Objective—To clone segments of the canine liver
alkaline phosphatase (LALP) and corticosteroidinduced
alkaline phosphatase (CIALP) genes and use
those clones to determine the tissue source of CIALP,
the kinetics of LALP and CIALP mRNA expression for
glucocorticoid-treated dogs, and the correlation
between LALP and CIALP transcript concentrations
and isoenzyme activities.
Sample Population—Tissues obtained from 7 dogs
treated with prednisone (1 mg/kg, SC, q 24 h) for up
to 32 days and 1 untreated (control) dog.
Procedure—Gene segments of LALP and CIALP
were obtained by reverse transcription-polymerase
chain reaction (RT-PCR) assay. The tissue source of
CIALP and IALP mRNA was determined by northern
blot analysis of tissues from 1 of the glucocorticoidtreated
dogs. Hepatic tissues and serum samples
were obtained from the 6 remaining glucocorticoidtreated
dogs on days 0, 2, 5, 10, and 32 of prednisone
treatment, and relative expression of LALP and CIALP
mRNA was correlated with LALP and CIALP activity.
Results—A 2,246-base pair (bp) segment of canine
LALP and a 1,338-bp segment of CIALP were cloned.
Northern blot analysis revealed CIALP mRNA expression
in hepatic tissues only after glucocorticoid treatment.
Kinetics of LALP and CIALP mRNA expression
in the liver of glucocorticoid-treated dogs paralleled
liver and serum activities of LALP and CIALP.
Conclusions and Clinical Relevance—The liver is
the most likely source for CIALP in dogs. Analysis of
kinetics of serum and hepatic LALP and CIALP mRNA
suggests that after glucocorticoid treatment, both are
regulated by modification of mRNA transcript concentrations,
possibly through differing mechanisms.
(Am J Vet Res 2002;63:1089–1095)
Objective—To determine the effect of glucocorticoids
on the induction of alkaline phosphatase (ALP)
isoenzymes in the liver, kidneys, and intestinal
mucosa, 3 tissues that are principally responsible for
ALP synthesis in dogs.
Sample Population—Tissues from the liver, kidneys,
and intestinal mucosa of 6 dogs treated with 1 mg of
prednisone/kg/d for 32 days and 6 untreated control
Procedure—Using canine-specific primers for the
ALP isoenzymes, a reverse transcription-polymerase
chain reaction assay was designed to measure liver
ALP (LALP) and intestinal ALP (IALP) mRNA and heterogeneous
nuclear RNA (hnRNA) expression in tissues
from the liver and kidneys and intestinal mucosa
of glucocorticoid-treated and control dogs. Tissue ALP
isoenzyme activities were compared between the
Results—The LALP activity and mRNA concentrations
increased in tissues of the liver and kidneys in
dogs treated with prednisone, whereas LALP hnRNA
increased only in liver tissues. The IALP activity and
mRNA expression increased in intestinal mucosa and
liver tissues in prednisone-treated dogs. We did not
detect an increase in IALP hnRNA expression in these
Conclusions and Clinical Relevance—Synthesis of
ALP is increased in the liver, kidneys, and intestinal
mucosa of dogs in response to prednisone treatment.
This response appears to be regulated at the transcriptional
level, but mechanisms may differ between
LALP and IALP. (Am J Vet Res 2002;63:1083–1088)
Objective—To map canine mitochondrial proteins and identify qualitative and quantitative differences in heart mitochondrial protein expression between healthy dogs and dogs with naturally occurring and induced dilated cardiomyopathy (DCM).
Sample Population—Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with induced DCM.
Procedures—Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by ≥ 2-fold between groups were analyzed by matrixassisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry.
Results—Within narrow pH gradients of control canine heart mitochondrial samples, a total of 1,528 protein spots were revealed. Forty subunits of heart mitochondrial proteins that differ significantly from control tissues were altered in tissue specimens from dogs with naturally occurring and induced forms of DCM. The most affected heart mitochondrial proteins in both groups were those of oxidative phosphorylation (55%). Upregulation of manganese superoxide dismutase was suggestive of heart oxidative injury in tissue specimens from dogs with both forms of DCM. Evidence of apoptosis was associated with overexpression of the heart mitochondrial voltage-dependent anion channel-2 protein and endonuclease G in tissue specimens from dogs with induced DCM.
Conclusions and Clinical Relevance—Alterations of heart mitochondrial proteins related to oxidative phosphorylation dysfunction were more prevalent in tissue specimens from dogs with induced or naturally occurring DCM, compared with those of control dogs.
