Influence of lβ-, dα- and dβ-Asp isomers of the Asp-76 residue on the properties of αA-crystallin 70–88 peptide

Proteins have been considered to consist exclusively of l-amino acids in living tissues. However, our previous studies showed that two specific aspartyl (Asp) residues in αA- and αB-crystallins from human eye lenses invert to the d-isomers to a high degree during aging. The reaction is also accompanied by isomerization into a form containing β-Asp (isoaspartate) residues. The appearance of d- and β-Asp in a protein potentially induces large changes to the higher order structure of the protein as well as to its function. However, it remains unclear whether the formation of the Asp isomer is the direct trigger of the change to the higher order structure and function. In this study, in order to clarify the effect of the inversion to d-isomers in a protein, we synthesized peptides corresponding to the 70–88 (KFVIFLDVKHFSPEDLTVK) fragment of human αA-crystallin and its corresponding diastereoisomers in which lα-Asp was replaced with lβ-Asp, dα-Asp, and dβ-Asp at position 76 and compared their biochemical properties with that of normal peptide. The peptides containing abnormal isomers (lβ-Asp, dα-Asp, and dβ-Asp residues, respectively) were more hydrophilic than the normal peptide (containing lα-Asp), lost β-sheet structure and changed to random structures. The normal peptide promoted the aggregation of insulin while the other three isomers suppressed the aggregation of insulin. This is the first evidence that a single substitution of an Asp isomer in a peptide induces a large change to the properties of the peptide.     

<Emphasis Type="SmallCaps">d</Emphasis>-Aspartate affects secretory activity in rat Harderian gland: molecular mechanism and functional significance

In this paper, the role of d-aspartate in the rat Harderian gland (HG) was investigated by histochemical, ultrastructural, and biochemical analyses. In this gland, substantial amounts of endogenous d-Asp were detected, along with aspartate racemases that convert d-Asp to l-Asp and vice versa. We found that the gland was capable of uptaking and accumulating exogenously administered d-Asp. d-Asp acute treatment markedly increased lipid and porphyrin secretion and induced a powerful hyperaemia in inter-acinar interstitial tissue. Since d-Asp is known to be recognized by NMDA receptors, the expression of such receptors in rat HG led us to the hypothesis that d-Asp acute treatment induced the activation of the extracellular signal-regulated protein kinase (ERK) and nitric oxide synthase (NOS) pathways mediated by NMDA. Interestingly, as a result of enhanced oxidative stress due to increased porphyrin secretion, the revealed activation of the stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pro-apoptotic pathway was probably triggered by the gland itself to preserve its cellular integrity.     

Role of the active site residues arginine-216 and arginine-237 in the substrate specificity of mammalian <Emphasis Type="SmallCaps">d</Emphasis>-aspartate oxidase

d-Aspartate oxidase (DDO) and d-amino acid oxidase (DAO) are flavin adenine dinucleotide-containing flavoproteins that catalyze the oxidative deamination of d-amino acids. Unlike DAO, which acts on several neutral and basic d-amino acids, DDO is highly specific for acidic d-amino acids. Based on molecular modeling and simulated annealing docking analyses, a recombinant mouse DDO carrying two substitutions (Arg-216 to Leu and Arg-237 to Tyr) was generated (R216L-R237Y variant). This variant and two previously constructed single-point mutants of mouse DDO (R216L and R237Y variants) were characterized to investigate the role of Arg-216 and Arg-237 in the substrate specificity of mouse DDO. The R216L-R237Y and R216L variants acquired a broad specificity for several neutral and basic d-amino acids, and showed a considerable decrease in activity against acidic d-amino acids. The R237Y variant, however, did not show any additional specificity for neutral or basic d-amino acids and its activity against acidic d-amino acids was greatly reduced. The kinetic properties of these variants indicated that the Arg-216 residue is important for the catalytic activity and substrate specificity of mouse DDO. However, Arg-237 is, apparently, only marginally involved in substrate recognition, but is important for catalytic activity. Notably, the substrate specificity of the R216L-R237Y variant differed significantly from that of the R216L variant, suggesting that Arg-237 has subsidiary effects on substrate specificity. Additional experiments using several DDO and DAO inhibitors also suggested the involvement of Arg-216 in the substrate specificity and catalytic activity of mouse DDO and that Arg-237 is possibly involved in substrate recognition by this enzyme. Collectively, these results indicate that Arg-216 and Arg-237 play crucial and subsidiary role(s), respectively, in the substrate specificity of mouse DDO.     

