cDNA sequence of the horse (Equus caballus) LAMA3 gene and characterization of two intronic SNP markers.

Laminins are large heterotrimeric basement membrane glycoproteins composed of alpha, beta and gamma chains. The Laminin 5 isoform has an alpha3beta3gamma2 composition and is essential for the adhesion of basal keratinocytes to the underlying epithelial basement membrane where it is mainly located. Mutations in the genes coding for the 3 chains have been associated with a severe skin blistering disease, Herlitz's junctional epidermolysis bullosa (JEB), observed in different species as man, dog, cat and horse. In this study, we report the sequence of the 5.2 kb horse laminin alpha 3 cDNA (LAMA3) as well as the detection of two intronic SNPs. These data will be useful to further identify causal mutations for the disease in this gene.     

Plasma lipid transport in the horse (Equus caballus)

• 1.1. Equine plasma contains lipoproteins corresponding to very low density (VLDL), low density (LDL) and high density lipoproteins (HDL).
• 2.2. HDL accounts for approximately 60% of plasma lipoprotein mass and consists of a single population of particles.
• 3.3. LDL is heterogeneous comprising three discrete subfractions.
• 4.4. Two proteins are found in the region of apolipoprotein (apo) B-100 in VLDL and LDL and a third similar to apo B-48 is in VLDL.
• 5.5. Lecithin:cholesterol acyl transferase is active in plasma and hepatic lipase and lipoprotein lipase are evident in post-heparin plasma.
• 6.6. There is no significant cholesteryl ester transfer protein activity.

     

Ciliates from the intestine of Yakut horse (Equus caballus)

Endobiotic ciliates of native Yakut horse are investigated. 57 ciliate species have been found. From 17 up to 43 species of ciliates have been revealed in each host specimen. The specimens of Alloiozona trizona Hsiung, 1930 and Triadinium caudatum Fiorentini, 1890 were present in 100 % of the horses. The endemic genus and species of allantosomides, Strelkowella urunbasiensis Kornilova, 2004, has been found in 11 horses. Allantoxena japonensis (Imai, 1979) and Cycloposthium ishikawai Gassovsky, 1919 being recorded previously in Japan only were found for the first time in Russia. The species Cycloposthium ponomarevi Kornilova, 2001 peculiar to the Turkmenistan koulan has been found in the Yakut horses.     

Characterization of horse (Equus caballus) immunoglobulin mu chain-encoding genes

 Horse (Equus caballus) immunoglobulin mu chain-encoding (IgM) variable, joining, and constant gene segments were cloned and characterized. Nucleotide sequence analyses of 15 cDNA clones from a mesenteric lymph node library identified 7 unique variable gene segments, 5 separate joining segments, and a single constant region. Based on comparison with human sequences, horse variable segments could be grouped into either family 1 of immunoglobulin (Ig) clan I or family 4 of Ig clan II subclan IV. All horse sequences had a relatively conserved 16 base pair (bp) segment in framework 3 which was recognized with high specificity in polymerase chain reaction by a degenerate oligonucleotide primer. Horse complementarity determining regions (CDR) had considerable variability in predicted amino acid content and length but also included the presence of relatively conserved residues and several canonical sequences that may be necessary in formation of the β chain main structure and conformation of antigen-binding sites through interaction with light chain CDR. Sequence analysis of joining regions revealed the presence of nearly invariant 3′ regions similar to those found in human and mouse genes. A single horse IgM constant region comprising 1472 bp and encoding 451 residues was also identified. Direct comparison of the horse constant region predicted amino acid sequence with those from eleven other species revealed the presence of 53 invariant residues with particularly conserved sequences within the third and fourth exons. Phylogenetic analysis using a neighbor-joining algorithm showed closest similarity of the horse mu chain-encoding constant region gene to human and dog sequences. Together, these findings provide insights into the comparative biology of IgM as well as data for additional detailed studies of the horse immune system and investigation of immune-related diseases. Received: 14 October 1996 / Revised: 10 December 1996     

