骨骼肌纤维烟酰胺腺嘌呤二核苷酸四唑氧化还原酶组化染色的检测

本文应用烟酰胺腺嘌呤二核苷酸四唑氧化还原酶（NADH-TR）组化染色，将大鼠与家兔小腿胫前肌与腓肠肌的肌纤维型分成染色最深的快缩氧化FO型、染色较深的慢缩氧化SO型、染色浅的快缩氧化酵解FOG型及染色最浅的快缩酵解FG型纤维。这与定量分析结果相吻合。并对低温液氮保存骨骼肌标本进行观察，发现小块肌组织＋生理盐水和小块肌组织＋5％二甲基亚砜（DMSO）冷冻保护剂的玻璃化冻存一起浸入液氮制成切片，都未见冰晶形成；而在用5％二甲基亚砜玻璃化冻存大块肌组织切片见有少量冰晶出现；未使用5％二甲基亚砜玻璃化冻存大块肌组织切片见有大量冰晶出现。提示小块标本超快速浸入液氮内能减少冰晶形成，保证酶组化染色切片的质量。并进一步对烟酰胺腺嘌呤二核苷酸四唑氧化还原酶组化作用机理与孵育液的科学配制进行了探讨。     

Effects of exercise and adrenaline on equine erythrocyte ATP content

To investigate the claim that equine erythrocytes released from the spleen are older cells than those found at rest in the circulation, the 2,3, diphosphoglycerate (2,3 DPG), adenosine triphosphate (ATP) and creatine concentration of erythrocytes before and following splenic emptying were examined. Normal values for thoroughbreds (43) and ponies (10) at rest were established. Following either exercise or intravenous injection of adrenaline in six thoroughbreds, there was an increase in erythrocyte creatine content and a decrease in ATP concentration. Exercise produced a slight increase in 2,3 DPG while no change occurred with adrenaline. In two splenectomised ponies adrenaline only produced a decrease in erythrocyte ATP, with no change in creatine or 2,3 DPG content. It was concluded that erythrocytes released from the spleen are not aged cells. However, the stimuli used produced a decrease in ATP content which may affect the properties of the erythrocytes.     

Inherited erythrocyte pyruvate kinase deficiency in a beagle dog

A 1-year-old, female Beagle dog with minimal exercise intolerance was found to have a persistent, severe, and highly regenerative anemia, splenomegaly, and progressive osteosclerosis. Despite near-normal in vitro erythrocyte pyruvate kinase (PK) activity, the authors diagnosed PK deficiency by demonstrating a glycolytic block at the PK step, the lack of normal R-type PK isoenzyme, and the presence of M(2)-type PK in the animal's erythrocytes. The dam had half-normal erythrocyte PK activity, which supports an autosomal recessive mode of inheritance. We conclude from our studies that close similarities exist between erythrocyte PK deficiency in Beagle, Basenji, and West Highland White Terrier dogs and that this form of PK deficiency may be more widespread than previously thought.     

Quantitation of adenine nucleotides in equine colonic mucosal tissue using high performance liquid chromatography.

The objectives were to use high performance liquid chromatography (HPLC) to validate an established method for adenine nucleotide separation in equine colonic mucosal tissue, to determine the inherent variability in the tissue and extraction method, and to determine the stability of ATP, ADP, and AMP in the tissue with time. Equine colonic mucosal tissue obtained from a single horse was immediately submersed in liquid nitrogen, and stored at -70 degrees C. Samples were lyophilized, extracted, and separated by HPLC. The limit of quantitation was 0.05 microg/mL. The coefficient of variation for the instrument was less than 10% for all nucleotides measured. When the tissue was not homogenized prior to sampling, there were significant differences in adenine nucleotide content between samples. However, when the tissue was homogenized prior to analysis, these differences were no longer significant. There was no significant decrease in ATP, ADP, or AMP content over a 54-day analysis period.     

Effects of enrofloxacin and magnesium deficiency on matrix metabolism in equine articular cartilage

