Characterization of mouse glandular kallikrein 24 expressed in testicular Leydig cells

Mouse kallikrein 24 is thought to encode a functional serine protease belonging to the mouse glandular kallikrein gene family. Preliminary results suggest that this kallikrein may play a role in testis function in adult mice. In order to obtain insights into its physiological functions, we undertook molecular and biochemical analyses of this enzyme. We cloned a cDNA for kallikrein 24 from the adult mouse testis cDNA library. Kallikrein 24 was expressed in the kidney, submandibular glands, ovary, epididymis, and testis of the mouse. In the testis, kallikrein 24 mRNA was detectable at 4 weeks of postnatal development, and became more prominent thereafter. The kallikrein 24 gene was expressed exclusively in the Leydig cells of adult mice. When Leydig cells isolated from a 2-week-old mouse testis were cultured in the presence of testosterone, kallikrein 24 expression was induced. Active recombinant enzyme showed trypsin-like specificity, favorably cleaving Arg-X bonds of synthetic peptide substrates. The enzymatic activity was strongly inhibited by typical serine protease inhibitors. Mouse kallikrein 24 degraded casein, gelatin, fibronectin and laminin. These results suggest that the enzyme may play a role in the degradation of extracellular matrix proteins in the interstitial area surrounding the Leydig cells of the adult mouse testis. The present findings should contribute to future physiological studies of this mouse testis protease.     

Recognition of Bergmann glial and ependymal cells in the mouse nervous system by monoclonal antibody

A monoclonal antibody designated anti-Cl was obtained from a hybridoma clone isolated from a fusion of NS1 myeloma with spleen cells from BALB/c mice injected with homogenate of white matter from bovine corpus callosum. In the adult mouse neuroectoderm, C1 antigen is detectable by indirect immunohistology in the processes of Bergmann glial cells (also called Golgi epithelial cells) in the cerebellum and of Muller cells in the retina, whereas other astrocytes that express glial fibrillary acidic protein in these brain areas are negative for C1. In addition, C1 antigen is expressed in most, if not all, ependymal cells and in large blood vessels, but not capillaries. In the developing, early postnatal cerebellum, C1 antigen is not confined to Bergmann glial and ependymal cells but is additionally present in astrocytes of presumptive white matter and Purkinje cell layer. In the embryonic neuroectoderm, C1 antigen is already expressed at day 10, the earliest stage tested so far. The antigen is distinguished in radially oriented structures in telencephalon, pons, pituitary anlage, and retina. Ventricular cells are not labeled by C1 antibody at this stage. C1 antigen is not detectable in astrocytes of adult or nearly adult cerebella from the neurological mutant mice staggerer, reeler, and weaver, but is present in ependymal cells and large blood vessels. C1 antigen is expressed not only in the intact animal but also in cultured cerebellar astrocytes and fibroblastlike cells. It is localized intracellularly.     

Expression of fatty acid-CoA ligase 4 during development and in brain

Fatty acid utilization is initiated by fatty acid-CoA ligase, which converts free fatty acids into fatty acyl-CoA esters. We have cloned previously the human long-chain fatty acid-CoA ligase 4 (FACL4), which is a central enzyme in controlling the free arachidonic acid level in cells and thereby regulating eicosanoid production. We report here the expression of this gene in tissues, particularly in different parts of the brain. We found that FACL4 encoded a 75 kDa enzyme and that there was a modified translation product expressed in the brain. FACL4 was expressed in early stages of development with a significant amount of FACL4 mRNA detected in an E7 mouse embryo. In addition, FACL4 was highly expressed in both adult and newborn mouse brain especially in the granule cells of the dentate gyrus and the pyramidal cell layer of CA1 in hippocampus, and the granular cell layer and Purkinje cells of the cerebellum.     

