Changes in the skeletal dimensions of the digits of the thoracic limb of Holstein cows after maturation.

The 12 left digits of the thoracic limb of Holstein female cows were obtained from a slaughterhouse, and 13 linear measurements of skeletal specimens of the digits of the thoracic limb were used in this study. Bivariate plots, regressions and correlation analyses were performed to assess the relationships between osteometric data and age, and between osteometric data and body size. The geometric mean (GM) of the metacarpal size was used as a proxy of body weight. The results of the regression analysis demonstrated that the length and the width of the distal phalanx were significantly correlated with age, whereas those of other bones were not. In addition, the correlations of the distal phalanx tended to be higher with age than with the GM of the metacarpal size, and their skeletal measurements tended to be longer in older animals than in younger animals. These findings suggest that the distal phalanx is slightly elongated and enlarged in its solear aspect with increasing age in skeletally mature cows. Moreover, although the side difference in the distal phalanx between the lateral and medial digits did not increase significantly with age, the solear aspect of the distal phalanx tended to be rougher, and deformation of the flexor tubercle tended to be more pronounced in the medial digit compared with the lateral digit. Therefore, in the distal phalanx of the front feet, the skeletal deformation with age and asymmetrical bone development might be related to the pathogenesis of claw horn lesions, as in the hind feet.

Multivariate analysis of variations in intrinsic foot musculature among hominoids.

Comparative analysis of the foot muscle architecture among extant great apes is important for understanding the evolution of the human foot and, hence, human habitual bipedal walking. However, to our knowledge, there is no previous report of a quantitative comparison of hominoid intrinsic foot muscle dimensions. In the present study, we quantitatively compared muscle dimensions of the hominoid foot by means of multivariate analysis. The foot muscle mass and physiological cross-sectional area (PCSA) of five chimpanzees, one bonobo, two gorillas, and six orangutans were obtained by our own dissections, and those of humans were taken from published accounts. The muscle mass and PCSA were respectively divided by the total mass and total PCSA of the intrinsic muscles of the entire foot for normalization. Variations in muscle architecture among human and extant great apes were quantified based on principal component analysis. Our results demonstrated that the muscle architecture of the orangutan was the most distinctive, having a larger first dorsal interosseous muscle and smaller abductor hallucis brevis muscle. On the other hand, the gorilla was found to be unique in having a larger abductor digiti minimi muscle. Humans were distinguished from extant great apes by a larger quadratus plantae muscle. The chimpanzee and the bonobo appeared to have very similar muscle architecture, with an intermediate position between the human and the orangutan. These differences (or similarities) in architecture of the intrinsic foot muscles among humans and great apes correspond well to the differences in phylogeny, positional behavior, and locomotion.

Double Virus Vector Infection to the Prefrontal Network of the Macaque Brain.

To precisely understand how higher cognitive functions are implemented in the prefrontal network of the brain, optogenetic and pharmacogenetic methods to manipulate the signal transmission of a specific neural pathway are required. The application of these methods, however, has been mostly restricted to animals other than the primate, which is the best animal model to investigate higher cognitive functions. In this study, we used a double viral vector infection method in the prefrontal network of the macaque brain. This enabled us to express specific constructs into specific neurons that constitute a target pathway without use of germline genetic manipulation. The double-infection technique utilizes two different virus vectors in two monosynaptically connected areas. One is a vector which can locally infect cell bodies of projection neurons (local vector) and the other can retrogradely infect from axon terminals of the same projection neurons (retrograde vector). The retrograde vector incorporates the sequence which encodes Cre recombinase and the local vector incorporates the "Cre-On" FLEX double-floxed sequence in which a reporter protein (mCherry) was encoded. mCherry thus came to be expressed only in doubly infected projection neurons with these vectors. We applied this method to two macaque monkeys and targeted two different pathways in the prefrontal network: The pathway from the lateral prefrontal cortex to the caudate nucleus and the pathway from the lateral prefrontal cortex to the frontal eye field. As a result, mCherry-positive cells were observed in the lateral prefrontal cortex in all of the four injected hemispheres, indicating that the double virus vector transfection is workable in the prefrontal network of the macaque brain.

Urinary carbonic anhydrase VI as a biomarker for kidney disease in pigs.

