Cystathionine beta synthase deficiency affects mouse endochondral ossification.

Cystathionine beta synthase (CBS) is a crucial regulator of plasma concentrations of homocysteine. Severe hyperhomocysteinemia due to CBS deficiency confers diverse clinical manifestations, notably characteristic skeletal abnormalities. To investigate this aspect of hyperhomocysteinemia, we analyzed the skeleton of CBS-deficient mice, a murine model of severe hyperhomocysteinemia. Radiography, Alcian Blue/Alizarin Red S-stained whole skeletal preparations, and histological comparisons were used to determine the extent, pattern, and distribution of skeletal abnormalities in CBS-deficient mice. Disruption of the murine CBS gene leads to skeletal abnormalities, notably kyphoscoliosis, with temporal shortening of long bones due to impaired cartilage differentiation, albeit to differing degrees.

Expression of the cystathionine beta synthase (CBS) gene during mouse development and immunolocalization in adult brain.

Hyperhomocysteinemia, caused by a lack of cystathionine beta synthase (CBS), leads to elevated plasma concentrations of homocysteine. This is a common risk factor for atherosclerosis, stroke, and possibly neurodegenerative diseases. However, the mechanisms that link hyperhomocysteinemia due to CBS deficiency to these diseases are still unknown. Early biochemical studies describe developmental and adult patterns of transsulfuration and CBS expression in a variety of species. However, there is incomplete knowledge about the regional and cellular expression pattern of CBS, notably in the brain. To complete the previous data, we used in situ hybridization and Northern blotting to characterize the spatial and temporal patterns of Cbs gene expression during mouse development. In the early stages of development, the Cbs gene was expressed only in the liver and in the skeletal, cardiac, and nervous systems. The expression declined in the nervous system in the late embryonic stages, whereas it increased in the brain after birth, peaking during cerebellar development. In the adult brain, expression was strongest in the Purkinje cell layer and in the hippocampus. Immunohistochemical analyses showed that the CBS protein was localized in most areas of the brain but predominantly in the cell bodies and neuronal processes of Purkinje cells and Ammon's horn neurons.

Hyperkeratosis in cystathionine beta synthase-deficient mice: an animal model of hyperhomocysteinemia.

Cystathionine beta synthase (CBS) is a crucial regulator of plasma concentrations of homocysteine. Severe hyperhomocysteinemia due to CBS deficiency confers diverse clinical manifestations. Patients with severe hyperhomocysteinemia have fine hair and thin skin, but it is unclear whether these changes are related to CBS deficiency or are coincidental. To investigate these aspects of hyperhomocysteinemia, we characterized skin abnormalities of CBS-deficient mice, a murine model of severe hyperhomocysteinemia. Histological and histomorphometric analyses revealed that CBS-deficient mice have wrinkled skin with hyperkeratinosis of the epidermis and thinning of the dermis.

Chemical immobilization of raccoons (Procyon lotor) with ketamine-medetomidine mixture and reversal with atipamezole.

Safe and reliable capture techniques for wild animals are important for ecologic studies and management operations. We assessed the efficiency of ketamine-medetomidine (K:M) injection and reversal with atipamezole. We anesthetized 67 raccoons (Procyon lotor; 34 males, 33 females) 103 times (individuals captured between one and five times) from April 2009-October 2010 in Mont-Orford Provincial Park, Quebec, Canada. We administered a 1:1 mixture by volume of ketamine and medetomidine by intramuscular injection. Mean (±SD) induction times for males and females were 6.1±2.8 and 6.6±3.7 min, respectively. Mean induction time was 2 min longer for juveniles than for adults (7.8±3.9 and 5.8±2.9 min, respectively) and longer in autumn than in spring for adults (7.7±3.8 and 5.4±2.9 min, respectively). Recovery time after administration of atipamezole was 9.6±3.8 and 8.4±4.4 min for males and females, respectively. Recovery time was longer in spring than in autumn (10.2±4 and 7.4±3.8 min, respectively) for adults. Induction time increased by 166% after five captures of the same individual. Immobilization did not affect body mass, adult survival, or female reproductive success. We suggest the K:M mixture used is a safe and reliable method for anesthetizing raccoons in field conditions.

Genetic structure and rabies spread potential in raccoons: the role of landscape barriers and sex-biased dispersal.

Identifying natural barriers to movements of hosts associated with infectious diseases is essential for developing effective control strategies. Raccoon rabies variant (RRV) is a zoonosis of concern for humans because its main vector, the raccoon (Procyon lotor), is found near residential areas. In Québec, Canada, all cases of RRV found in raccoons since 2006 were detected on the eastern side of the Richelieu River, suggesting that this river acts as a barrier to gene flow and thus the potential for RRV to spread. The objectives of this study were to characterize the genetic structure of raccoon populations and assess the effect of the Richelieu River on the population structure in southern Québec, Canada. We also evaluated whether RRV spread potential differed between sex and at a larger spatial scale. Our analyses revealed a weak signal of genetic differentiation among individuals located on each side of the Richelieu River. At a larger spatial scale, genetic structuring was weak. Our results suggest that rivers might not always efficiently restrain raccoon movements and spread of RRV. We suggest that the difference in genetic structure found between sexes can be partly explained by male movements during the breeding season in winter, when ice bridges allow passage over most rivers in Québec.