The cloning and characterization of a second brain enzyme with NAAG peptidase activity

The peptide neurotransmitter N-acetylaspartylglutamate is inactivated by extracellular peptidase activity following synaptic release. It is speculated that the enzyme, glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse enzyme (tentatively identified as glutamate carboxypeptidase III or GCPIII) that is homologous to an enzyme identified in a human lung carcinoma. The mouse peptidase was cloned from two non-overlapping EST clones and mouse brain cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild-type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound peptidase activity with similar V(max) and K(m) values (1.4 micro m and 54 pmol/min/mg; 3.5 micro m and 71 pmol/min/mg, respectively). Both enzymes are activated by a similar profile of metal ions and their activities are blocked by EDTA. GCPIII message was detected in brain and spinal cord by RT-PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous enzymes with similar pharmacological properties and affinity for NAAG.     

Characterization of central nervous system structures by magnetic resonance diffusion anisotropy

Diffusion-weighted magnetic resonance imaging (MRI) provides information about tissue water diffusion. Diffusion anisotropy, which can be measured with diffusion tensor MRI, is a quantitative measure of the directional dependence of the diffusion restriction that is introduced by biological structures such as nerve fibers. Diffusion tensor MRI data was obtained in the brain, brain stem, and cervical spinal cord. For each region, scans were performed in four normal volunteers. Fractional anisotropy (FA), an index of diffusion anisotropy, was measured within regions of interest located in the corpus callosum, capsula interna, thalamus, caudate nucleus, putamen, brain cortex, pyramidal tract of the medulla, accessory olivary nucleus, dorsal olivary nucleus, inferior olivary nucleus, spinal white and gray matter. The highest FA value was measured in the corpus callosum (81 +/- 3%). The values of the other areas decreased in the following order: pyramidal tract in the medulla (72 +/- 1%), spinal white matter (65 +/- 4%), capsula interna (62 +/- 3%), accessory olivary nucleus (36 +/- 2%), spinal gray matter (35 +/- 5%), dorsal olivary nucleus in the medulla (29 +/- 2%), thalamus (28 +/- 2%), inferior olivary nucleus (15 +/- 2%), putamen (13 +/- 2%), caudate nucleus (13 +/- 2%), and brain cortex (9 +/- 1%). Our results indicate that the underlying fiber architecture, fiber density, and uniformity of nerve fiber direction affect anisotropy values of the various structures. Characterization of various central nervous system structures with diffusion anisotropy is possible and may be useful to monitor degenerative diseases in the central nervous system.     

Distinct spatio-temporal expression of ABCA and ABCG transporters in the developing and adult mouse brain

Using in situ hybridization for the mouse brain, we analyzed developmental changes in gene expression for the ATP-binding cassette (ABC) transporter subfamilies ABCA1-4 and 7, and ABCG1, 2, 4, 5 and 8. In the embryonic brains, ABCA1 and A7 were highly expressed in the ventricular (or germinal) zone, whereas ABCA2, A3 and G4 were enriched in the mantle (or differentiating) zone. At the postnatal stages, ABCA1 was detected in both the gray and white matter and in the choroid plexus. On the other hand, ABCA2, A3 and A7 were distributed in the gray matter. In addition, marked up-regulation of ABCA2 occurred in the white matter at 14 days-of-age when various myelin protein genes are known to be up-regulated. In marked contrast, ABCA4 was selective to the choroid plexus throughout development. ABCG1 was expressed in both the gray and white matters, whereas ABCG4 was confined to the gray matter. ABCG2 was diffusely and weakly detected throughout the brain at all stages examined. Immunohistochemistry of ABCG2 showed its preferential expression on the luminal membrane of brain capillaries. Expression signals for ABCG5 and G8 were barely detected at any stages. The distinct spatio-temporal expressions of individual ABCA and G transporters may reflect their distinct cellular expressions in the developing and adult brains, presumably, to regulate and maintain lipid homeostasis in the brain.     

