Rediscovering TNAP in the Brain: A Major Role in Regulating the Function and Development of the Cerebral Cortex.

The presence of alkaline phosphatase (AP) activity in the neural tissue has been described decades ago. However, only recent studies clarified the isotype, regional distribution and subcellular localization of the AP expressed in the cerebral cortex of diverse mammalian species including the human. In the primate brain the discovery that the bone AP isotype (TNAP) is expressed provided the opportunity of a deeper understanding of the role of this enzyme in neuronal functions based on the knowledge acquired by studying the role of the enzyme in hypophosphatasia, mostly in bone mineralization. TNAP exhibits widespread substrate specificity and, in the brain, it is potentially involved in the regulation of molecules which play fundamental roles in signal transmission and development. In light of these observations, the localization of TNAP in the human cerebral cortex is of high significance when considering that epilepsy is often diagnosed in hypophosphatasia. Here we overview our results on the identification of TNAP in the primate cerebral cortex: TNAP exhibits a noticeably high activity in the synapses and nodes of Ranvier, is specifically present in layer 4 of the sensory cortices and additionally in layer 5 of prefrontal, temporal and other associational areas in human. Our studies also indicate that bone AP activity depends on the level of sensory input and that its developmental time-course exhibits characteristic regional differences. The relevance of our findings regarding human cortical physiology and brain disorders are discussed.

Loss of ß-carotene 15,15'-oxygenase in developing mouse tissues alters esterification of retinol, cholesterol and diacylglycerols.

We provide novel insights into the function(s) of ß-carotene-15,15'-oxygenase (CMOI) during embryogenesis. By performing in vivo and in vitro experiments, we showed that CMOI influences not only lecithin:retinol acyltransferase but also acyl CoA:retinol acyltransferase reaction in the developing tissues at mid-gestation. In addition, LC/MS lipidomics analysis of the CMOI-/- embryos showed reduced levels of four phosphatidylcholine and three phosphatidylethanolamine acyl chain species, and of eight triacylglycerol species with four or more unsaturations and fifty-two or more carbons in the acyl chains. Cholesteryl esters of arachidonate, palmitate, linoleate, and DHA were also reduced to less than 30% of control. Analysis of the fatty acyl CoA species ruled out a loss in fatty acyl CoA synthetase capability. Comparison of acyl species suggested significantly decreased 18:2, 18:3, 20:1, 20:4, or 22:6 acyl chains within the above lipids in CMOI-null embryos. Furthermore, LCAT, ACAT1 and DGAT2 mRNA levels were also downregulated in CMOI-/- embryos. These data strongly support the notion that, in addition to cleaving ß-carotene to generate retinoids, CMOI serves an additional function(s) in retinoid and lipid metabolism and point to its role in the formation of specific lipids, possibly for use in nervous system tissue.

Synergistic effects of bone mesenchymal stem cells and chondroitinase ABC on nerve regeneration after acellular nerve allograft in rats.

This study aimed to evaluate whether combination therapy of bone marrow stromal cells (BMSCs) transplantation and chondroitinase ABC (ChABC) treatment further enhances axonal regeneration and functional recovery after acellular nerve allograft repair of the sciatic nerve gap in rats. Eight Sprague-Dawley rats were used as nerve donors, and 32 Wistar rats were randomly divided into four groups: Group I: acellular rat sciatic nerve (ARSN) group; Group II: ChABC treatment; Group III: BMSCs transplantation; and Group IV: ChABC treatment and BMSCs transplantation. The results showed that compared with ARSN control group, BMSC transplantation promoted axonal regeneration, the secretion of neural trophic factors NGF, BDNF and axon angiogenesis in nerve graft. ChABC treatment degraded chondroitin sulfate proteoglycans in ARSN in vitro and in vivo and improved BMSCs survival in ARSN. The combination therapy caused much better beneficial effects evidenced by increasing sciatic function index, nerve conduction velocity, restoration rate of tibialis anterior wet muscle weight, and myelinated nerve number, but did not further boost the therapeutic effects on neurotrophic factor production, axon angiogenesis, and sensory functional recovery by BMSC transplantation. Taken together, for the first time, we demonstrate the synergistic effects of BMSC transplantation and BMSCs treatment on peripheral nerve regeneration, and our findings may help establish novel strategies for cell transplantation therapy for peripheral nerve injury.