Use of three-dimensional ultrasound in the detection of breast tumor bed displacement during radiotherapy.

PURPOSE: To evaluate the feasibility and usefulness of a three-dimensional ultrasound (3D-US) image-guided system in identifying and tracking the tumor bed (TB) for planning and daily localization before radiation delivery for breast cancer. METHODS AND MATERIALS: Twenty breast cancer patients underwent two CT scans at the time of simulation and just before their boost. Three-dimensional ultrasound images were acquired immediately after the CT scans, to which the images were automatically fused. Three-dimensional ultrasound images were also acquired immediately before treatment. Spatial and temporal TB differences between CT and US were evaluated. RESULTS: The TB was not visible on US and CT in 1 subject who had and 1 subject who had not received chemotherapy before whole-breast radiotherapy. The mean (SD) TB volume overlap was 78% (14%). The mean centroid position of the TB on CT vs. US differed by 0.1, 0.2, and 0.4 mm in the anterior-posterior, left-right, and superior-inferior directions. The mean (SD) absolute radial displacement of the TB on each fraction from the treatment plan was 10.8 (6.3) mm. CONCLUSIONS: The TB was well visualized by US for the majority of patients. Clinically insignificant differences in the displacements calculated by paired CT vs. paired US demonstrate the feasibility of using 3D-US. The present study suggests that a 10-mm planning target volume margin could result in undercoverage of the clinical target volumes in 50% of treatments. Multimodality planning and image-guided radiotherapy with US potentially offers an accurate and non-ionizing solution for the daily definition of the TB position during partial-breast irradiation and boost treatments.

Cystathionine beta synthase deficiency promotes oxidative stress, fibrosis, and steatosis in mice liver.

BACKGROUND & AIMS: Cystathionine beta-synthase (CBS) deficiency causes severe hyperhomocysteinemia, which confers diverse clinical manifestations, notably liver disease. To investigate this aspect of hyperhomocysteinemia, we performed a thorough investigation of liver pathology in CBS-deficient mice, a murine model of severe hyperhomocysteinemia. METHODS: The degree of liver injury and inflammation was assessed by histologic examination, by measurements of products of lipid peroxidation, and by formation of carbonyl groups on protein as a measure for the occurrence of protein oxidation. Analysis of profibrogenic, proinflammatory factors and cell apoptosis was performed by Western blots, real-time quantitative reverse-transcription polymerase chain reaction, caspase-3 activity, DNA laddering, and TUNEL assay. RESULTS: Histologic evaluation of liver specimens of 8- to 32-week-old CBS-deficient mice showed that CBS-deficient mice develop inflammation, fibrosis, and hepatic steatosis, concomitant with an enhanced expression of tissue inhibitor of metalloproteinase-1, alpha-smooth muscle actin, pro(alpha)1 collagen type I, transforming growth factor-beta1, and proinflammatory cytokines. Moreover, even if the proapoptotic protein Bax was dominantly expressed and Bcl-2 was down-regulated, caspase-3 was not activated, DNA laddering was not detected, and number of positive TUNEL cells was not increased in liver of CBS-deficient mice compared with wild-type mice. CONCLUSIONS: The results show that hyperhomocysteinemia in liver of CBS-deficient mice promotes oxidative stress, which may cause mitochondrial damage in association with activation of hepatic stellate cells, leading to liver injury. The absence of caspase-3 activation, DNA fragmentation, and TUNEL-positive cells shows that protective signals may counteract apoptotic signals in liver of CBS-deficient mice.

The neuronal SAPK/JNK pathway is altered in a murine model of hyperhomocysteinemia.

Deficiency in cystathionine beta synthase (CBS) leads to high plasma homocysteine concentrations and causes hyperhomocysteinemia, a common risk factor for vascular disease, stroke and possibly neurodegenerative diseases. Various neuronal diseases have been associated with hyperhomocysteinemia, but the molecular mechanisms of homocysteine toxicity are unknown. We investigated the pathways involved in the pathological process, by analyzing differential gene expression in neuronal tissues. We used a combination of differential display and cDNA arrays to identify genes differentially expressed during hyperhomocysteinemia in brain of CBS-deficient mice. In this murine model of hyperhomocysteinemia, both plasma and brain homocysteine concentrations were high. Several genes were found to be differentially expressed in the brains of CBS-deficient mice, and the identities of some of these genes suggested that the SAPK/JNK pathway was altered in the brains of CBS-deficient mice. We therefore investigated the activation of proteins involved in the SAPK/JNK cascade. JNK and c-Jun were activated in the hippocampal neurones of CBS-deficient mice, suggesting that the SAPK/JNK pathway may play an important role in the development of neuronal defects associated with hyperhomocysteinemia.