A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene

To search for genes involved in determining the morphology of individual neuronal types, a cDNA library was constructed from postnatal day 13 mouse cerebellum. From this library, 2 clones, L7 and L19, were isolated by a differential hybridization procedure and shown by in situ hybridization to be Purkinje cell-specific within the cerebellum. Both RNAs appear between postnatal days 4 and 8 and continue into adulthood, coinciding with terminal differentiation of the Purkinje cells. L7 seems to be expressed exclusively in the cerebellum, whereas L19 is expressed throughout the brain. Consistent with the RNA localization, L7 protein is found only in the cerebellum and is confined to the Purkinje cells. The L7 amino acid sequence has been deduced from the cDNA sequence, and a pseudo-repeat within the L7 protein sequence is homologous to the amino acids sequence in the primary translation product of the gene for human sis/PDGF.     

cDNA cloning of putative rat acetyl-CoA transporter and its expression pattern in brain

Rat acetyl-CoA transporter gene (Acatn) encodes a hydrophobic multi-transmembrane protein involved in the O-acetylation of gangliosides. O-acetylated gangliosides have been found to play important roles in the embryonic development of the nervous system. We have isolated rat Acatn cDNA by PCR cloning. The amino acid sequence of rat Acatn exhibited 92% and 96% homology with human and mouse sequences, respectively. The mRNA was expressed in brain at all developmental stages. Acatn expression was higher in embryonic and postnatal rats than in adult rats. Cellular localization of Acatn mRNA in adult rat brain was also analyzed by in situ hybridization. Acatn mRNA expression was detected in the neuronal cells of cerebellum, hippocampus, hypothalamus, cortex, olfactory bulb, and dorsal and ventral anterior olfactory nucleus in adult rat brain.     

Cloning, genomic organization, chromosomal assignment and expression of a novel mosaic serine proteinase: epitheliasin

We report the isolation of a cDNA encoding a novel murine serine proteinase, epitheliasin. The cDNA spans 1753 bp and encodes a mosaic protein with a calculated molecular mass of 53529 Da. Its domains include a cytoplasmic tail, a type II transmembrane domain, a low-density lipoprotein receptor class A domain, a cysteine rich scavenger receptor-like domain and a serine proteinase domain. The proteinase portion domain shows 46-53% identity with mouse neurotrypsin, acrosin, hepsin and enteropeptidase. The gene, located in the telomeric region in the long arm of mouse chromosome 16, consists of 14 exons and 13 introns and spans approximately 18 kb. Epitheliasin is expressed primarily in the apical surfaces of renal tubular and airway epithelial cells.     

Regional distribution of adenosine deaminase in the human neuraxis

Adenosine deaminase was determined in 28 different areas of the human neuraxis in 5 adult male cadavers, with no known disease of the nervous system, using a very sensitive colorimetric method. The enzyme was highest in the frontal lobe white matter, and lowest in the medulla and all levels of the spinal cord. Enzyme content was about twice as great in the white matter of the frontal and temporal lobes and cerebellum as it was in the cortical gray matter of these areas, but only slightly higher in the white matter of the parietal and occipital lobes as compared to gray. Average values of the enzyme were found in the remaining areas of the brain, with the exception of the pons and cerebellar white matter, where a higher than average value was noted.     

Expression of the Oligodendrocyte-Myelin Glycoprotein by Neurons in the Mouse Central Nervous System

Abstract: The oligodendrocyte-myelin glycoprotein (OMgp) is a 110-kDa glycosylphosphatidylinositol-linked protein that was initially identified as a myelin-specific protein but whose precise function remains unknown. In this study, immunohistochemistry, western blots, in situ hybridization, and northern blots were used to determine the distribution of OMgp in the mouse brain. OMgp is present in a concentration detectable on western blots in the brains of newborn mice, and its concentration gradually increases until day 24 of life. OMgp mRNA is also present in amounts detectable on northern blots in the brains of newborn mice, and its concentration gradually increases until day 21 of life, after which the concentration diminishes a little. Most of the OMgp in the mouse brain appears to be expressed in diverse groups of neurons, but it is particularly prominent in large projection neurons such as the pyramidal cells of the hippocampus, the Purkinje cells of the cerebellum, motoneurons in the brainstem, and anterior horn cells of the spinal cord. However, OMgp is not confined to these cells and is expressed in cells in the white matter as well. The OMgp gene is placed within an intron of the neurofibromatosis type I gene and on the opposite strand. This organization raises the possibility that there may be a relationship between the functions of the products of the two genes. In support of this possibility, we show that within the mouse CNS OMgp and neurofibromin are expressed in the same cell types.     