OBJECTIVE: To investigate the effects of enrofloxacin and magnesium deficiency on explants of equine articular cartilage. SAMPLE POPULATION: Articular cartilage explants and cultured chondrocytes obtained from adult and neonatal horses. PROCEDURE: Full-thickness explants and cultured chondrocytes were incubated in complete or magnesium-deficient media containing enrofloxacin at concentrations of 0, 1, 5, 25, 100, and 500 microg/ml. Incorporation and release of sulfate 35S over 24 hours were used to assess glycosaminoglycan (GAG) synthesis and degradation. An assay that measured binding of dimethylmethylene blue dye was used to compare total GAG content between groups. Northern blots of RNA from cultured chondrocytes were probed with equine cDNA of aggrecan, type-II collagen, biglycan, decorin, link protein, matrix metalloproteinases 1, 3, and 13, and tissue inhibitor of metalloproteinase 1. RESULTS: A dose-dependent suppression of 35S incorporation was observed. In cartilage of neonates, 35S incorporation was substantially decreased at enrofloxacin concentrations of 25 mg/ml. In cartilage of adult horses, 35S incorporation was decreased only at enrofloxacin concentrations of > or =100 microg/ml. Magnesium deficiency caused suppression of 35S incorporation. Enrofloxacin or magnesium deficiency did not affect GAG degradation or endogenous GAG content. Specific effects of enrofloxacin on steady-state mRNA for the various genes were not observed. CONCLUSION AND CLINICAL RELEVANCE: Enrofloxacin may have a detrimental effect on cartilage metabolism in horses, especially in neonates.     

Levels of nicotinamide adenine dinucleotide in extracellular body fluids of pigs may be growth-limiting for Actinobacillus pleuropneumoniae and Haemophilus parasuis

During infection, nutrient deprivation can alter bacterial phenotype. This, in turn, may have implications for pathogenesis and prophylaxis. Actinobacillus pleuropneumoniae (biotype 1) and Haemophilus parasuis, respiratory tract pathogens of swine, are both V-factor-dependent. The concentrations of V factor in the extracellular fluids of pigs are unknown and may limit the growth of these bacteria in vivo. The aim of this study was to determine the concentrations of nicotinamide adenine dinucleotide (NAD) in select porcine body fluids and to compare the availability of NAD in vivo with the affinities of the organisms for this compound. Levels in plasma, tissue fluids (peritoneal, pleural, synovial, and cerebrospinal), and laryngeal, tracheal, and lung washings were determined with an enzymatic cycling assay. We concluded that, although the NAD supply in the respiratory tract is probably not growth-limiting, it may become limiting if the organisms are disseminated.     

Determination of erythrocyte pyruvate kinase deficiency in Basenjis with chronic hemolytic anemia

Erythrocyte pyruvate kinase (PK) deficiency was first described in Basenjis 20 years ago. Although the approach to diagnosis had not been well established, a screening program to detect heterozygotes was thought to have eliminated PK deficiency from the Basenjis of the United States. Four not closely related Basenjis with severe chronic hemolytic anemia, from various parts of the United States, were studied. Their PCV ranged from 16 to 25% and their reticulocyte count was always above 15%. A progressive osteosclerosis was seen in all of the Basenjis and hepatic failure developed in 2 of them. The erythrocyte intermediary metabolite patterns indicated a glycolytic defect at the PK step. Erythrocyte PK activities were markedly increased in the anemic Basenjis, compared with those of a control group, but the enzyme in these Basenjis had abnormal kinetic properties and was thermolabile. An antibody against R-type PK, the regular erythrocyte PK form, did not neutralize the PK activity of affected Basenjis, and results of electrophoretic studies suggested the expression of M2-type PK, a leukocyte and fetal erythroid PK-type. Clinically healthy heterozygous Basenjis had half-normal R-type PK activity and did not express the M2-type in their erythrocytes. We conclude that severe chronic hemolytic anemia, caused by erythrocyte PK deficiency, and associated osteosclerosis still develop in Basenjis. A definitive diagnosis cannot be reached by simply measuring erythrocyte PK activity; rather, diagnosis requires measurement of glycolytic substrate accumulation and enzyme stability and immunologic or electrophoretic studies of erythrocyte PK.     

Histochemical study of the distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive neurons in the chicken caecum

The distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive neurones was investigated in the chicken caecum. Double staining combined NADPHd histochemistry with immunohistochemistry for neural nitric oxide synthase (nNOS) indicated that NADPHd-positive neurones also showed immunoreactivity for nNOS. NADPHd-positive nerve cell bodies were observed in both the myenteric and the submucous plexuses. Nerve fibres showing enzyme activity were mainly distributed in the circular muscle layer, but only a few fibres in the mucosal layer. Fine nerve fibres showing NADPHd activity were found running between germinal centres in the caecal tonsil. Quantitative analysis showed no significant differences in the number of enzyme-positive nerve cell bodies per ganglion of the myenteric and the submucous plexuses among three different caecal regions; proximal, middle and distal regions. Larger numbers of ganglia were detected in the submucous plexus than the myenteric plexus at all three regions. These data indicate that nitrergic neurones in the submucous plexus mainly project to the circular muscle layer in the chicken caecum. It is possible that nitrergic nerves regulate the motility of the chicken caecum.     