Vasoactive intestinal peptide (VIP) receptors in the canine gastrointestinal tract

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter in both the brain and peripheral tissues. To define possible target tissues of VIP we have used quantitative receptor autoradiography to localize and quantify the distribution of ¹²⁵I-VIP receptor binding sites in the canine gastrointestinal tract. While the distribution of VIP binding sites was different for each segment examined, specific VIP binding sites were localized to the mucosa, the muscularis mucosa, the smooth muscle of submucosal arterioles, lymph nodules, and the circular and longitudinal smooth muscle of the muscularis externa. These results identify putative target tissues of VIP action in the canine gastrointestinal tract. In correlation with physiological data, VIP sites appear to be involved in the regulation of a variety of gastrointestinal functions including epithelial ion transport, gastric secretion, hemodynamic regulation, immune response, esophageal, gastric and intestinal motility.     

Effects of Z-FA.FMK on d-galactosamine/tumor necrosis factor-alpha-induced kidney injury and oxidative stress in mice

Background The aim of this investigation was to demonstrate that benzyloxicarbonyl-l-phenylalanyl-alanine-fluoromethylketone (Z-FA.FMK), which is a pharmacological inhibitor of cathepsin B, has protective role on the kidney injury that occurs together with liver injury. Methods BALB/c male mice used in this study were divided into four groups. The first group was given physiologic saline only, the second group was administered Z-FA.FMK alone, the third group received d-galactosamine and tumor necrosis factor-alpha (d-GalN/TNF-α), and the fourth group was given both d-GalN/TNF-α and Z-FA.FMK. One hour after administration of 8 mg/kg Z-FA.FMK by intravenous injection, d-GalN (700 mg/kg) and TNF-α (15 μg/kg) were given by intraperitoneal injection. Results In the group given d-GalN/TNF-α, the following results were found: severe degenerative morphological changes in the kidney tissue, a significant increase in the number of activated caspase-3-positive tubular epithelial cell, an insignificant increase in the number of proliferating cell nuclear antigen (PCNA)-positive tubular epithelial cell, a decrease in the kidney glutathione (GSH) levels, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, an increase in the kidney lipid peroxidation (LPO) levels, lactate dehydrogenase (LDH) activity, serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities, uric acid and urea levels. In contrast, in the group given d-GalN/TNF-α and Z-FA.FMK, a significant decrease in the d-GalN/TNF-α-induced degenerative changes, a decrease in the number of activated caspase-3-positive tubular epithelial cell, a insignificant decrease in the number of PCNA-positive tubular epithelial cell, an increase in the kidney GSH levels, CAT, SOD and GPx activities, a decrease in the kidney LPO levels, LDH activity, serum AST and ALT activities, uric acid and urea levels were determined. Conclusion These results suggest that pretreatment with Z-FA.FMK markedly lessens the degree of impairment seen in d-GalN/TNF-α-induced kidney injury, which occurred together with liver injury in mice.     

Characterization of Na+-Coupled Glutamate/Aspartate Transport by a Rat Brain Astrocyte Line Expressing GLAST and EAAC1

d-Aspartate (d-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. d-Asp influx is Na⁺-dependent with K _m = 5 μm and V _max= 0.7 nmoles · min⁻¹· mg protein⁻¹. Influx is sigmoidal as f[Na⁺] with _Na+ K _m ∼ 12 μm and Hill coefficient of 1.9. The cells establish steady-state d-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in V _max, with no change in K _m . At initial [d-Asp] = 10 μm, RBA take up more than 90% of total d-Asp, and extracellular levels are reduced to levels below 1 μm. Ionophores that dissipate the Δμ_Na+ inhibit gradient formation. Genistein (GEN, 100 μm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4α-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na⁺-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), l-Asp, and l-Glu, but not by d-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells. Received: 11 January 2001/Revised: 26 March 2001     

Heterogenous staining ofd-amino acid oxidase in peroxisomes of rat liver and kidney