[Cholinesterase activity in tissues of some crab species from the Japan Sea].

The comparative study of the cholinesterase activity in some crab species was carried out for the first time with use of a set of thiocholine substrates. The substrate specificity was studied in stellar nerve, heart, and hemolymph of three crab species. The crab hemolymph was shown to be characterized by the highest enzyme activity. The enzyme from various crab organs has different structure o substrate specificity. Properties of crab enzymes was compared with acetylcholinesterase (AChE) of human blood erythrocytes, butyrylcholinesterase (BuChE) of horse blood serum, enzyme o squids and bivalve molluscs. The obtained data allow the conclusion to be made on differences in properties of enzymes both at the interspecies and at the tissue levels.

[Localization of acetylcholinesterase in the nervous system of Cotylophoron indicum].

The nervous system of Cotylophoron indicum was studied by using acetylcholine esterase histochemical staining techniques. Cranial ganglia and transverse commissure situate at dorso-lateral body between oral sucker and genital sucker. From the cranial ganglia four pairs of nerves proceed cephalad and connect with nerve network of the oral sucker. The posterior nerve cords from the cranial ganglia consist of 3 pairs and the ventral ones are the stoutest and longest nerves. A few branches from the 3 pairs of nerve cords connect to ventral sucker. There is a developed nerve network distributed in its genital sucker. The nerve fibers on body surface in pairs and parallel are diagonal and cross to form a nerve network on body surface. Three kinds of neurocytes distribute at the prosomal region. Results show that the nervous system structure of C. indicum is consistent with the essential features of Digenea, but more special and complicated around genital sucker.

Adenosine triphosphatase activity in the membranes of the squid nerve fiber.

This investigation deals with the localization of sites of ATPase activity, especially of transport ATPase, in nerve fibers of the squid Doryteuthis plei, at the subcellular level. Splitting of ATP liberates inorganic phosphate which reacts with lead to form a precipitate in the tissue. The reaction was made on nerve fibers fixed with glutaraldehyde. Frozen slices were incubated in Wachstein-Meisel medium containing ATP and Pb(NO(3))(2). Deposits of reaction product were found in the axolemma (towards its axoplasmic side), Schwann cell membranes (mainly at the channels crossing the layer), and mitochondria. Control experiments revealed that no deposits were observed in nerve fibers fixed in osmium tetroxide prior to incubation in the medium containing ATP, or in nerve fibers incubated without substrate or with adenosine monophosphate, adenosine diphosphate, glycerophosphate, or guanosine triphosphate as substrate. For evaluation of transport ATPase activity, these findings were compared with results obtained with nerve fibers treated with G-strophanthin or K-strophanthoside before or after glutaraldehyde fixation. The cardiac glycosides produced a disappearance or diminution of the deposits. The largest inhibitory effect was observed in the axolemma. The findings indicate that the highest ATPase activity is localized in the axolemma and may be due primarily to transport ATPase.

Diisopropylphosphorofluoridate and Tabun: enzymatic hydrolysis and nerve function.

Squid nerve contains an enzyme that hydrolyzes the nerve gas Tabun at about one-tenth the rate it hydrolyzes diisopropylphosphorofluoridate (DFP), and at about one-third to one-fourth the rate it hydrolyzes Sarin and Soman. Tabun is a more potent inhibitor of acetylcholinesterase than is DFP, is both lipid-and water-soluble, and penetrates readily into the squid giant axon in its inhibitory form. The failure of Tabun to block or markedly decrease the conducted action potential in the squid axon makes it likely that the blocking of conduction caused by DFP is probably not due to inhibition of acetylcholinesterase. Sub-strate specificity with regard to organophosphate metabolism by squid enzyme has possible implications for the disposal and detoxication of nerve gases in the ocean.

