Therapeutic efficacy of topical hydrocortisone aceponate in experimental flea-allergy dermatitis in dogs.

OBJECTIVE: To evaluate the treatment efficacy of a topical spray containing hydrocortisone aceponate (HCA) on dogs with flea-allergy dermatitis (FAD). DESIGN: A controlled clinical study was conducted on dogs with experimentally induced FAD. Sixteen laboratory beagles with mild to moderate clinical signs were divided into two groups. The test group received HCA by topical spray once daily for 7 days, while the control group did not. Pruritic events (time and frequency) were videotaped and then scored. Clinical signs (erythema, papules, excoriation and alopecia) present on four anatomical regions were monitored and their severity directly assessed. RESULTS: After 2 days, pruritus was reduced by 94% in the treatment group and by 24% in the control group (P = 0.014) in cumulative time, and by 86% versus 34% (P = 0.034) in frequency. The HCA spray also resulted in significant improvements in overall clinical signs: 23% versus 0% in the control group (P = 0.0006) on day 3 and 43% versus 15% in the control group (P = 0.0006) on day 7. During the 7-day trial, no drug-related adverse effects were observed. CONCLUSIONS: Topical treatment with HCA showed a rapid and potent antipruritic effect on dogs with FAD. HCA also demonstrated significant overall therapeutic effects on FAD-associated skin lesions.

Efficacies of osaterone and delmadinone in the treatment of benign prostatic hyperplasia in dogs.

A multicentre randomised clinical trial was performed to compare the therapeutic potential of osaterone acetate with that of delmadinone acetate in the treatment of benign prostatic hyperplasia in dogs. The osaterone was administered orally at 0.25 mg/kg bodyweight once a day for seven days to 73 dogs. The delmadinone was administered by a single intramuscular or subcutaneous injection at 3 mg/kg bodyweight to 69 dogs. During the 180-day trial, the dogs were monitored five times for their clinical signs and prostate volume. The two drugs were similarly effective in reducing the clinical signs and inducing complete clinical remission, and both induced a similar level of minor, mostly transitory adverse effects. Osaterone reduced the volume of the prostate glands of the dogs significantly more quickly than delmadinone.

Treatment of canine parvoviral enteritis with interferon-omega in a placebo-controlled field trial.

The clinical efficacy of a recombinant feline interferon (IFN) (type omega) was evaluated under field conditions for the treatment of dogs with parvoviral enteritis. In this multicentric, double-blind, placebo-controlled trial, 94 dogs from one to 28 months old were randomly assigned to two groups which were treated intravenously either with IFN (2.5 million units/kg) or placebo once a day for three consecutive days, and monitored for clinical signs and mortality for 10 days. Each dog received individual supportive treatment The data from 92 interpretable cases (43 IFN-treated and 49 placebo) showed that the clinical signs of the IFN-treated animals improved significantly in comparison with the control animals, and that there were only three deaths in the IFN group compared with 14 deaths in the placebo group (P = 0.0096) corresponding to a 4.4-fold reduction. Alternative analyses of the data taking into account the prior vaccination status of the dogs against canine parvovirus suggested that the IFN therapy resulted in a 6.4-fold reduction in mortality (P = 0.044) in the unvaccinated cohort, a significant reduction when compared with the vaccinated cohort.

Two amino acids, Phe 16 and Ala 776, on the polyprotein are most likely to be responsible for the diabetogenicity of encephalomyocarditis virus.

The diabetogenic D variant of encephalomyocarditis virus (EMC-D) was previously shown to differ from the non-diabetogenic B variant (EMC-B) by 14 nucleotides out of 7829 bases. Similar approaches with a new nondiabetogenic variant, EMC-DV1, obtained by plaque purification of the EMC-D variant stock pool, enabled us to narrow down further the possible genomic area responsible for the diabetogenicity of EMC virus. EMC-DV1 does not induce interferon in vitro, differing from the highly interferon-inducing EMC-B. The complete nucleotide sequence of EMC-DV1 was determined by RNA-dependent DNA sequencing and cDNA sequencing. The genomic size and organization of EMC-DV1 are similar to those of EMC-D and EMC-B, with a long open reading frame encoding a polyprotein of 2292 amino acids. Comparative analyses of sequence information as well as biological activities of EMC-DV1 with EMC-D and EMC-B suggest that (i) the diabetogenicity is apparently distinct from the ability to induce interferon, which is probably due to the single U base insertion at position 765 in EMC-B, and (ii) the diabetogenicity of EMC virus is most probably controlled by one or both of two amino acids, Phe 16 (on the leader peptide) and Ala 776 (152nd amino acid on the VP1) on the polyprotein.

