QIL1-dependent assembly of MICOS complex-lethal mutation in C19ORF70 resulting in liver disease and severe neurological retardation.

Seven subunits of the mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) in humans have been recently described in function and structure. QIL1 (also named MIC13) is a small complex that is crucial for the maintenance and assembling of MICOS. A novel mutation of an essential splice site in the C19orf70 gene encoding QIL1 induces severe mitochondrial encephalopathy, hepatopathy and lactate acidosis consistent with psychomotor retardation. In addition, bilateral kidney stones were observed. Disassembly of MICOS complex subunits displays lack of MIC10-MIC26-MIC27-QIL1 subcomplex, resulting in aberrant cristae structure and a loss of cristae junctions and contact sites. In liver and muscle tissue, the activity of the respiratory chain complexes (OXPHOS) was severely impaired. Defects in MICOS complex do not only affect mitochondrial architecture, but also mitochondrial fusion, metabolic signalling, lipid trafficking and cellular electric homeostasis.

Xenografting of isolated equine (Equus caballus) testis cells results in de novo morphogenesis of seminiferous tubules but not spermatogenesis.

The study of spermatogenesis in the horse is challenging because of the absence of an in vitro system that is capable of reproducing efficient spermatogenesis and because of the difficulties and costs associated with performing well-controlled studies in vivo. In an attempt to develop novel methods for the study of equine spermatogenesis, we tested whether cells from enzymatically digested pre-pubertal equine testicular tissue were capable of de novo tissue formation and spermatogenesis following xenografting under the back skin of immunocompromised mice. Testes were obtained from normal pre-pubertal colts and dissociated into cell suspensions using trypsin/collagenase digestion. Resulting cell pellets, consisting of both somatic and germ cells, were injected into fascial pockets under the back skin of immunocompromised, castrated mice and maintained for between 1 and 14 months. Mice were killed and grafts were recovered and analyzed. As has been reported for testis cell suspensions from pigs, mice, cattle, and sheep, de novo formation of equine testicular tissue was observed, as evidenced by the presence of seminiferous tubules and an interstitial compartment. There was an increased likelihood of de novo testicular formation as grafting period increased. Using indirect immunofluorescence, we confirmed the presence of spermatogonia in de novo formed seminiferous tubules. However, we found no evidence of meiotic or haploid cells. These results indicate that dissociated pre-pubertal equine testis cells are capable of reorganizing into the highly specialized endocrine and spermatogenic compartments of the testis following ectopic xenografting. However, in spite of the presence of spermatogonia within the seminiferous tubules, spermatogenesis does not occur. Although this technique does allow access to the cells within the seminiferous tubule and interstitial compartments of the equine testis prior to reaggregation, the absence of spermatogenesis will limit its use as a method for the study of testicular function in the horse.

Inhibition of neointima formation by a nonpeptide alpha(v)beta(3) integrin receptor antagonist in a rabbit cuff model.

This study was performed to determine whether a highly selective nonpeptide alpha(v)beta(3) antagonist (SH306) would prove effective in inhibiting neointima formation in a rabbit cuff model. The animals were dosed with SH306, 5 mg/kg i.v., followed by 10 mg/kg s. c., 3 times daily for 3 days, or with vehicle (10% DMAC). Rabbits were sacrificed and perfused on days 1, 3, and 21; the vessels were paraffin embedded. A reduction in the intima/media (I/M) of the SH306-treated rabbits, as compared with the vehicle-treated control group, was noted (0.20 vs 0.36 [n = 4]). A significant increase in the area of the media was observed in the SH306-treated group versus the control group (0.20 vs 0.13). No difference was observed in cell proliferation between SH306 and vehicle after 1-day and 3-day dosing. Thrombi were found in 43% of the control vessels and in only 14% of the drug-treated vessels. No anticoagulant was used during the surgical procedure. No increase in inhibition of GPIIb/IIIa was observed in SH306-treated animals, as compared with the vehicle control group. We conclude that selective inhibition of alpha(v)beta(3) reduced neointima formation in a rabbit model at 3 weeks.

alphavbeta3, alphavbeta5, and osteopontin are coordinately upregulated at early time points in a rabbit model of neointima formation.