Kinetics of enzyme inhibition by active molluscicidal agents ferulic acid, umbelliferone, eugenol and limonene in the nervous tissue of snail Lymnaea acuminata.

Ferulic acid, umbelliferone (Ferula asafoetida), eugenol (Syzygium aromaticum) and limonene (Carum carvi) are active molluscicidal components that inhibited the activity of alkaline phosphatase and acetylcholinesterase in in vivo and in vitro exposure of Lymnaea acuminata. It was observed that ferulic acid, umbelliferone and eugenol are competitive and limonene is a competitive-non-competitive inhibitor of alkaline phosphatase. Ferulic acid and umbelliferone are competitive, whereas eugenol and limonene are competitive-non-competitive and uncompetitive inhibitors of acetylcholinesterase, respectively.

Expression patterns of protein kinase D 3 during mouse development.

BACKGROUND: The PKD family of serine/threonine kinases comprises a single member in Drosophila (dPKD), two isoforms in C. elegans (DKF-1 and 2) and three members, PKD1, PKD2 and PKD3 in mammals. PKD1 and PKD2 have been the focus of most studies up to date, which implicate these enzymes in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, immune responses, apoptosis and cell proliferation. Concerning PKD3, a role in the formation of vesicular transport carriers at the trans-Golgi network (TGN) and in basal glucose transport has been inferred from in vitro studies. So far, however, the physiological functions of the kinase during development remain unknown. RESULTS: We have examined the expression pattern of PKD3 during the development of mouse embryos by immunohistochemistry. Using a PKD3 specific antibody we demonstrate that the kinase is differentially expressed during organogenesis. In the developing heart a strong PKD3 expression is constantly detected from E10 to E16.5. From E12.5 on PKD3 is increasingly expressed in neuronal as well as in the supporting connective tissue and in skeletal muscles. CONCLUSION: The data presented support an important role for PKD3 during development of these tissues.

Roles of steroid sulfatase in brain and other tissues.

Steroid sulfatase (EC 3.1.6.2) is an important enzyme involved in steroid hormone metabolism. It catalyzes the hydrolysis of steroid sulfates into their unconjugated forms. This action rapidly changes their physiological and biochemical properties, especially in brain and neural tissue. As a result, any imbalance in steroid sulfatase activity may remarkably influence physiological levels of active steroid hormones with serious consequences. Despite that the structure of the enzyme has been completely resolved there is still not enough information about the regulation of its expression and action in various tissues. In the past few years research into the enzyme properties and regulations has been strongly driven by the discovery of its putative role in the indirect stimulation of the growth of hormone-dependent tumors of the breast and prostate.

Manduca sexta hemolymph protease-2 (HP2) activated by HP14 generates prophenoloxidase-activating protease-2 (PAP2) in wandering larvae and pupae.

Melanization is a universal defense mechanism of insects against microbial infection. During this response, phenoloxidase (PO) is activated from its precursor by prophenoloxidase activating protease (PAP), the terminal enzyme of a serine protease (SP) cascade. In the tobacco hornworm Manduca sexta, hemolymph protease-14 (HP14) is autoactivated from proHP14 to initiate the protease cascade after host proteins recognize invading pathogens. HP14, HP21, proHP1*, HP6, HP8, PAP1-3, and non-catalytic serine protease homologs (SPH1 and SPH2) constitute a portion of the extracellular SP-SPH system to mediate melanization and other immune responses. Here we report the expression, purification, and functional characterization of M. sexta HP2. The HP2 precursor is synthesized in hemocytes, fat body, integument, nerve and trachea. Its mRNA level is low in fat body of 5th instar larvae before wandering stage; abundance of the protein in hemolymph displays a similar pattern. HP2 exists as an active enzyme in plasma of the wandering larvae and pupae in the absence of an infection. HP14 cleaves proHP2 to yield active HP2. After incubating active HP2 with larval hemolymph, we detected higher levels of PO activity, i.e. an enhancement of proPO activation. HP2 cleaved proPAP2 (but not proPAP3 or proPAP1) to yield active PAP2, responsible for a major increase in IEARpNA hydrolysis. PAP2 activates proPOs in the presence of a cofactor of SPH1 and SPH2. In summary, we have identified a new member of the proPO activation system and reconstituted a pathway of HP14-HP2-PAP2-PO. Since high levels of HP2 mRNA were present in integument and active HP2 in plasma of wandering larvae, HP2 likely plays a role in cuticle melanization during pupation and protects host from microbial infection in a soil environment.