Aspartoacylase-lacZ knockin mice: an engineered model of Canavan disease

Canavan Disease (CD) is a recessive leukodystrophy caused by loss of function mutations in the gene encoding aspartoacylase (ASPA), an oligodendrocyte-enriched enzyme that hydrolyses N-acetylaspartate (NAA) to acetate and aspartate. The neurological phenotypes of different rodent models of CD vary considerably. Here we report on a novel targeted aspa mouse mutant expressing the bacterial β-Galactosidase (lacZ) gene under the control of the aspa regulatory elements. X-Gal staining in known ASPA expression domains confirms the integrity of the modified locus in heterozygous aspa lacZ-knockin (aspa(lacZ/+)) mice. In addition, abundant ASPA expression was detected in Schwann cells. Homozygous (aspa(lacZ/lacZ)) mutants are ASPA-deficient, show CD-like histopathology and moderate neurological impairment with behavioural deficits that are more pronounced in aspa(lacZ/lacZ) males than females. Non-invasive ultrahigh field proton magnetic resonance spectroscopy revealed increased levels of NAA, myo-inositol and taurine in the aspa(lacZ/lacZ) brain. Spongy degeneration was prominent in hippocampus, thalamus, brain stem, and cerebellum, whereas white matter of optic nerve and corpus callosum was spared. Intracellular vacuolisation in astrocytes coincides with axonal swellings in cerebellum and brain stem of aspa(lacZ/lacZ) mutants indicating that astroglia may act as an osmolyte buffer in the aspa-deficient CNS. In summary, the aspa(lacZ) mouse is an accurate model of CD and an important tool to identify novel aspects of its complex pathology.     

Topographical Atlas of the Gangliosides of the Adult Human Brain

Forty different brain samples, consisting of neocortical, archicortical, and paleocortical areas; telencephalic, diencephalic, and mesencephalic subcortical nuclei; and the cerebellum as well as some of the corresponding white matter bundles were analyzed with respect to total content of ganglioside-sialic acid and the ganglioside pattern. The total content of gangliosides seems to depend mainly on the proportions of gray and white matter. Thus, neocortical areas, which are rich in gray matter, have a four- to fivefold higher ganglioside content (per milligram of protein) than white matter-rich samples such as optic chiasm, capsula interna, or corpus callosum. White matter-rich regions, although very heterogeneous in ganglioside composition, are further characterized by appreciable amounts of the myelin-enriched GM4. In the neocortex a remarkable degree of regional pattern differences was revealed. In the frontal and parietal areas there is a moderate, and in the temporal region a strong preponderance of sialic acid bound to gangliosides of the a-pathway (GD1a, GM1). In contrast, the occipital cortex favors the b-pathway of ganglioside synthesis (GQ1b, GT1b, GD1b). A predominance of "b-gangliosides" was found in all structures that are related to the visual system (optic chiasm, pulvinar-thalamus, superior colliculi, visual cortex) as well as in the cerebellum and the nucleus ruber. All diencephalic nuclei tend to favor slightly "b-gangliosides," while the mesencephalic nuclei are very heterogeneous in their ganglioside composition. A preponderance of "a-gangliosides" was found in the periamygdalar cortex, putamen, inferior colliculi, substantia nigra, frontal white matter, internal capsule, globus pallidus, basal nucleus of Meynert, and corpus callosum as well as in the frontal, parietal, and temporal cortices. An exceptional predominance of GM1 and GD1a was revealed for the hippocampal archicortex and the amygdala, suggesting a possible functional correlation to glutaminergic synaptic transmission.