A simple and versatile method for the preparation of vector-primers by adapter-end-primer ligation.

A group of efficient cDNA cloning strategies employs vector-primers where cDNA synthesis starts from the oligo(dT)-primer tail, which is conventionally attached to cloning vectors by use of terminal deoxynucleotidyl transferase. An alternative, efficient and more versatile method of vector-primer preparation is to directly ligate, by use of T4 DNA ligase, a double-digested vector, e.g., pTZ18R/Pst I/Bam HI, to a synthetic (Bam HI)-adapter-end-primer, 5'-pGATCC-Tn or 5'-pGATCC-site-specific sequence. The use of a utility-vector containing a sizable spacer between the two selected restriction sites enables unambiguous separation on agarose gels of the double-digested vector precursors from single-digested ones, further simplifying the vector preparation. The adapter-end-primer ligation method can be applied to any suitable vectors with multiple cloning sites for the preparation of not only oligo(dT)-tailed, but also site-specific sequence-tailed vectors. Thus, the method enables the cDNA cloning of total poly (A+)-mRNAs, as well as specific RNA or mRNA species with or without poly(A)-tail.

Genomic differences between the diabetogenic and nondiabetogenic variants of encephalomyocarditis virus.

Plaque purification of the M variant of encephalomyocarditis (EMC-M) virus resulted in the isolation of two stable variants. One is a highly diabetogenic D variant (EMC-D) and the other is a nondiabetogenic B variant (EMC-B). The cDNA of EMC-D and EMC-B genomes were cloned and seven overlapping cDNA clones were selected to cover the entire genome except the 5'-end 310 bases which were determined by RNA-dependent DNA sequencing and enzymatic RNA sequencing. Each clone was restriction-mapped, subcloned, and sequenced. The genomes of EMC-D and EMC-B are composed of 7829 and 7825 bases, respectively. Both genomes contain a long open reading frame of 6876 nucleotides starting at position 830 on the consensus sequence, which encodes a polyprotein of 2292 amino acids. The sequences of EMC-D and EMC-B differ by two deletions, one insertion, and eight point mutations. The first deletion of 3 nucleotides is located in the 5' poly(C) tract where EMC-B has 127 nucleotides compared with 130 nucleotides in EMC-D. The second deletion in EMC-B involves 2 nucleotides at the 3'-end polyadenylation site. A single base insertion of U occurs at the 5' noncoding region of EMC-B. The eight point mutations are located in the polyprotein coding region. Two are silent and are each located in the structural gene 1B and in the nonstructural gene 2B. The remaining six mutations, one on the L gene and the other five on the 1D gene, introduce respective amino acid changes. It is concluded that the diabetogenic EMC-D viral genome (7829 bases) differs from the nondiabetogenic EMC-B viral genome (7825 bases) by 14 nucleotides out of 7829.

An efficient and site-specific gene trimming method.

Trimming a DNA strand into a precisely determined fragment can be carried out efficiently by an improved method involving a site-specific trim-primer and a single-stranded DNA template which is generated from a multifunctional vector, pTZ18R, and linearized by using an Eco RI-pTZ18R splinter. A complementary DNA strand is synthesized by DNA polymerase I (Klenow), and the 3'-end of the template upstream from the annealed primer is trimmed by subsequent T4 DNA polymerase reaction. An ATG translation initiator codon or a termination codon can be incorporated into the trim-primer, providing versatility to this single-stranded DNA-initiated gene trimming method that can be applied to subcloning and expression of any DNA fragment with known terminal sequences.

Molecular identification of diabetogenic viral gene.