Modulating longevity in Drosophila by over- and underexpression of glutamate-cysteine ligase.

A notable extension of life span (up to 50%) was achieved in Drosophila melanogaster when the catalytic subunit of glutamate-cysteine ligase (GCLc) was overexpressed in neuronal tissue, while a moderate increase (up to 24%) was observed when the modulatory subunit of GCL (GCLm) was overexpressed globally. We sought to identify specific tissue domains that are particularly sensitive to the beneficial effects of GCLc overexpression. Overexpression of GCLc using the mushroom body driver (OK107-GAL4) had a small but significant beneficial effect on longevity (approximately 12%) while overexpression in serotonergic (MZ360-GAL4) neurons or dopaminergic and serotonergic neurons (Ddc-GAL4) had small, nonsignificant effects on longevity. A significant beneficial effect (12-13%) was also observed using the C23-GAL4 transverse muscle driver. Finally, a low-level global driver (armadillo) was shown to increase life span significantly (15%). A series of mutant and knockdown studies were also carried out. Reduction of GCLm by > 95% had no discernable effect on longevity or resistance to oxidative stress. In contrast, knockdown of GCLc by 30-70% using an RNAi-hairpin strategy had a significant effect, resulting in greater sensitivity to H(2)O(2) and reduced survivorship under normal conditions varying from a 50% reduction in median life span to lethality. A GCLc null allele was identified and shown to be recessive lethal. Overall, this study demonstrates that the longevity effects of GCLc are dependent on dosage and that there are specific tissues (mushroom bodies, motor neurons, and transverse muscle cells) particularly sensitive to the benefits of GCLc overexpression.

Doñana National Park survey using crayfish (Procambarus clarkii) as bioindicator: esterase inhibition and pollutant levels.

Utility of carboxylesterase and acetylcholinesterase inhibition as pesticide exposure biomarker was studied at Doñana National Park (SW Spain) in crayfish (Procambarus clarkii). Activities were measured in animals from reference sites or potentially exposed to pesticides, and their reactivation studied after dilution or 2-PAM treatment. Crayfish from affected sites had significantly less carboxylesterase and acetylcholinesterase activity than reference ones. No significant differences were found after dilution or 2-PAM treatment, showing that inhibition was irreversible. High pesticide levels were found in water and/or soil at rice growing sites, and lower levels at other affected places. High metal levels existed at rice growing sites and lower at other affected and at both reference sites. A combined effect on esterase inhibition of pesticides and metals is proposed. This field study suggest that the rice growing areas near Guadiamar stream are most polluted, followed by strawberry and citrics growing zones near Partido and Rocina streams. However, no correlation exist between the pesticide concentration at different sites and the extent of esterase inhibition, indicating that other factors could affect esterase response of animals from polluted sites.

Separate development of nitric oxide synthase- and vasoactive intestinal polypeptide-immunoreactive nerves arising from the vertebral artery in the rat.