This study investigated whether carbonic anhydrase (CA)-VI has utility as a biomarker in swine kidney disease. Serum chemistry, histopathology, immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) analyses were performed. In the kidney of normal healthy pigs, CA-VI was localized in the epithelial cells of the renal distal straight tubules. CA-VI levels were 16 ± 35 ng/g wet tissue and 50 ± 66 ng/mL in normal pig kidney and urine, respectively, and 136 ± 173 ng/mL in the urine of pigs with kidney disease. CA-VI urinary concentration was not correlated with urinary urea nitrogen (UUN), urinary creatinine (Cre), or urinary albumin levels in pigs with kidney disease. However, UUN and Cre levels were positively correlated in the urine of pigs with kidney disease. These data suggest that urinary CA-VI may represent a biomarker for kidney disease in pigs, particularly for disorders affecting distal straight tubules.

Muscle dimensions of the foot in the orangutan and the chimpanzee.

The hindlimbs of two orangutans and four chimpanzees were dissected, and muscle parameters (mass, fascicle length, and physiological cross-sectional area: PCSA) were determined to explore possible interspecies variation in muscle dimensions. Muscle mass and PCSA were divided by the total mass and total PCSA of the entire foot muscles for normalization. The results indicate that the pedal interosseous and the intrinsic pedal digital extensor muscles in the orangutans probably have higher capacity for force production due to their relatively larger PCSAs than in chimpanzees. Moreover, the medial components of the intrinsic muscles exhibited relatively larger mass and PCSA ratios in orangutans. The mass and PCSA ratios of the hallucal muscles were larger in chimpanzees. These differences in foot muscle dimensions of the two species suggest that the orangutan is more specialized for hook-like digital gripping without involvement of the rudimentary hallux, while the chimpanzee is adapted to hallux-assisted power gripping in arboreal locomotion.

Binding affinity of anti-xylitol antibodies to canine hepatic vessels.

Xylitol is used as a sugar substitute in food products. Dogs have been reported to experience lethal liver injury after accidental ingestion of xylitol. Because liver injury may be a serious consequence of canine immune-mediated reactions, antibodies produced against xylitol may attack the liver. Therefore, in the present study, we evaluated whether binding sites for xylitol antibodies are located at the liver or not. Anti-xylitol antibodies were generated by immunization of rabbits with a xylose-bovine serum albumin conjugate. Immunohistological examination showed that binding sites for the anti-xylitol antibodies were located in the hepatic arteries and the portal veins. Western blotting analyses by using a canine liver homogenate showed 4 protein bands with different molecular weights which reacted with anti-xylitol antibodies. Therefore, binding of anti-xylitol antibodies to the vessels may be the first step in an immune-mediated pathogenic response in xylitol toxicity. Further studies are necessary to determine the effects of anti-xylitol antibodies on the liver in the pathogenesis of xylitol toxicity.

Muscle carbonic anhydrase III levels in normal and muscular dystrophia afflicted chickens.

BACKGROUND: The levels and immunohistochemical localization of muscle carbonic anhydrase III (CA-III) in healthy chickens and in muscular dystrophia affected (DA) chickens show that the muscles of diseased animal undergo a progressive increase of enzyme activity. METHODS: An enzyme-linked immunoassay was used to assess the CA-III levels in the muscles and other tissues from eight normal White Leghorn chickens and in two chickens with muscular dystrophy. Immunohistochemical localization of the enzyme in the muscles of these animals was also determined. RESULTS: The levels of CA-III in the tensor fasciae latae and the superficial pectoral muscles of the DA chickens were higher than the level in normal chickens. The concentrations of CA-III in erythrocytes and plasma from diseased chickens were approximately 15-fold and 1.4-fold higher than in the normal chickens, respectively. In the superficial pectoral and the tensor fasciae latae muscles of diseased chickens, the numbers of strongly stained and weakly stained fibers were greater than that in the normal chickens. CONCLUSION: The levels of CA-III in the superficial pectoral muscle, the tensor fasciae latae muscle, plasma and erythrocytes from the chickens with muscular dystrophy were higher than found in normal chickens.

Biochemical and developmental characterization of carbonic anhydrase II from chicken erythrocytes.