The best evidence that viruses have a causative role in the pathogenesis of insulin-dependent diabetes mellitus comes from experiments in mice infected with encephalomyocarditis (EMC) virus. When SJL/J male mice were inoculated with a highly diabetogenic EMC-D virus, diabetes developed in 95% of the animals. In contrast, none of the mice inoculated with a nondiabetogenic EMC-B virus became diabetic. Tissue culture experiments showed that EMC-B induces considerable amounts of interferon, whereas EMC-D does not. Despite these differences, EMC-D and EMC-B could not be distinguished antigenically by a sensitive plaque-neutralization assay. Furthermore, the buoyant density in CsCl density gradients and the capsid proteins of these two variants on polyacrylamide gels could not be distinguished. Molecular-hybridization studies with radiolabeled DNA complementary to EMC-D and EMC-B RNAs failed to distinguish them. Determination of complete nucleotide sequences of EMC-D and EMC-B revealed that EMC-D (7829 bases) differs from EMC-B (7825 bases) by only 14 nucleotides. The differences consist of two deletions of five nucleotides, one base insertion, and eight point mutations. The first deletion of three nucleotides and the second deletion of two nucleotides are located in the 5'-poly(C) tract and the 3'-end polyadenylation site, respectively. One base insertion in EMC-B occurs in the 5'-noncoding region. The eight point mutations are located in the polyprotein-coding region. Two of them are silent, whereas the other six mutations, one located on the L gene and five on the VP1 gene, introduce amino acid changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine modification by phenylglyoxal and (p-hydroxyphenyl)glyoxal: reaction rates and intermediates.

Reaction rates and pathways of two commonly used arginine-modifying reagents, phenylglyoxal (PGO) and (p-hydroxyphenyl)glyoxal (HPGO), were investigated by spectrophotometry. The initial rate at pH 9.0 of PGO with arginyl compounds was found to be 15 to 20 times greater than that of HPGO in the absence of borate but only 1.6 times greater in the presence of borate. Time-resolved spectra of HPGO reactions with arginines revealed, in contrast to the relatively simple spectra of PGO, at least two spectrophotometrically identifiable intermediates, one non-absorbing at 336 nm and the other absorbing at 458 nm. The 458 nm absorbing intermediate species was no longer detectable in the presence of borate.

Association of cytomegalovirus infection with autoimmune type 1 diabetes.

The lymphocytes from 59 newly diagnosed type 1 diabetic patients and 38 normal control subjects were examined for the presence of human cytomegalovirus (CMV) genome by molecular hybridizations with human CMV specific probe. The CMV specific viral genome was found in 13 (22%) of 59 diabetic patients, but in 1 (2.6%) of 38 control subjects. Of the patients, 39% had islet cell antibody (ICA) and 41% had cytotoxic beta cell surface antibody (CBSA) in their serum; of the controls the corresponding rates were 2.6% and 2.6%. 62% and 69% of CMV genome-positive patients had ICA and CBSA, respectively, compared with 33% and 33% of CMV genome-negative patients. The single CMV genome-positive control subject did not have either ICA or CBSA whereas only 1 of the 37 CMV genome-negative control subjects had ICA. The strong correlation between CMV genome and islet cell autoantibodies detected in diabetic patients suggests that persistent CMV infections may be relevant to pathogenesis in some cases of type 1 diabetes.

Identification of antigenic differences between the diabetogenic and non-diabetogenic variants of encephalomyocarditis virus using monoclonal antibodies.

The M variant of encephalomyocarditis (EMC) virus consists of two biologically distinct variants: one, diabetogenic D variant (EMC-D) and the other, non-diabetogenic B variant (EMC-B). These two variants cannot be distinguished by hyperimmune sera. Monoclonal antibodies were generated against EMC-D or EMC-B to identify antigenic differences between these two variants. Fourteen independent hybrid cell lines, selected from seven separate fusions of mouse myeloma cells to spleen cells isolated from mice immunized with EMC-D, consisted of 12 hybrids which produced monoclonal antibodies that neutralized both EMC-D and EMC-B, and two hybrids (ED-HJ-23 and ED-HJ-31) which produced monoclonal antibodies that neutralized EMC-D but not EMC-B. Similarly, 16 independent hybrid cell lines, selected from eight separate fusions using spleen cells prepared from mice immunized with EMC-B, consisted of 15 hybrids which produced monoclonal antibodies neutralizing both EMC-D and EMC-B, and one hybrid (EB-48A-F1) which produced antibody that neutralized EMC-B, but not EMC-D. The specificities of these monoclonal antibodies (ED-HJ-23, ED-HJ-31, EB-48A-F1) were further confirmed using an immunofluorescent technique. The D variant-specific monoclonal antibodies reacted with cells infected with EMC-D but not EMC-B. In contrast, the B variant-specific monoclonal antibody reacted with the cells infected with EMC-B but not EMC-D. It is concluded that the EMC-D- and EMC-B-specific monoclonal antibodies are able to identify antigenic differences between diabetogenic and non-diabetogenic variants of EMC virus which cannot be distinguished by hyperimmune sera.