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-α-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H₂O₂ into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly ₁₂−Gly₁₃−Gly ₁₄−Ile₁₅−Ile₁₆−Gly ₁₇. Recombinant GlpO lacking these six amino acids (GlpOΔFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpOΔFAD, similarly to anti-GlpO antibodies, neutralised H₂O₂ production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.     

Modulation of nicotinamide adenine dinucleotide phosphate oxidase activity through sequential posttranslational modifications of p22 phagocytic oxidase during capacitation and acrosome reaction in goat spermatozoa

Superoxide anion radical, produced in low quantities, plays a positive role in sperm function. Spermatozoa produce superoxide anion radical during posttesticular development, which shows an abrupt increase during capacitation. The NAD phosphate oxidase (NOX) family members NOX2 and NOX5 are the 2 enzymes implicated in superoxide production in spermatozoa. We examined the organization of NOX2 in goat spermatozoa during epididymal maturation, capacitation, and acrosome reaction. Spermatozoa from testis, caput epididymidis, corpus epididymidis, and cauda epididymidis possessed components of the phagocytic oxidase (PHOX; i.e., gp91phox, p22phox, p67phox, p47phox, p40phox), and ras-related C3 botulinum toxin substrate 1/2 (Rac1/2) on spermatozoa, and their concentrations did not show significant alterations during epididymal maturation. During capacitation in vitro, p22phox underwent Thr-phosphorylation, which resulted in a mobility shift of the corresponding band toward greater molecular mass. The Rac1/2 also showed a mobility shift from 32 to 23 kDa during capacitation. During progesterone-induced acrosome reaction, the spermatozoa experienced a total loss of p22phox and p47phox. The p47phox, but not p22phox, was detected in the exocytic vesicles of the acrosome. The Thr-phosphorylated form of p22phox was ubiquitinated and degraded through proteasome-mediated pathways in goat sperm cell lysates. Thus, Thr phosphorylation of p22phox acts as a regulatory switch in goat spermatozoa that transiently activates the NOX2 system during capacitation and subsequently directs it for degradation through the ubiiquitin-proteasomal pathway during progesterone-induced acrosome reaction.     

Sarcocystis myositis and vitamin E deficiency in a Gypsy Vanner stallion suspected of having equine motor neuron disease

A 3-year-old Gypsy Vanner stallion was presented for evaluation of intermittent recumbency, muscle fasciculations, weakness, low head carriage, shifting of weight between the hindlimbs and an elevated tail head. History, physical examination and serum alpha tocopherol concentrations were suggestive of vitamin E deficiency and equine motor neuron disease (EMND). Sacrocaudalis dorsalis medialis muscle biopsy identified myositis secondary to sarcocystosis. Treatment with alpha tocopherol, ponazuril and sulfadiazine/pyrimethamine resulted in significant improvement in muscle weakness and body condition with resolution of sarcocystosis and inflammation on repeat muscle biopsy. This case illustrates the importance of muscle biopsy in horses with neuromuscular disease as concurrent diseases may be present that require specific treatment for a positive outcome.     

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.     

Identification of a 6 base pair insertion in West Highland White Terriers with erythrocyte pyruvate kinase deficiency.

OBJECTIVE: To define the disease-causing mutation in West Highland White Terriers (WHWT) with erythrocyte pyruvate kinase (R-PK) deficiency and to design a genetic test capable of recognizing affected (homozygous) and carrier (heterozygous) dogs. ANIMALS: 3 anemic WHWT littermates and 1 unaffected littermate; 16 dogs from the same kennel, including 4 unrelated, phenotypically normal dogs (control dogs), and 12 for which PK activity was not known; 2 PK-deficient Basenjis; 2 PK-deficient Beagles; 4 unaffected English Springer Spaniels; and 1 mixed-breed dog. PROCEDURES: cDNA was cloned and sequenced, and cDNA sequences were compared with the published sequence for canine R-PK cDNA to identify the putative disease-causing mutation. Genomic DNA spanning the affected region was cloned and sequenced to verify the mutation. Subsequently, polymerase chain reaction primers were designed to amplify the section of the gene containing the mutation from DNA in blood or buccal swab samples. Gel electrophoresis allowed assignment of genotypes on the basis of allele separation. RESULTS: 4 single base polymorphisms attributable to sequencing errors in the published sequence were identified, along with a 6 base pair (bp) insertion in exon 10 that was recognized as a putative disease-causing mutation. An identical insertion was found in genomic DNA. Amplification of genomic DNA yielded a 117 bp product for genotypically normal dogs and a 123 bp product for WHWT homozygous for PK deficiency. Carriers had 1 copy of each allele and variable heteroduplex structures. CONCLUSIONS AND CLINICAL RELEVANCE: A 6 bp insertion in the C domain of R-PK was identified in WHWT with PK deficiency. Affected and carrier dogs could be distinguished with a genetic test.