BACKGROUND: Carbonic anhydrase (CA) of the chicken has attracted attention for a long time because it has an important role in the eggshell formation. The developmental profile of CA-II isozyme levels in chicken erythrocytes has not been determined or reported. Furthermore, the relations with CA-II in erythrocyte and egg production are not discussed. In the present study, we isolated CA-II from erythrocytes of chickens and determined age-related changes of CA-II levels in erythrocytes. METHODS: Chicken CA-II was purified by a combination of column chromatography. The levels of CA-II in the hemolysate of the chicken were determined using the ELISA system in blood samples from 279 female chickens, ages 1 to 93 weeks, 69 male chickens, ages 3 to 59 weeks and 52 weeks female Araucana-chickens. RESULTS: The mean concentration of CA-II in hemolysate from 1-week-old female was 50.8 ± 11.9 mg/g of Hb. The mean levels of CA-II in 25-week-old (188.1 ± 82.6 mg/g of Hb), 31-week-old (193.6 ± 69.7 mg/g of Hb) and 49-week-old (203.8 ± 123.5 mg/g of Hb) female-chickens showed the highest level of CA-II. The levels of CA-II in female WL-chickens significantly decreased at 63 week (139.0 ± 19.3 mg/g of Hb). The levels of CA-II in female WL-chicken did not change from week 63 until week 93.The mean level of CA-II in hemolysate of 3-week-old male WL-chickens was 78.3 ± 20.7 mg/g of Hb. The levels of CA-II in male WL-chickens did not show changes in the week 3 to week 59 timeframe. The mean level of CA-II in 53-week-old female Araucana-chickens was 23.4 ± 1.78 mg/g of Hb. These levels of CA-II were about 11% of those of 49-week-old female WL-chickens. Simple linear regression analysis showed significant associations between the level of CA-II and egg laying rate from 16 week-old at 63 week-old WL-chicken (p<0.01). CONCLUSIONS: Developmental changes and sexual differences of CA-II concentration in WL-chicken erythrocytes were observed. The concentration of CA-II in the erythrocyte of WL-chicken was much higher than that in Araucana-chicken (p<0.01).

Accumulation of caveolin-3 protein is limited in damaged muscle in chicken muscular dystrophy.

Members of the caveolin family are the main component of caveolae, and caveolin-3 is a muscle-specific protein. Caveolin-3 deficiency induces a muscular dystrophic phenotype, while its overexpression is also harmful to muscle cells. Increased caveolae were observed in chicken muscular dystrophy; however, the underlying mechanism causing the onset remains unclear. Therefore, the current study analyzes the expression of caveolin-3 and other caveola-related proteins in dystrophic chickens. Western blotting and semi-quantitative RT-PCR analysis revealed that (1) caveolin-3 is highly expressed in the damaged muscle of dystrophic chickens (7.12-fold); (2) the amount of caveolin-3 protein is regulated in posttranslational modification, since no significant increase is observed at the mRNA level (1.09-fold); and (3) the expression pattern of other caveola-related proteins is similar to that of caveolin-3. These results suggest that the accumulation of caveolin-3 protein may be associated with the causative process of chicken muscular dystrophy.

Immunohistochemical localization and gene expression of carbonic anhydrase isoenzymes CA-II and CA-VI in canine lower airways and lung.

The immunohistolocalization and gene expression of carbonic anhydrase (CA) isoenzymes CA-II and CA-VI in the canine lower airways and lung were examined using specific canine CA-II and CA-VI antisera and the RT-PCR method. Laryngeal, tracheal and bronchial epithelia, serous acinar and bronchiolar secretory cells and pulmonary great alveolar cells showed immunopositive reactions to anti-CA-II and anti-CA-VI antisera. However, all mucous cells showed immunonegative reactions. The physiological roles of CA-II and CA-VI in the lower airways and lung may involve the maintenance of pH balance and the protection of mucosal surfaces against the acidic milieu.

Determination of full-length cDNA nucleotide sequence of equine carbonic anhydrase VI and its expression in various tissues.

A full-length cDNA clone of an equine carbonic anhydrase (CA)-VI was obtained from the equine parotid gland. The cDNA sequence was 1338 bp long and was predicted to encode a 319 amino acid polypeptide with a putative signal peptide of 18 amino acids. The deduced amino acid sequence of mature CA-VI showed the similarity of 70% to those of other mammalians reported. Westernblot analysis using anti-horse CA-VI peptide detected the single band in parotid gland, and the band reduced its size by treatment with N-glycosidase F. Additionally, CA-VI protein expression was confirmed in submandicular gland and weakly in liver. In contrast, RT-PCR analysis revealed signals in the digestive tract including duodenum, jejunum, ileum, cecum and colon as well as the salivary glands. In addition, certain signals were detected in testis, thyroid gland and liver, but not in nerve tissue, skeletal muscle, spleen or lymph node.

Ubiquitin C-terminal hydrolase-L3-knockout mice are resistant to diet-induced obesity and show increased activation of AMP-activated protein kinase in skeletal muscle.