Development of nitric oxide synthase (NOS)-and vasoactive intestinal polypeptide (VIP)-immunoreactive (-IR) nerves supplying the basilar and vertebral arteries (BA and VA) was investigated in White Wistar rats, using double immunohistochemistry. NOS-IR and VIP-IR nerves via the anterior circulation (AC), which mostly expressed NO(+)/VIP(+), extended to the BA during the second postnatal week, and usually reached as far as the rostral two third of the BA on PND 20. NOS-IR nerves were completely lack in the cBA and the VA on PND10, and often absent from these arterial regions even at PND 20. Nevertheless, a small number of VIP(+)/NOS(-) nerves were localized in the walls from the caudal BA (cBA) to the VA on PND 5. On PND 20, they frequently met with the descending NOS-IR and VIP-IR nerves via the AC around the lower portion of the middle BA. Fiber bundles containing NOS(+)/VIP(+) axons were first visualized on the caudal VA at PND 30 and observed frequently at PND 80, with a distinct increase in number of NOS-IR and VIP-IR nerves supplying the cBA and the VA. Thus, NOS-IR nerves coming from the VA develop through its own characteristic sequence that lags markedly behind the time of appearance for VIP-IR nerves from the same vascular route and for NOS-IR and VIP-IR nerves via the AC.

Galactosyltransferase activities during embryonic development of chick neural tissue.

Galactosyltransferase specific activities in embryonic chick retina, optic tectum, and telencephalon were found to decline during embryonic development. Incorporation of galactose from nucleotide sugar into exogenously added glycoprotein acceptor was measured in the presence of excess of glycoprotein acceptor. This ensured that the specific activity measurements were reflections of tru enzyme specific activity rather than availability of acceptor. Moreover, we have shown that the decline in specific activity is not due to degradation of the nucleotide sugar, UDP-galactose, under our in vitro assay conditions. Enzymatic specific activity declined sharply with embryonic age for all tissues tested. This decline was not affected by the presence of 5-bromodeoxyuridine during in vitro culture of embryonic chick neural retina above that caused by the culturing alone. Galactosyltransferase activity was not found to be associated with the plasma membrane fraction from homogenized tissue but rather with the microsomal fraction. Thus, the changes in galactosyltransferase specific activity detected here do not reflect changes at the cell surface.

Metabolism of acetylcholine in the nervous system of Aplysia californica. II. Reginal localization and characterization of choline uptake.

The choline required for synthesis of acetylcholine is derived exogenously by Aplysia ganglia. Under physiological conditions choline was taken up primarlily by neuropile and nerves and not by cholinergic cell bodies. In addition, compared with their contents of choline acetyltransferase, those components of nervous tissue which contain nerve terminals and axons synthesized acetylcholine far more efficiently. Choline was accumulated by high and low affinity uptake processes; the high affinity process appeared to be characteristic of cholinergic nuerons (Swartz, J. H., M. L. Eisenstadt, and H. Cedar.1975. J. Gen. Physiol. 65:255). The two uptake processes were similarly affected by temperature with a Q10 of 2.8. Both were dependent on a variety of ions in a complicated manner. High affinity uptake seemed to be more dependent on Na+, showed greater inhibition by ouabain, and was selectively inhibited by oxotremorine. We found that the functional state of neurons did not alter uptake of radioactive choline by either process, nor did it change the conversion to radioactive acetylcholine.

Neuronal function and alpha3 isoform of the Na/K-ATPase.

The Na/K-ATPase is a complex of integral membrane proteins that carries out active transport of sodium and potassium across the cell plasma membrane, and maintains chemical gradients of these ions. The alpha subunit of the Na/K-ATPase has several isoforms that are expressed in a cell type- and tissue-dependent manner. In adult vertebrates, while kidney cells express mostly alpha1, muscle and glial cells -- alpha1 and alpha2, and sperm cells -- alpha1 and alpha4 isoforms of Na/K-ATPase, neurons may express alpha1, alpha2, alpha3 or any combination of these isoforms, and evidence suggests that neuronal type is the determining factor. The functional significance of multiple isoforms of the Na/K-ATPase and their non-uniform expression, and the link between neuron function and expression of a given isoform of the Na/K-ATPase in particular, remains unknown. Several hypotheses on this account were introduced, and in this work we will review the present status of these hypotheses, and their standing in application to recent data on the expression of isoforms of the Na/K-ATPase in the peripheral nervous system of vertebrate animals.

Dynamic expression of a glutamate decarboxylase gene in multiple non-neural tissues during mouse development.