Amino acid differences in capsid protein, VP1, between diabetogenic and nondiabetogenic variants of encephalomyocarditis virus.

The genes for the major capsid protein, VP1(1D), of both diabetogenic D variant (EMC-D) and nondiabetogenic B variant (EMC-B) of encephalomyocarditis virus were cloned by using two synthetic primers which are common to both EMC-D and EMC-B. The cloned genes were mapped for major restriction enzyme sites including AccI, BamHI, EcoRI, HincII, KpnI, PvuII, SstI, TaqI, and XbaI. Among those nine restriction enzyme sites, only the TaqI site distinguished EMC-D genome from the counterpart of EMC-B genome. The complete nucleotide sequences (831 bases) of the VP1 genes revealed five amino acid differences between the two variants. Three of the changes, at positions 41, 58, and 152, were Thr (EMC-B) to Ala (EMC-D). The additional two changes occurred at positions 63 [Gln (EMC-B) to Glu (EMC-D)] and 181 [Thr (EMC-B) to Ser (EMC-D)]. All of these amino acid changes were due to point mutations at the first base of each codon.

An apparent deletion of an oligonucleotide detected by RNA fingerprint in the nondiabetogenic B variant of encephalomyocarditis virus is caused by a point mutation.

The diabetogenic D variant of encephalomyocarditis virus (EMC-D) was previously shown to be different from the nondiabetogenic B variant of encephalomyocarditis virus (EMC-B) by a single spot in an oligonucleotide fingerprint after RNase T1 digestion of their genomic RNAs. An oligoribonucleotide was missing from EMC-B but was present in EMC-D. The oligoribonucleotide specific to EMC-D was isolated from a two-dimensional polyacrylamide gel and sequenced as 5'-ACAAUCUCACUUUUCCAACAACAG-3'. Molecular hybridizations of EMC-D and EMC-B genomic RNAs with a DNA primer complementary to the EMC-D-specific oligoribonucleotide revealed that the absence of a corresponding spot in EMC-B was due to a point mutation rather than a deletion. By sequencing a cloned cDNA of EMC-B corresponding to the EMC-D-specific oligoribonucleotide, the point mutation was identified as a G for EMC-B and an A for EMC-D transversion at base 9 of the oligonucleotide. Comparative sequence analysis of eight randomly picked RNA segments around the EMC-D-specific oligoribonucleotide revealed that there were no base changes between EMC-D and EMC-B. It is concluded that the diabetogenic EMC-D viral genome differs from the nondiabetogenic EMC-B viral genome by at least a point mutation.

Possible mechanisms in the pathogenesis of virus-induced diabetes mellitus.

Insulin-dependent diabetes mellitus results from destruction of pancreatic beta cells. Viruses and autoimmunity have been implicated as possible causes of beta cell destruction in genetically predisposed individuals. The evidence for viruses comes largely from experiments in animals, but several studies in humans point to viruses as triggers in the pathogenesis of diabetes in some cases. In animal models, at least 4 different possible mechanisms for virus-induced diabetes have been proposed. The first mechanism is direct cytolytic infection of pancreatic beta cells. One group of viruses, including encephalomyocarditis virus, Mengovirus 2T, and Coxsackie B viruses, can directly infect and destroy pancreatic beta cells independent of autoimmune processes. The second mechanism is triggering of autoimmune responses. In contrast to the encephalomyocarditis virus-induced diabetes, reovirus type 1 and rubella virus seem to be somehow associated with autoimmunity in the genesis of a diabetes-like syndrome in a certain strain of suckling mice and hamsters, respectively. The third mechanism is cumulative environmental insults. The cumulative environmental insults with viruses and beta cell toxic chemicals can result in diabetes in genetically predisposed non-human primates and certain inbred strains of mice. The fourth mechanism is persistent infection. A certain virus, such as lymphocytic choriomeningitis virus, persistently infects murine pancreatic beta cells and produces hyperglycemia. The evidence that viruses cause diabetes in humans is more circumstantial.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of cyclosporin A in macromolecular synthesis of beta-cells.