Obesity results from the dysregulation of energy balance throughout the entire body. Although the ubiquitin system participates in many cellular processes, its contribution to the balance of energy in the body remains poorly understood. Here, we show that ubiquitin C-terminal hydrolase (UCH)-L3, one of the deubiquitinating enzymes, contributes to the regulation of metabolism. Uchl3(-/-) mice displayed a reduction of adipose tissue mass and were protected against high-fat diet (HFD)-induced obesity and insulin resistance. Uchl3(-/-) mice given both a normal chow and an HFD had an increased whole-body energy expenditure accounting for the reduction of adipose tissue mass. Activation of AMP-activated protein kinase (AMPK) in skeletal muscle has been reported to increase fatty acid beta-oxidation, leading to the elevation of the whole-body energy expenditure. Consistently, increased activation of AMPK and fatty acid beta-oxidation was observed in skeletal muscle of Uchl3(-/-) mice. Mouse embryonic fibroblasts derived from Uchl3(-/-) mice also showed increased activation of AMPK, indicating that UCH-L3 is involved in a cell-autonomous down-regulation of AMPK. These results suggest a role for UCH-L3 in the regulation of AMPK activity and whole-body energy metabolism.

Dimensions of forelimb muscles in orangutans and chimpanzees.

Eight forelimbs of three orangutans and four chimpanzees were dissected and the muscle mass, fascicle length and physiological cross-sectional area (PCSA) of all forelimb muscles were systematically recorded to explore possible interspecies variation in muscle dimensions. Muscle mass and PCSA were divided by the total mass and total PCSA of the entire forelimb muscles for normalization. The results indicate that the mass and PCSA ratios of the monoarticular elbow flexors (M. brachialis and M. brachioradialis) are significantly larger in orangutans. In contrast, the mass ratios of the biarticular muscles in the upper arm (the short head of M. biceps brachii and the long head of M. triceps brachii) are significantly larger in chimpanzees. For the rotator cuff muscles, the force-generating capacity of M. subscapularis is significantly larger in orangutans, whereas the opposite rotator cuff muscle, M. infraspinatus, is larger in chimpanzees. These differences in forelimb muscle dimensions of the two species may reflect functional specialization for their different positional and locomotor behaviors.

Dimensions of the foot muscles in the lowland gorilla.

We dissected the hindlimb of a female western lowland gorilla and determined the muscle dimensions (mass, fascicle length, and physiological cross-sectional area: PCSA). Comparisons of the muscle parameters of the measured gorilla with corresponding reported human data demonstrated that the triceps surae muscles were larger and had more capacity to generate force than the other muscle groups in both species, but this tendency was more prominent in the human, probably as an adaptation to strong toe-off during bipedal walking. On the other hand, PCSAs of the extrinsic pedal digital flexors and digiti minimi muscles were larger in the western lowland gorilla, suggesting that the foot, particularly the fifth toe, has a relatively high grasping capability in the lowland gorilla.

Pathomechanism of cellular infiltration in the perivascular region of several organs in SAMP1/Yit mouse.

We investigated the histological changes of extra-intestinal organs, such as the liver, kidney, lung and pancreas in SAMP1/Yit mice, a human Crohn's disease model, using immunohistochemical techniques. The perivascular cellular infiltration was detected around the small vessels after 30 weeks. These infiltrating cells consisted of many CD4-positive T-lymphocytes, and small numbers of CD8- positive T-lymphocytes and IgG-positive B-lymphocytes. MAdCAM-1 and VCAM-1 were detected in vascular endothelial cells in non-affected regions of 13 and 20 week-old, as well as in the affected regions showing perivascular cellular infiltration after 30 weeks. In addition, integrin alpha4beta7 was detected on these infiltrating cells in the perivascular regions after 30 week-old. LT-beta and IL-12, cytokines of the Th-1-type immune response, were not observed in these affected regions. However, IL-4, one of the cytokines of the Th-2-type immune response, was detected on the perivascular infiltrating cells after 30 week-old. These results revealed that the changes in extra-intestinal organs were mainly caused by infiltration of CD4-positive T-lymphocytes into the perivascular regions in SAMP1/Yit mice. These cellular infiltrations were thought to be initiated by adhesion of CD4-positive T-lymphocytes to the endothelial cells mediated by MAdCAM-1 and integrin beta7. Immunohistochemistry for Th related cytokines indicated that the perivascular cellular infiltration was developed by the Th-2-type immune response in the extra-intestinal organs of SAMP1/Yit mouse.

Immunohistolocalization and gene expression of secretory carbonic anhydrase isoenzyme CA-VI in canine nasal cavity.

