Normal copper metabolism in quaking mice

This research measured lipid peroxidation products in rats of varying age by a new method. Wistar male rats, 4, 12, 22 and 32 months old, were examined for lipid peroxidation in vivo by measurement of ethane, ethylene, butane and pentane in breath gases of intact animals. In the older rats, the amounts of the four hydrocarbons exhaled were greater than those in younger rats. All hydrocarbons tested were related to age by an exponential relationship, and quantitatively, ethane and ethylene were related to age with a linear regression fit correlation coefficient, 0.466–0.622 (p<0.01-0.001). When hydrocarbon gas production of 32 month-old rats was compared with that of 4 month-old rats, the greatest ratio was that for pentane (1.99). The following order was ethylene >ethane>butane. There were significant differences in the production of all hydrocarbons between 32 month-old rats and 4 month-old rats.Thiobarbituric acid reactants in serum exhibited an increasing tendency with age, but the values of 32 month-old rats were lower than those of 22 month-old rats. However, the differences between the age groups were not significant.These results showed that the measurement of hydrocarbons in the rats'breath was a sensitive index of in vivo peroxidation during aging.     

Studies of brain myelin in the "quaking mouse"

Myelin was isolated from the brains of "quaking" and littermate control animals and its composition was determined. The brains of quaking animals contained approximately one-fourth as much myelin as the control animals. There were qualitative as well as quantitative differences between the myelin from the two groups. By continuous cesium chloride gradient flotation it was shown that the myelin from the quaking animals consisted solely of a band corresponding to the heavier and smaller of the two bands found in normal controls. Cholesterol and glycolipids were lower and phospholipids (mainly phosphatidylcholine) and protein were higher in quaking animals than in controls. Also, phosphatidal-ethanolamine was decreased, and several consistent differences in the fatty acids (both unsubstituted and hydroxy) and aldehydes of the component lipids were found. In general there were smaller amounts of monounsaturated fatty acids in quaking animals. We suggest from these findings that myelin in the quaking mouse has certain compositional similarities with juvenile myelin, but it may be an abnormal type of myelin.     

Lethal phenotype of mice carrying a Sept11 null mutation

Septins are cytoskeletal GTP-binding proteins involved in processes characterized by active membrane movement, such as cytokinesis, vesicle trafficking and exocytosis. Most septins are expressed ubiquitously, however, some septins accumulate in particular tissues. The ubiquitous SEPT11 also shows high expression levels in the central nervous system and in platelets. Here, SEPT11 is involved in vesicle trafficking and may play a role in synaptic connectivity. Interestingly, mice that harbor a homozygous Sept11 null mutation, die in utero. From day 11.5 post coitum onwards, development of homozygous embryos seems to be retarded and the embryos from day 13.5 onwards were dead.     

Nuclear retention of MBP mRNAs in the quaking viable mice

Quaking viable (qk(v)) mice fail to properly compact myelin in their central nervous systems. Although the defect in the qk(v) mice involves a mutation affecting the expression of the alternatively spliced qk gene products, their roles in myelination are unknown. We show that the QKI RNA binding proteins regulate the nuclear export of MBP mRNAs. Disruption of the QKI nucleocytoplasmic equilibrium in oligodendrocytes results in nuclear and perikaryal retention of the MBP mRNAs and lack of export to cytoplasmic processes, as it occurs in qk(v) mice. MBP mRNA export defect leads to a reduction in the MBP levels and their improper cellular targeting to the periphery. Our findings suggest that QKI participates in myelination by regulating the mRNA export of key protein components.     

Proteolysis in quaking mouse brain and spinal cord

Six proteolytic enzymes were assayed for activity in quaking CNS in examining the hypothesis that increased proteolytic activity contributes to the hypomyelination characteristic of this mutant. Cathepsin B-like enzyme, cathepsin D, neutral proteinase, calcium-activated neutral proteinase, prolyl endopeptidase, and diaminopeptidase II were assayed in whole homogenate of brain or spinal cord and each was found to have activity similar to that in normal mice. These results do not support a relationship between proteolysis and the genetic defect and suggest that other factors should be investigated to delineate the pathogenesis of this mutant.     