Summary The localization ofd-amino acid oxidase (d-AAOX) in rat liver and kidney has been investigated using the cerium technique for electron microscopy and a recent modification of it for light microscopy. In the liver a mosaic pattern with strongly and weakly stained cells together with some completely negative hepatocytes is observed. The staining is stronger and more uniform in periportal than in perivenous regions of the liver lobule. In the kidney the reaction is confined to the proximal tubules of the renal cortex with the rest of the nephron being negative. At the ultrastructural level in both liver and kidney a marked heterogencity is obseved in the intensity of reaction in peroxisomes of some neighbouring cells. Moreover, in some cells heavily and weakly stained peroxisomes are seen side by side. When Pipes buffer is used in the incubation medium thed-AAOX reaction in kidney peroxiosomes is aggregated in the central region of the matrix with weaker staining of the periphery. A similar result is obtained when the enzyme is localized by immunocytochemistry confirming a recent report by Usuda et al. (1986). The heterogeneous staining of peroxisomes ford-AAOX suggests that subpopulation of this organelle with specialized functions may exist not only in different tissues and cells but even within the same cell.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

d-Aspartic acid induced oxidative stress and mitochondrial dysfunctions in testis of prepubertal rats

Previously we demonstrated the potential of d-aspartic acid (d-Asp), an acidic amino acid to induce oxidative response in prepubertal rat testis in vitro. In the present study, we determined the extent of oxidative stress in the testis of prepubertal rats that were administered d-Asp (100 and 500 mg/kg bw/d, i.p. 7 days). d-Asp treatment significantly elevated the levels of reactive oxygen species, malondialdehyde and hydroperoxide in cytosol and mitochondria of testis, which were accompanied by enhanced glutathione levels, elevated activities of glutathione-dependent enzymes and catalase suggesting a state of oxidative stress. Further, the activities of d-aspartate oxidase and 3β-hydroxy steroid dehydrogenase were elevated in the testis. The testis mitochondria of d-Asp-treated rats showed altered citric acid and complex enzyme activities, reduction in membrane potential, increased permeability and intracellular Ca²⁺ levels. Collectively, these findings suggest the potential of d-Asp to induce oxidative perturbations in the testis of prepubertal rats and this mechanism may in part be responsible for the observed physiological effects.     

Comparison of Different Forms of Dietary Selenium Supplementation on Growth Performance,Meat Quality,Selenium Deposition,and Antioxidant Property in Broilers

This study was conducted to investigate the effects of different sources of dietary selenium (Se) supplementation on growth performance, meat quality, Se deposition, and antioxidant property in broilers. A total of 600 one-day-old Ross 308 broilers with an average body weight (BW) of 44.30 ± 0.49 g were randomly allotted to three treatments, each of which included five replicates of 40 birds. These three groups received the same basal diet containing 0.04 mg Se/kg, supplemented with 0.15 mg Se/kg from sodium selenite (SS) or from l-selenomethionine (l-Se-methionine (Met)) or from d-selenomethionine (d-Se-Met). The experiment lasted 42 days. Both Se source and time significantly influenced (p < 0.01) drip loss of breast muscle. Supplementation with l-Se-Met and d-Se-Met were more effective (p < 0.05) in decreasing drip loss than SS. Besides, the pH value of breast muscle was also significantly influenced (p < 0.05) by time. The SS-supplemented diet increased more (p < 0.05) liver, kidney, and pancreas glutathione peroxidase (GSH-Px) activities than the d-Se-Met-supplemented diet. In addition, l-Se-Met increased more (p < 0.01) liver and pancreas GSH-Px activities than d-Se-Met. The antioxidant status was greatly improved in broilers of l-Se-Met-treated group in comparison with the SS-treated group and was illuminated by the increased glutathione (GSH) concentration in serum, liver, and breast muscle (p < 0.05); superoxide dismutase (SOD) activity in liver (p < 0.01); total antioxidant capability (T-AOC) in kidney, pancreas, and breast muscle (p < 0.05) and decreased malondialdehyde (MDA) concentration in kidney and breast muscle (p < 0.05) of broilers. Besides, supplementation with d-Se-Met was more effective (p < 0.01) in increasing serum GSH concentration and decreasing breast muscle MDA concentration than SS. l-Selenomethionine supplementation significantly increased GSH concentration in liver and breast muscle (p < 0.05); SOD activity in liver (p < 0.01); and T-AOC in liver, pancreas, and breast muscle (p < 0.05) of broilers, compared with broilers fed d-Se-Met diet. The addition of l-Se-Met and d-Se-Met increased (p < 0.01) Se concentration in serum and different organs studied of broilers in comparision with broilers fed SS diet. Therefore, dietary l-Se-Met and d-Se-Met supplementation could improve antioxidant capability and Se deposition in serum and tissues and reduce drip loss of breast muscle in broilers compared with SS. Besides, l-Se-Met is more effective than d-Se-Met in improving antioxidant status in broilers.     