BACKGROUND: Glutamate decarboxylase (GAD) is the biosynthetic enzyme for the neurotransmitter gamma-aminobutyric acid (GABA). Mouse embryos lacking the 67-kDa isoform of GAD (encoded by the Gad1 gene) develop a complete cleft of the secondary palate. This phenotype suggests that this gene may be involved in the normal development of tissues outside of the CNS. Although Gad1 expression in adult non-CNS tissues has been noted previously, no systematic analysis of its embryonic expression outside of the nervous system has been performed. The objective of this study was to define additional structures outside of the central nervous system that express Gad1, indicating those structures that may require its function for normal development. RESULTS: Our analysis detected the localized expression of Gad1 transcripts in several developing tissues in the mouse embryo from E9.0-E14.5. Tissues expressing Gad1 included the tail bud mesenchyme, the pharyngeal pouches and arches, the ectodermal placodes of the developing vibrissae, and the apical ectodermal ridge (AER), mesenchyme and ectoderm of the limb buds. CONCLUSIONS: Some of the sites of Gad1 expression are tissues that emit signals required for patterning and differentiation (AER, vibrissal placodes). Other sites correspond to proliferating stem cell populations that give rise to multiple differentiated tissues (tail bud mesenchyme, pharyngeal endoderm and mesenchyme). The dynamic expression of Gad1 in such tissues suggests a wider role for GABA signaling in development than was previously appreciated.

Immunodetection of Na,K-ATPase alpha 3-isoform in renal and nerve tissues.

At least three types of mRNA of the catalytic subunit of Na,K-ATPase namely alpha-,alpha+- and alpha 3-isoforms are identified in different tissues. Only two of them alpha and alpha+ have well known structural and catalytic properties. Here we present immunochemical data indicating that the alpha 3 protein really exists in pig and human kidney, and human brain. Crude membrane fractions and purified membrane-bound Na,K-ATPases were immunoblotted with alpha 3-specific antibodies raised against the synthetic peptide corresponding to the unique sequence of this isoform. The mature alpha 3-subunit is shown to include the sequence GDKKDDKSSPK followed by the initiating methionine residue. Nephron collecting tubules are proposed to specifically contain Na,K-ATPase alpha 3-isoform.

Expression of glutamic acid decarboxylase in nervous tissue structures targeted by autoantibodies in patients with diabetic autonomic neuropathy.

We have previously identified an association between symptomatic diabetic autonomic neuropathy (DAN) and autoantibodies to sympathetic and parasympathetic nervous structures. The antigens identified by these autoantibodies are not known, but glutamic acid decarboxylase (GAD) has been suggested as a candidate target, since anti-GAD autoantibodies are present in patients with long-term diabetes and GAD is expressed in a variety of cell types and structures in the nervous system. The aim of this study was to examine GAD expression in sympathetic ganglia and vagus nerve and to compare the distribution of GAD within these tissues with that of anti-sympathetic ganglia and anti-vagus nerve autoantibodies from patients with DAN, using single and double indirect immunofluorescence on tissue sections. The monoclonal antibody GAD-6, specific for GAD65, gave a granular, peripheral, cytoplasmic staining pattern in sympathetic ganglion cells. Dual immunofluorescence demonstrated that serum from a patient with anti-sympathetic ganglion autoantibodies stained the same cells, but homogeneously throughout the cytoplasm. In the vagus nerve, patient's serum stained the fibres only; GAD-6 stained the cytoplasm of parasympathetic ganglion cells but only occasional fibres. In addition, GAD enzymatic activity was detectable in both sympathetic ganglia and vagus nerve. Incubation of sera or GAD-6 overnight with a crude homogenate of human brain as an antigen source abolished staining of the nervous tissues by GAD-6, but not by patients' sera. The different localisation of GAD and the autoantigens targeted by patients' sera indicates that GAD is not the target of the autoantibodies characteristic of DAN. Moreover, absorption studies using human brain homogenate suggest that the targets of anti-sympathetic ganglion and anti-vagus nerve autoantibodies are absent or represented only at low levels in the central nervous system and may be confined to the periphery.