Abnormal glycosylation of myelin-associated glycoprotein in quaking mouse brain

Brain slices from actively myelinating (26–28 days) quaking and normal littermates were dual-labeled with radioactive mannose and fucose for 2 h. Following the incubation myelin was isolated by sucrose density gradient centrifugation and the incorporation of sugars into the major myelinassociated glycoprotein (MAG) determined. The incorporation of mannose (an internal monosaccharide) and fucose (a terminal monosaccharide) was impaired in quaking by approximately 70 and 83% respectively as compared to control. The mannose/fucose ratio in quaking myelin was approximately 70% higher than in control. The results indicate an abnormal processing of the N-linked oligosaccharide moiety of MAG in quaking oligodendrocytes.     

Esterase alterations in the liver and kidneys of ducky,a neurological mutation in mice

We have investigated qualitative and quantitative changes which occur with development and disease progression in nonspecific esterases of livers and kidneys of ducky mice. Total activity in both organs is reduced compared with normal mice, and permanent and temporary deletions of isozyme bands occur. The isozyme profile depends upon the stage of the disease.Supported in part by NIH Research Grant NB 06448 from the National Institutes of Neurological Diseases and Stroke, and grants from the National Foundation for Neuromuscular Diseases, Inc., the Southwaite Foundation, and the United Health Services of North Carolina.Visiting Scientist, The Jackson Laboratory, Bar Harbor, Maine. On leave of absence from the Department of Physiology, Wakayama Medical College, Wakayama, Japan.     

Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene

Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apc(Min/+)) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca(2+) activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin-luciferase in isolated erythrocytes from apc(Min/+) mice and wild-type mice (apc(+/+)). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apc(Min/+) mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Extracellular Ca(2+) removal or inhibition of Ca(2+) entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca(2+) -entry by treatment with Ca(2+) -ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Following retrieval and injection into the circulation of the same mice, apc(Min/+) erythrocytes were more rapidly cleared from circulating blood than apc(+/+) erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apc(Min/+) mice. The observations point to accelerated eryptosis and subsequent clearance of apc(Min/+) erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.     

Myelin subfractions isolated from mouse brain. Studies of normal mice during development,quaking mutants,and three brain regions

Myelin isolated from three areas of mouse brain, from whole brain at several ages in normal mice, and from whole brain of adult quaking mutant mice was separated into seven bands and a pellet on discontinuous density gradients using 0.32, 0.45, 0.55, 0.60, 0.70, 0.75, and 0.85 M sucrose. The distribution of myelin in the subfractions was independent of homogenization and shocking conditions employed to isolate the myelin preparations, but was related to the type of myelin applied to the gradient. Compared to myelin isolated from older animals, myelin isolated from 18–24 day old mice displayed a distribution pattern with greater proportions of material banding at lesser sucrose densities. Similarly, myelin obtained from hindbrain contained proportionately more material layering at lesser sucrose densities compared to myelin isolated from cerebral cortex. Myelin subfraction patterns observed for 8–12 day old control mice and quaking mutants were unlike each other or any other myelin preparation examined. In the 18–90 day old animals, the markers studied were not uniformly distributed among the myelin subfractions. The pellet and the layer banding at the 0.75/0.85 M sucrose interface contained the highest specific concentrations of sialic acid, nucleic acid, and total adenosine triphosphatase activity. In contrast, the specific activity of 2′,3′ -cyclicnucleotide-3′-phosphohydrolase was lowest in the pellet as well as the three bands obtained above 0.60 M sucrose and was highest in the fraction banding at the 0.65/0.70 M sucrose interface. The results obtained were not consistent with an artifactual origin of the myelin subfractions, but instead suggested that the subfractions have physiological significance. One explanation for the different banding patterns observed between young and mature myelin may be the different amount of myelin in various brain regions during development.     

Chronic ketamine administration modulates midbrain dopamine system in mice

Ketamine is an anesthetic and a popular abusive drug. As an anesthetic, effects of ketamine on glutamate and GABA transmission have been well documented but little is known about its long-term effects on the dopamine system. In the present study, the effects of ketamine on dopamine were studied in vitro and in vivo. In pheochromocytoma (PC 12) cells and NGF differentiated-PC 12 cells, ketamine decreased the cell viability while increasing dopamine (DA) concentrations in a dose-related manner. However, ketamine did not affect the expression of genes involved in DA synthesis. In the long-term (3 months) ketamine treated mice, significant increases of DA contents were found in the midbrain. Increased DA concentrations were further supported by up-regulation of tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. Activation of midbrain dopaminergic neurons could be related to ketamine modulated cortical-subcortical glutamate connections. Using western blotting, significant increases in BDNF protein levels were found in the midbrain, suggesting that perhaps BDNF pathways in the cortical-subcortical connections might contribute to the long-term ketamine induced TH upregulation. These data suggest that long-term ketamine abuse caused a delayed and persistent upregulation of subcortical DA systems, which may contribute to the altered mental status in ketamine abusers.     