Wistar rats developed hypoinsulinemia and hyperglycemia within 7 days when treated daily with 40 mg/kg body wt of cyclosporin A (CsA) and recovered from the metabolic alteration within 1 wk when CsA treatment was terminated. By light microscopy, there was no lymphocytic infiltration, but cytoplasmic vacuolization in the islets of Langerhans from the CsA-treated rats was seen. By electron microscopy, severe degranulation, cytoplasmic vacuolization, and dilation of endoplasmic reticulum were clearly seen in the pancreatic beta-cells. Islet cells isolated from the CsA-treated rats showed greater than 50% reduction in mRNA synthesis. A similar inhibitory pattern of mRNA synthesis was observed in in vitro CsA-treated (10 micrograms/ml) human pancreatic islet cells from one biopsy sample and in similarly treated rat insulinoma cells (RINm5F). The inhibitory effect of CsA on mRNA synthesis in RINm5F cells was dose dependent, with a 50%-inhibiting dose of 5 micrograms/ml. In addition to the inhibition of mRNA synthesis, CsA also inhibited protein and DNA syntheses, although the inhibitory effect on these macromolecular syntheses was significantly less than that on mRNA synthesis. However, there was only a minor effect of CsA on in vitro transcription and translation compared with that on RINm5F and islet cells. It is concluded that CsA-induced degranulation of the beta-cells in Wistar rats, accompanied by hypoinsulinemia and hyperglycemia, may be due to indirect, reversible interference of the cellular function primarily involved in mRNA synthesis.

Reaction of phenylglyoxal and (p-hydroxyphenyl) glyoxal with arginines and cysteines in the alpha subunit of tryptophan synthase.

The alpha subunit of tryptophan synthase from Escherichia coli is inactivated by phenylglyoxal and by (p-hydroxyphenyl)glyoxal. The use of these chemical modification reagents to determine the role of arginyl residues in the alpha subunit of tryptophan synthase has been complicated by our finding that these reagents react with sulfhydryl groups of the alpha subunit, as well as with arginyl residues. Analyses of the data for incorporation of phenyl[2-14C]glyoxal, for inactivation, and for sulfhydryl modification in the presence and absence of indole-3-glycerol phosphate indicate that two sulfhydryl groups and one arginine are essential for the activity. Our finding that the substrate protects the single essential arginyl residue but not the two sulfhydryl groups is consistent with the observed kinetics of partial protection by substrate or by a substrate analogue, indole-3-propanol phosphate. In contrast to phenylglyoxal, (p-hydroxyphenyl)glyoxal modifies two to three sulhydryl groups that are not protected by indole-3-glycerol phosphate and modifies none of the arginyl residues that are modified by phenylglyoxal.

DD-Carboxypeptidase activity of membrane fragments of Mycobacterium smegmatis. Enzymatic properties and sensitivity to beta-lactam antibiotics.

A DD-carboxypeptidase activity is present in membrane fragments of Mycobacterium smegmatis. Kinetic parameters of the enzymatic activity have been studied using UDP-N-glycolylmuramyl-L-alanyl-gamma-D-glutamyl-meso-2,2'-diaminopimelyl-D-[14C]alanyl-D-[14C]alanine as substrate. The DD-carboxypeptidase can be solubilized by Triton X-100 and Genapol X-100. It is inhibited by beta-lactam antibiotics although intact cells of M. smegmatis are insensitive to that kind of antibiotics. Inhibition by penicillin G is slowly reversible. By storage, at -20degrees C, kinetic parameters and sensitivity to penicillin G vary non-concomittantly, suggesting a penicillin binding site different from the substrate binding site.