box to initiate an ordered pathway of assembly of the basic transcription factors. Biochemical analyses have shown that human and Saccharomyces cerevisiae TFIID are functionally interchangeable in vitro. To study further the functional conservation of this critical factor, we are surveying proteins from divergent organisms that can substitute in vivo for the S. cerevisiae TFIID. We report here the isolation of a unique gene from Schizosaccharomyces pombe that fully complements a null mutation in SPT15, the gene that encodes TFIID in S. cerevisiae. The Schiz. pombe gene encodes a protein 93% identical (166/178) to S. cerevisiae TFIID in a region consisting of a direct repeat.

Toxicology of selected pesticides, drugs, and chemicals. Organophosphorus and carbamate insecticides.

Organophosphorus and carbamate insecticides produce acute toxicosis via a common mechanism--the inhibition of acetylcholinesterase--and, therefore, these categories of insecticides are considered together. This article discusses the mechanisms, toxicity, clinical signs, lesions, and diagnosis of, as well as treatment for, toxicoses produced by these compounds.

Maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo. Sequence requirements for efficient processing and demonstration of an alternate cleavage site.

Comparative analyses of a number of secretory proteins processed by eukaryotic and prokaryotic signal peptidases have identified a strongly conserved feature regarding the residues positioned -3 and -1 relative to the cleavage site. These 2 residues of the signal peptide are thought to constitute a recognition site for the processing enzyme and are usually amino acids with small, neutral side chains. It was shown previously that the substitution of aspartic acid for alanine at -3 of the Escherichia coli maltose-binding protein (MBP) signal peptide blocked maturation by signal peptidase I but had no noticeable effect or MBP translocation across the cytoplasmic membrane of its biological activity. This identified an excellent system in which to undertake a detailed investigation of the structural requirements and limitations for the cleavage site. In vitro mutagenesis was used to generate 14 different amino acid substitutions at -3 and 13 different amino acid substitutions at -1 of the MBP signal peptide. The maturation of the mutant precursor species expressed in vivo was examined. Overall, the results obtained agreed fairly well with statistically derived models of signal peptidase I specificity, except that cysteine was found to permit efficient processing when present at either -3 and -1, and threonine at -1 resulted in inefficient processing. Interestingly, it was found that substitutions at -1 which blocked processing at the normal cleavage site redirected processing, with varying efficiencies, to an alternate site in the signal peptide represented by the Ala-X-Ala sequence at positions -5 to -3. The substitution of aspartic acid for alanine at -5 blocked processing at this alternate site but not the normal site. The amino acids occupying the -5 and -3 positions in many other prokaryotic signal peptides also have the potential for constituting alternate processing sites. This appears to represent another example of redundant information contained within the signal peptide.

Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide.

Oligonucleotide-directed mutagenesis was employed to investigate the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the protein export process. The three basic residues residing at the amino terminus of the signal peptide were systematically substituted with neutral or acidic residues, decreasing the net charge in a stepwise fashion from +3 to -3. It was found that a net positive charge was not absolutely required for MBP export to the periplasm. However, export was most rapid and efficient when the signal peptide retained at least a single basic residue and a net charge of +1. The nature of the adjacent hydrophobic core helped to determine the effect of charge changes in the hydrophilic segment on MBP export, which suggested that these two regions of the signal peptide do not have totally distinct functions. Although the stepwise decrease in net charge of the signal peptide also resulted in a progressive decrease in the level of MBP synthesis, the data do not readily support a model in which MBP synthesis and export are obligately coupled events. The export defect resulting from alterations in the hydrophilic segment was partially suppressed in strains harboring certain prl alleles but not in strains harboring prlA alleles that are highly efficient suppressors of signal sequence mutations that alter the hydrophobic core.

Factors influencing the in vitro translocation of the Escherichia coli maltose-binding protein.

An in vitro system has been utilized to study the translocation of newly synthesized Escherichia coli maltose-binding protein (MBP) into inverted membrane vesicles. Approximately 40% of precursor MBP (pMBP) synthesized with a wild-type signal peptide was imported into vesicles. However, MBP species with even minor alterations in the signal peptide hydrophobic core were imported into vesicles with an efficiency much lower than predicted from in vivo studies. Posttranslational import of wild-type pMBP into vesicles could be demonstrated if membranes were added after the termination of protein synthesis. However, if vesicles were present throughout the synthesis reaction, most pMBP import occurred either cotranslationally or very soon after completion of synthesis. The wild-type pMBP rapidly became incompetent for posttranslational translocation upon continued incubation in the absence of membranes, whereas pMBP species with altered folding properties remained competent for significantly longer periods. The rate of in vitro pMBP folding was affected by the nature of the signal peptide. The evidence suggests that one or more soluble factors may interact with the newly synthesized pMBP to help maintain it in a translocation-competent state and to promote its entrance into the export pathway.

Dinoseb toxicosis in two dogs.

Two male English Setters were noticed to be breathing rapidly, hyperexcitable, and atactic after roaming a rural area for 2 hours. Both dogs' cost were stained with yellow liquid. One dog died while en route to the veterinarian. Treatment was begun for the surviving dog for what was initially diagnosed to be organophosphorus or carbamate insecticide toxicosis. Before the diagnosis could be confirmed, the second dog died. The yellow liquid on the dogs' skin was identified as dinoseb in high concentrations. Dinoseb is an acutely toxic, substituted dinitrophenolic herbicide believed to act as an uncoupler of electron transport from oxidative phosphorylation.

Novel secA alleles improve export of maltose-binding protein synthesized with a defective signal peptide.

Mutations previously designated prlD were described that suppressed malE signal sequence mutations and were located in the vicinity of the secA gene on the Escherichia coli chromosome. In this study, we demonstrated that four such independently isolated prlD mutations represented three unique single-base substitutions in secA, resulting in alterations at residues 111, 373, and 488 of the 901-residue SecA protein. Heretofore, the only mutations that had been described for secA were located early in the gene and resulted in a general protein export defect. Insertion mutations in the cloned gene X-secA operon that reduced or eliminated suppression by a prlD mutation also have been obtained. The properties of these suppressor and insertion mutations provide some insight into the role of SecA in the protein export process.