Immunolocalization of the secretory form of carbonic anhydrase isoenzyme, CA-VI were studied using a specific canine CA-VI antiserum, and CA-VI mRNA signals were also investigated using the reverse-transcriptase polymerase chain reaction (RT-PCR) in canine nasal mucosal epithelia and glands. Immunoreactivity to CA-VI was positive throughout the mucosal epithelial cells and in the cytoplasm of serous acinar and ductal epithelial cells of the nasal mucosa and glands, including the vestibule of the nose, but the mucous acinar cells of the glands were immunonegative. We detected CA-VI gene transcripts in the same regions as the CA-VI immunoreactivity. The physiological roles of CA-VI in the nasal mucosal epithelium and glands might maintain bicarbonate levels in nasal secretions and protect the mucosa against acid.

The ubiquitin ligase gene (WWP1) is responsible for the chicken muscular dystrophy.

Chicken muscular dystrophy with abnormal muscle (AM) has been studied for more than 50 years, but the gene responsible for it remains unclear. Our previous studies narrowed down the AM candidate region to approximately 1Mbp of chicken chromosome 2q containing seven genes. In this study, we performed sequence comparison and gene expression analysis to elucidate the responsible gene. One missense mutation was detected in AM candidate genes, while no remarkable alteration of expression patterns was observed. The mutation was identified in WWP1, detected only in dystrophic chickens within several tetrapods. These results suggested WWP1 is responsible for chicken muscular dystrophy.

A macroscopic examination of M. biceps femoris and M. gluteus maximus in the orangutan.

The musculature of the hip and thigh in the orangutan has been described previously. Anatomically, there are various descriptions among primates in those structures, in particular, the relationship between M. biceps femoris and M. gluteus maximus, their derivatives, and the muscle segment. However, a detailed innervation system to this ischiofemoral part has not been described, thus there is still uncertainty as to with which muscle it is associated. In this analysis, we examined the gross anatomy of the hip and thigh muscles of the orangutan and chimpanzee, including their innervation. Also, a comparison was made with documented data of other primates. As a result of these observations, it was found that the ischiofemoral part in the orangutan is innervated by the same sciatic nerve branch (the common peroneal nerve) as the long head of M. biceps femoris, but not by the same nerve as M. gluteus maximus. Therefore, the ischiofemoral part is appropriately considered as a part of the long head of M. biceps femoris. It appears that this morphologic feature is an adjustment to the arboreal life of the orangutan. The development of the flexor complex of the thigh is necessary for this arboreal adaptation, resulting in a unique musculature of M. biceps femoris in the orangutan.

Dopaminergic neuronal loss in transgenic mice expressing the Parkinson's disease-associated UCH-L1 I93M mutant.

The I93M mutation in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) was reported in one German family with autosomal dominant Parkinson's disease (PD). The causative role of the mutation has, however, been questioned. We generated transgenic (Tg) mice carrying human UCHL1 under control of the PDGF-B promoter; two independent lines were generated with the I93M mutation (a high- and low-expressing line) and one line with wild-type human UCH-L1. We found a significant reduction in the dopaminergic neurons in the substantia nigra and the dopamine content in the striatum in the high-expressing I93M Tg mice as compared with non-Tg mice at 20 weeks of age. Although these changes were absent in the low-expressing I93M Tg mice, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment profoundly reduced dopaminergic neurons in this line as compared with wild-type Tg or non-Tg mice. Abnormal neuropathologies were also observed, such as silver staining-positive argyrophilic grains in the perikarya of degenerating dopaminergic neurons, in I93M Tg mice. The midbrains of I93M Tg mice contained increased amounts of insoluble UCH-L1 as compared with those of non-Tg mice, perhaps resulting in a toxic gain of function. Collectively, our data represent in vivo evidence that expression of UCHL1(I93M) leads to the degeneration of dopaminergic neurons.

Comparative observation of skeletal-dental abnormalities in wild, domestic, and laboratory rabbits.

Dietary habits must be considered as one of the major potential factors resulting in acquired malocclusions in rabbits. Although the dentition of the wild rabbit and the domesticated laboratory rabbit are basically identical, dietary habits are noticeably different. Therefore, the prevalence of tooth problems between these lagomorph species were investigated anatomically and radiographically. Mean measurements of the skull and dental arches suggested that wild rabbits have slightly shorter and wider skulls and dental arches compared with domestic laboratory rabbits. Root elongation of incisors and check teeth, and periodontal disease were more frequently observed in domestic laboratory rabbits. Diagnostic radiographs from domestic pet rabbits showed relatively higher crowns, severe root elongation, and advanced periodontitis. These results do not provide definitive evidence that dietary habits cause malocclusions, however they suggest that diet is a major factor in the initiation of malocclusions in rabbits.