Tissue distribution and immunohistochemical localization of the collagen-binding heat-shock protein gp46 in neonatal rats

Summary Collagen-binding heat-shock proteins ofM _r 46–47 kDa have been postulated to function as putative molecular chaperones in the biosynthesis of collagen in several species. The rat homologue of this family of heat-shock proteins is called gp46. In the present study, we employed Western blotting and immunohistochemical methods to determine the tissue distribution and cellular localization of gp46 in the thoracic aorta, heart, kidney, liver and lung of eight-day-old Wistar rats. Highest levels of gp46 were detected in the thoracic aorta and lung, followed by the kidney and heart. Gp46 levels were low to undetectable by Western blot analysis in the liver. Immunohistochemistry revealed that gp46 labelling was observed almost exclusively in three distinct cell types: fibroblasts, muscle cells, and some epithelial cells. Gp46 was detected in the fibroblasts of the hepatic triad, in the interstitium of the alveolar wall and in the tunica adventitia of blood vessels in the majority of tissues examined, in atrial and ventricular cardiomyocytes, in vascular smooth muscle cells of the abluminal portion of the tunica media, in parietal epithelial cells and mesangial cells of the glomerulus, in epithelial cells of the distal tubules and collecting ducts in the kidney and clusters of immature renal tubules, in epithelial cells of the bile duct, and in mesodermal cells surrounding the liver. These results demonstrate that gp46 is present in collagen producing cells and cells undergoing rapid growth and development, suggesting that gp46 may play a significant role in these processes.     

The expression, activity and localisation of the secretory pathway Ca-ATPase (SPCA1) in different mammalian tissues

The distribution of the secretory pathway Ca²⁺-ATPase (SPCA1) was investigated at both the mRNA and protein level in a variety of tissues. The mRNA and the protein for SPCA1 were relatively abundant in rat brain, testis and testicular derived cells (myoid cells, germ cells, primary Sertoli cells and TM4 cells; a mouse Sertoli cell line) and epididymal fat pads. Lower levels were found in aorta (rat and porcine), heart, liver, lung and kidney.SPCA activities from a number of tissues were measured and shown to be particularly high in brain, aorta, heart, fat pads and testis. As the proportion of SPCA activity compared to total Ca²⁺ ATPase activity in brain, aorta, fat pads and testis were relatively high, this suggests that SPCA1 plays a major role in Ca²⁺ storage within these tissues. The subcellular localisation of SPCA1 was shown to be predominantly around the Golgi in both human aortic smooth muscle cells and TM4 cells.     

Evidence for the involvement of d-aspartic acid in learning and memory of rat

d-Aspartic acid (d-Asp) is an endogenous amino acid present in neuroendocrine systems. Here, we report evidence that d-Asp in the rat is involved in learning and memory processes. Oral administration of sodium d-aspartate (40 mM) for 12–16 days improved the rats’ cognitive capability to find a hidden platform in the Morris water maze system. Two sessions per day for three consecutive days were performed in two groups of 12 rats. One group was treated with Na-d-aspartate and the other with control. A significant increase in the cognitive effect was observed in the treated group compared to controls (two-way ANOVA with repeated measurements: F _{(2, 105)} = 57.29; P value < 0.001). Five further sessions of repeated training, involving a change in platform location, also displayed a significant treatment effect [F _{(2, 84)} = 27.62; P value < 0.001]. In the hippocampus of treated rats, d-Asp increased by about 2.7-fold compared to controls (82.5 ± 10.0 vs. the 30.6 ± 5.4 ng/g tissue; P < 0.0001). Moreover, 20 randomly selected rats possessing relatively high endogenous concentrations of d-Asp in the hippocampus were much faster in reaching the hidden platform, an event suggesting that their enhanced cognitive capability was functionally related to the high levels of d-Asp. The correlation coefficient calculated in the 20 rats was R = −0.916 with a df of 18; P < 0.001. In conclusion, this study provides corroborating evidence that d-aspartic acid plays an important role in the modulation of learning and memory.     