Objective—To determine reference ranges for results
of hematologic analyses of healthy Belgian Tervuren,
to compare results of hematologic analyses for
healthy Belgian Tervuren with results for healthy dogs
of other breeds, and to determine prevalence of physiologic
leukopenia in Belgian Tervuren.
Animals—180 healthy Belgian Tervuren and 63
healthy dogs of other breeds.
Procedure—Blood samples were analyzed by use of
an automated device. Reference ranges were calculated
for Belgian Tervuren by use of standard methods.
Results—Total WBC counts of Belgian Tervuren ranged
from 2.61 to 16.90 X 103/µl. Mean WBC count of
Belgian Tervuren (mean ± SEM, 7.04 ± 0.16 X 103/µl)
was significantly lower than mean count for control
dogs. Significantly more Belgian Tervuren (65/180;
36%) than control dogs (2/63; 3%) had WBC counts
< 6.00 X 103/µl. Percentage of Belgian Tervuren with
WBC count < 6.00 X 103/µl was low for dogs ≤ 2 years
old, increased sharply for dogs between 2 and 4 years
old, and was approximately 65% for dogs > 4 years
old. Neutrophil, lymphocyte, and monocyte counts
were significantly lower, and RBC count, hematocrit,
and eosinophil fraction were significantly higher in
Belgian Tervuren than in control dogs.
Conclusions and Clinical Relevance—Results suggest
that physiologic leukopenia, resulting from low
numbers of neutrophils, lymphocytes, and monocytes,
may be a typical finding in a large percentage
of healthy Belgian Tervuren and is not of clinical
importance in otherwise healthy dogs. Healthy
Belgian Tervuren may also have RBC counts and
hematocrits higher than expected for healthy dogs.( J
Am Vet Med Assoc 2000;216:866–871)
Objective—To identify qualitative and quantitative differences in cardiac mitochondrial protein expression in complexes I to V between healthy dogs and dogs with natural or induced dilated cardiomyopathy (DCM).
Sample Population—Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with DCM induced by rapid right ventricular pacing.
Procedures—Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by 2-fold or greater between groups were analyzed by matrixassisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry.
Results—A total of 22 altered mitochondrial proteins were identified in complexes I to V. Ten and 12 were found in complex I and complexes II to V, respectively. Five were mitochondrial encoded, and 17 were nuclear encoded. Most altered mitochondrial proteins in tissue specimens from dogs with naturally occurring DCM were associated with complexes I and V, whereas in tissue specimens from dogs subjected to rapid ventricular pacing, complexes I and IV were more affected. In the experimentally induced form of DCM, only nuclear-encoded subunits were changed in complex I. In both disease groups, the 22-kd subunit was downregulated.
Conclusions and Clinical Relevance—Natural and induced forms of DCM resulted in altered mitochondrial protein expression in complexes I to V. However, subcellular differences between the experimental and naturally occurring forms of DCM may exist.
Objective—To determine the nucleotide and amino
acid sequence of atrial natriuretic peptide (ANP) in
cats and its typical regions of cardiac expression.
Animals—5 healthy adult mixed-breed cats.
Procedure—Total RNA was extracted from samples
obtained from the left and right atrium, left and right
ventricle, and interventricular septum of each cat. The
RNA was used to produce cDNA for sequencing and
northern blot analysis. Genomic DNA was extracted
from feline blood samples. Polymerase chain reaction
primers designed from consensus sequences of
other species were used to clone and sequence the
feline ANP gene.
Results—The feline ANP gene consists of 1,072
nucleotides. It consists of 3 exons (123, 327, and 12
nucleotides) separated by 2 introns (101 and 509
nucleotides). It has several typical features of eukaryotic
genes and a putative steroid-response element
located within the second intron. Preprohormone
ANP consists of 153 amino acids. The amino acid
sequence of the active form of feline ANP (ANP-30) is
identical to that of equine, bovine, and ovine ANP-30
and differs from that of human, canine, and porcine
ANP-28 only by 2 carboxy-terminal arginine residues.
The ANP mRNA was detected only in the left and
Conclusions and Clinical Relevance—The genetic
and protein structure and principal regions of cardiac
expression of feline ANP are similar to those of other
species. Results of this study should be helpful in
future studies on the natriuretic response in cats to
diseases that affect cardiovascular function.
(Am J Vet Res 2002;63:236–240)