Remodeling of Na,K-ATPase, and membrane fluidity after atrial fibrillation in sheep.

UNLABELLED: Atrial fibrillation (AF) is accompanied by various changes in ion channels that cause atrial electrophysiological remodeling. The enzyme Na,K-ATPase is also a major cellular mechanism for the regulation of ion homeostasis. During AF, Na,K-ATPase may be regulated by synthesis of its alpha- and beta-subunits as well as changes in membrane fluidity. To test this hypothesis, we studied the effect of pacing-induced AF in sheep on atrial Na,K-ATPase alpha- and beta-subunits and on membrane fluidity as well. METHODS: A group of six sheep (AF group) was subjected to overdrive electrical stimulation of the right atrium in order to induce AF. A group of six sham operated sheep served as control. All paced sheep developed multiple episodes of sustained AF with a mean total duration of 110 min over a 2-hours period. Protein expression of Na,K-ATPase alpha- and beta-subunits in atrial microsomal membranes was assayed by Western blotting analysis. When significant changes in membrane expression were observed, transcriptional regulation was analysed by Northern blotting. Membrane fluidity was assessed on atrial microsomal fractions by anisotropy measurements using the fluorescent probe diphenylhexatriene. RESULTS: Atrial fibrillation enhanced the expression of the Na,K-ATPase beta1-subunit at both membrane and mRNA levels. Anisotropy values were higher in AF group than in control group, indicating a decreased fluidity of the membranes isolated from paced sheep atria. CONCLUSION: These data are the first evidence for an enhanced Na,K-ATPase beta1-subunit expression in membrane during AF. Membrane rigification represents a new factor of tachycardia-induced atrial remodeling.

Altered gene expression in liver from a murine model of hyperhomocysteinemia.

Cystathionine beta-synthase (CBS) deficiency causes severe hyperhomocysteinemia and other signs of homocystinuria syndrome, in particular a premature atherosclerosis with multiple thrombosis. However, the molecular mechanisms by which homocysteine could interfere with normal cell function are poorly understood in a whole organ like the liver, which is central to the catabolism of homocysteine. We used a combination of differential display and cDNA arrays to analyze differential gene expression in association with elevated hepatic homocysteine levels in CBS-deficient mice, a murine model of hyperhomocysteinemia. Expression of several genes was found to be reproducibly abnormal in the livers of heterozygous and homozygous CBS-deficient mice. We report altered expression of genes encoding ribosomal protein S3a and methylthioadenosine phosphorylase, suggesting such cellular growth and proliferation perturbations may occur in homozygous CBS-deficient mice liver. Many up- or down-regulated genes encoded cytochromes P450, evidence of perturbations of the redox potential in heterozygous and homozygous CBS-deficient mice liver. The expression of various genes involved in severe oxidative processes was also abnormal in homozygous CBS-deficient mice liver. Among them, the expression of heme oxygenase 1 gene was increased, concomitant with overexpression of heme oxygenase 1 at the protein level. Commensurate with the difference in hepatic mRNA paraoxonase 1 abundance, the mean hepatic activity of paraoxonase 1, an enzyme that protects low density lipoprotein from oxidation, was 3-fold lower in homozygous CBS-deficient mice. Heterozygous CBS-deficient mice, when fed a hyperhomocysteinemic diet, have also reduced PON1 activity, which demonstrates the effect of hyperhomocysteinemia in the paraoxonase 1 activity.

Ginkgo biloba extract (EGb 761) protects Na,K-ATPase isoenzymes during cerebral ischemia.

Disturbances of Na,K-ATPase activity are implicated in the pathophysiology of cerebral ischemia. Previous experiments have shown that EGb 761 protects NaK-ATPase activity against one hour of cerebral ischemia. In the brain however, the 3 isoenzymes responsible for Na,K-ATPase activity may be differentially affected by various times of ischemia. In the present study, we investigated the effect of a longer period of ischemia, and the protection provided by a pre-treatment with EGb 761 on each of the 3 cerebral NaK-ATPase isoenzymes. In control and EGb 761 pre-treated mice exposed to a 6 hr unilateral occlusion of the middle cerebral artery, Na,K-ATPase activity was decreased by 60% and lipid peroxidation was increased by 40% in the ipsilateral (ischemic) cortex compared to the contralateral one. In parallel, membrane integrity was altered. The alteration of NaK-ATPase activity, as a whole, resulted from a decrease in the activity of the 3 isoenzymes. The two isoenzymes of high ouabain affinity however, had their affinities decreased while the sensitivity of the lowest affinity isoenzyme was increased. Pre-treatment with EGb 761 abolished the differences observed between ipsi- and contralateral cortex, with the exception of the change in ouabain affinity of the low affinity isoenzyme. Ischemia also induced changes in Na,K-ATPase isoenzyme ouabain affinities in the contralateral cortex that where not prevented by EGb 761.