Both smooth muscle cell migration and replication are known to be responsible for neointima formation. Recent reports based on in vitro studies and animal models of neointima formation highlight the possible importance of alphavbeta3 and alphavbeta5 integrins in mediating neointima formation. Clinical data suggest that specific alphavbeta3 blockade may limit restenosis. The aim of this study was to identify the expression of alphavbeta3 and alphavbeta5 and their ligand osteopontin in the very early phases of neointima formation in a rabbit model. A non-occlusive cuff placed around the rabbit femoral artery resulted in a complete, concentric neointima that formed by 14 days. Antibodies specific for the integrin heterodimers and for osteopontin, along with a probe specific for osteopontin mRNA, were used to identify expression at early time points (6 h, 1 day, 3 days, 5 days) post-cuffing. Immunohistochemistry and in situ hybridization expression results were quantitated by image analysis and tested for statistical significance by a two-tailed t-test. The data demonstrated the rapid (within 6 h) and abundant upregulation of alphavbeta3 and alphavbeta5 integrins and their ligand during very early time points of neointima formation. The very early (6 h) upregulation of alphavbeta3 underscores a potentially important clinical intervention point in limiting restenosis following clinical angioplasty procedures.

Hepatic and adrenal changes in rabbits associated with hyperlipidemia caused by a semi-synthetic diet.

Several investigators have reported that feeding a semi-synthetic diet of casein and dextrose to New Zealand White (NZW) rabbits will increase total serum cholesterol concentration, principally through an increase in the beta-lipoprotein fractions, thereby creating a useful model for atherosclerosis research. Although there is evidence to suggest that the dextrose/casein diet alters low-density lipoprotein receptor and bile acid clearance of cholesterol, the underlying mechanism is not completely understood. The effects of the diet on the overall physiology of the rabbit have received little attention. In this study feeding a diet of casein and dextrose of male NZW rabbits for 4 weeks resulted in changes in the serum lipid concentrations. During that time the rabbits fed the dextrose/casein diet gained less weight than did control rabbits. In the test diet rabbits, liver aspartate and alanine transaminase activities were increased from baseline values of 27 +/- 2 U/L and 89 +/- 9 U/L respectively to 112 +/- 21 U/L and 281 +/- 34 U/L respectively, then returned to the high end of the reference range. Necropsy findings included hepatomegaly caused by vacuolar hepatopathy in 19 or 20 experimental rabbits; rabbits fed the control diet had no hepatic lesions. Ultrastructural analysis revealed that enlargement of the liver cells was due to glycogen deposition. Adrenal glands from animals fed the experimental diet had a minimal change in the size of the adrenocortical cells consisting of slight ballooning and rarefaction of the cytoplasm. In a second study the level of dietary fiber was doubled. This resulted in a three-fold increase in lipid concentrations, compared with the fivefold increase in the first study. The liver enzyme activities were increased to the same extent as in the first study. Histologic changes were comparable to those in the first study. The activity of hepatic cholesterol 7alpha-hydroxylase was 3.7 +/- 0.4 pmol/min/mg of protein, compared with the control value of 7.7 +/- 1.1 pmol/min/mg of protein (P < 0.05) in the second study. The improved rate of weight gain and the lesser increase in total serum cholesterol concentration in the second study with increased dietary fiber suggest that two separate activities may be involved. Although the level of dietary fiber may be related to weight gain and total serum cholesterol values, the relation to the decrease in liver transaminase activities in study 1 was probably coincidental. It appears that the dextrose/casein diet causes decreased activity of hepatic cholesterol 7alpha-hydroxylase, which could cause a decrease in the biliary excretion of cholesterol.