Breathing strategy of the adult horse (Equus caballus) at rest

To investigate the mechanism underlying the polyphasic airflow pattern of the equine species, we recorded airflow, tidal volum, rib cage and abdominal motion, and the sequence of activation of the diaphragm, intercostal, and abdominal muscles during quiet breathing in nine adult horses standing at rest. In addition, esophageal, abdominal, and transdiaphragmatic pressures were simultaneously recorded using balloon-tipped catheters. Analysis of tidal flow-volume loops showed that, unlike humans, the horse at rest breathes around, rather than from, the relaxed volume of the respiratory system (Vrx). Analysis of the pattern of electromyographic activities and changes in generated pressures during the breathing cycle indicate that the first part of expiration is passive, as in humans, with deflation toward Vrx, but subsequent abdominal activity is responsible for a second phase of expiration: active deflation to below Vrx. From this end-expiratory volume, passive inflation occurs toward Vrx, followed by a second phase of inspiration: active inflation to above Vrx, brought about by inspiratory muscle contraction. Under these conditions the abdominal muscles appear to share the principal pumping duties with the diaphragm. Adoption of this breathing strategy by the horse may relate to its peculiar thoracoabdominal anatomic arrangement and to its very low passive chest wall compliance. We conclude that there is a passive and active phase to both inspiration and expiration due to the coordinated action of the respiratory pump muscles responsible for the resting adult horse's biphasic inspiratory and expiratory airflow pattern. This unique breathing pattern perhaps represents a strategy of minimizing the high elastic work of breathing in this species, at least at resting breathing frequencies.     

Purification of horse (Equus caballus) serum lecithin:cholesterol acyltransferase

1. A method for the purification of horse serum lecithin:cholesterol acyltransferase has been established. 2. The method involves the adsorption of the enzyme from diluted horse serum on DEAE-Sephadex A-50, (NH4)2SO4 fractionation, 1-butanol treatment, and chromatographic techniques of DEAE-Sepharose CL-6B, DEAE-Sephadex A-50, Affi-Gel blue and hydroxylapatite. 3. The resultant enzyme preparation essentially formed a single main band when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. 4. The final purification of the enzyme was 20,000-fold with 7% yield. 5. The apparent mol. wt of the enzyme was 64,000. 6. The activity of the enzyme was stable for 3 days at 0 degree C.     

Characterization of horse (Equus caballus) T-cell receptor beta chain genes

Genes encoding the horse (Equus caballus) T-cell receptor beta chain (TCRB) were cloned and characterized. Of 33 cDNA clones isolated from the mesenteric lymph node, 30 had functionally rearranged gene segments, and three contained germline sequences. Sixteen unique variable segments (TCRBV), 14 joining genes (TCRBJ), and two constant region genes (TCRBC) were identified. Horse TCRBV were grouped into nine families based on similarity to human sequences. TCRBV2 and TCRBV12 were the most commonly represented horse families. Analysis of predicted protein structure revealed the presence of conserved regions similar to those seen in TCRB of other species. A decanucleotide promoter sequence homologous to those found in humans and mice was located in the 5 untranslated region of one horse gene. Germline sequences included the 5 region of the TCRBD2 gene with flanking heptamer/nonamer recombination signals and portions of the TCRBJ-C2 intron. Southern blot hybridizations demonstrated restriction fragment length polymorphisms at the TCRBC locus among different horse breeds.     

Mass spectral measurements of the apoHDL in horse (Equus caballus) cerebrospinal fluid

As a continuation of our proteogenomic studies of equine apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with the HDL in horse cerebrospinal fluid (CSF). Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I and A-II, as well as acylated apoA-I. In comparison with our previously published data on equine plasma apolipoproteins, there appears to be a higher percentage of acylated apoA-I in the CSF than in plasma. As was the case in plasma, apoA-II circulates as a homodimer. These studies also revealed a protein with a mass of 34,468 Da that we are speculating is the value for horse apoE.     

Nucleotide sequence of a cDNA clone for human aldolase B

Two specific clones for human aldolase B were isolated from a human liver cDNA library using a rat aldolase B cDNA probe. The clones were identified by positive hybridization-selection and one of them was sequenced. The 127 C-terminal residues of the human protein were deduced from this nucleotide sequence analysis. They showed 92% homology with the corresponding previously published amino-acid sequence of rat liver aldolase B.     

Nucleotide sequence of a cDNA clone encoding mouse protamine 1

The nucleotide sequence of a 404-base cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine has been determined. This insert, isolated from a mouse testis cDNA library, encodes a polypeptide of 50 amino acids of which 28 are arginine, 9 are cysteine, and 3 are tyrosine. The insert contains the complete 3' noncoding region of 151 bases and most of the 5' noncoding region. The predicted amino acid sequence of mouse protamine 1 is about 80% homologous to boar protamine and 67% homologous to bull protamine and contains the central, highly basic domain of four arginine clusters found in the trout protamines. The identification of a cDNA clone for a mouse protamine will facilitate studies of the evolution, regulation, and protein-DNA interaction of this nuclear protein unique to haploid spermatogenic cells.