Myelin subfractions isolated from mouse brain. Studies of normal mice during development, quaking mutants, and three brain regions.

Myelin isolated from three areas of mouse brain, from whole brain at several ages in normal mice, and from whole brain of adult quaking mutant mice was separated into seven bands and a pellet on discontinuous density gradients using 0.32, 0.45, 0.55, 0.60, 0.70, 0.75 and 0.85 M sucrose. The distribution of myelin in the subfractions was independent of homogenization and shocking conditions employed to isolate the myelin preparations, but was related to the type of myelin applied to the gradient. Compared to myelin isolated from older animals, myelin isolated from 18-24 day old mice displayed a distribution pattern with greater proportions of material banding at lesser sucrose densities. Similarly, myelin obtained from hindbrain contained proportionately more material layering at lesser sucrose densities compared to myelin isolated from cerebral cortex. Myelin subfraction patterns observed for 8-12 day old control mice and quaking mutants were unlike each other or any other myelin preparation examined. In the 18-90 days old animals, the markers studied were not uniformly distributed among the myelin subfractions. The pellet and the layer banding at the 0.75/0.85 M sucrose interface contained the highest specific concentrations of sialic acid, nucleic acid, and total adenosine triphosphatase activity. In contrast, the specific activity of 2',3'-cyclicnucleotide-3'-phosphohydrolase was lowest in the pellet as well as the three bands obtained above 0.60 M sucrose and was highest in the fraction banding at the 0.65/0.70 M sucrose interface. The results obtained were not consistent with an artifactual origin of the myelin subfractions, but instead suggested that the subfraction have physiological significance. One explanation for the different banding patterns observed between young and mature myelin may be the different amount of myelin in various brain regions during development.     

Moderate G6PD deficiency increases mutation rates in the brain of mice

Mice that harbored the x-ray-induced low efficiency allele of the major X-linked isozyme of glucose-6-phospate dehydrogenase (G6PD), Gpdx(a-m2Neu), and, in addition, harbored the transgenic shuttle vector for the determination of mutagenesis in vivo, pUR288, were employed to further our understanding of the interdependence of general metabolism, oxidative stress control, and somatic mutagenesis. The Gpdx(a-m2Neu) mutation conferred moderate G6PD deficiency in hemizygous males (Gpdx(a-m2Neu/y)) displaying residual enzyme activities of 27% in red blood cells and 13% in brain (compared to wild-type controls, Gpdx(a/y) males). In spite of this mild phenotype, the brains of G6PD-deficient males exhibited a significant distortion of redox control ( approximately 3-fold decrease in the ratio of reduced glutathione to oxidized glutathione), a considerable accumulation of promutagenic etheno DNA adducts ( approximately 13-fold increase in ethenodeoxyadenosine and approximately 5-fold increase in ethenodeoxycytidine), and a substantial elevation of somatic mutation rates ( approximately 3-fold increase in mutant frequencies in lacZ, the target and reporter gene of mutagenesis in the shuttle vector, pUR288). The mutation pattern in the brain was dominated by illegitimate genetic recombinations, a presumed hallmark of oxidative mutagenesis. These findings suggested a critical function for G6PD in limiting oxidative mutagenesis in the mouse brain.     

Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations.

We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one-step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with 'null' mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR-related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement ('null') and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild-type CFTR activity. The long-term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype-phenotype correlations.     

Proteins from sciatic-nerve myelin in quaking and jimpy mice.

Myelin from two neurological mutants in mice was isolated from sciatic nerves and its protein composition analysed. In Quaking mice, two intrinsic myelin proteins P1 and P2 were drastically decreased, whereas the major myelin protein P0 was unaffected. A normal protein composition was found in sciatic myelin from Jimpy mice.     

Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.     

Neuroprotective effects of semax in MPTP-induced disturbances of brain dopamine system

Effects of an ACTH (4-10) analogue Semax (MEHFPGP) on behaviour of white rats with MPTP-induced disturbances of brain DA-system have been studied. It was shown that MPTP administration (25 mg/kg) reduced motor activity and auhmented the anxiety level in rats. Semax administration (daily intranasal 0.2 mg/kg) attenuated behaviour disturbances induced by neurotoxin. The observed protective action of Semax in rats with MFTP-induced DA system disturbances may be due to both its modulating influence on the brain DA system and peptide neuroprotective effects.