Immunoelectron microscopic localization of D-amino acid oxidase in rat kidney and liver

Summary The intracellular localization ofd-amino acid oxidase in rat kidney and liver has been investigated using the indirect immunogold postembedding technique. Different fixation and embedding conditions for optimal preservation of antigenicity and fine structure have been tested. Immunolabelling was possible only in tissues embedded in polar resins (glycol methacrylate and Lowicryl K4M). In kidney the enzyme was demonstrable only in the peroxisomes of the proximal tubule, where it was associated with the peroxisome core. The enzyme was present in all the peroxisomes of the proximal tubule and appeared to be codistributed with catalase. Control experiments and quantitative analysis confirmed the specificity of thed-amino acid oxidase immunolocalization. All the other cells in kidney failed to demonstrate any labelling. In liver, the immunolabelling was present in the matrix of the hepatocyte peroxisomes, whereas no traces of the enzyme were found in the nucleoid. The intensity of the immunolabelling in liver peroxisomes was lower than in kidney. No specific labelling was observed in cells other than hepatocytes.     

First evidence on induced topological changes in supercoiled DNA by an aluminium <Emphasis Type="SmallCaps">d</Emphasis>-aspartate complex

Aluminium is a debatable and suspected etiological factor in neurodegenerative disorders. Aluminium–amino acid complexes also play an important role in the complex biology of the metal. Recent reports indicate the presence of d-aspartate and d-glutamate in aging brain, human breast tumors, core amyloid plaques and neurofibrillary tangles of Alzheimer's brain. This stereoinversion from the l- to the d-enantiomer is enhanced by Al. Further, the observation that Al is localized in the chromatin region encouraged the present study of the interaction of Al–amino acid complexes with DNA. This study used circular dichroism of supercoiled DNA and showed that Al–d-Asp caused a native B-DNA to C-DNA conformational change, while Al–l-Asp, Al–l-Glu and Al–d-Glu did not alter the native B-DNA conformation. This differential DNA binding property of Al–amino acid complexes is assigned to the stereoisomerism and chirality of the complexes. Interestingly, polyamines like spermine further induced an asymmetric condensation of the "limit C-motif" induced by Al–d-Asp to a -DNA. The results were supported by computer modeling, gel studies and ethidium bromide binding. We also propose a mechanism of Al–d-Asp binding and its ability to modulate DNA topology.     

Griffonia simplicifolia lectins bind specifically to endothelial cells and some epithelial cells in mouse tissues

Summary The binding ofGriffonia simplicifolia agglutinin-I (GSA—I) and the isolectins GSA-I-AB₃ and GSA-I-B₄, having affinity for some -d-galactosyl andN-acetyl galactosaminyl residues was studied in different mouse tissues. In brain, cardiac muscle and skeletal muscle, the GSA-I-lectin conjugates showed prominent binding only to blood vessel endothelia. Similarly, in the liver and kidney cortex the GSA-I-conjugates selectively reacted with endothelial cells of the sinusoids and with intertubular and glomerular capillaries, respectively. However, a strong reactivity with the GSA-I-conjugates was additionally seen in the acinar cells of the pancreas, in the stratified squamous epithelia of skin and tongue, and in transitional epithelium. SDS—PAGE electrophoresis combined with the lectin-blotting technique indicated that a similar set of glycoproteins are responsible for the GSA-I binding, even in different tissues. Another lectin with specificity for -d-galactose, theMaclura pomifera agglutinin, displayed a distinctly different distribution of binding sites, mainly in the basement membranes, of all mouse tissues studied. The results suggest that some -d-galactosyl residues, recognized by the binding of GSA-I lectins, are preferentially expressed in endothelial cells of mouse tissues, and also provide further evidence that endothelial cells can present a highly specific surface glycosylation pattern.     

Folates of rat tissue: Bioassay of tissue folypolyglutamates and a relationship of liver folypolyglutamates to nutritional folate sufficiency

The folate content of young rat tissues extracted into boiling ascorbate was assayed by Lactobactillus casei both without and after treatment by a folate-free preparation of conjugase. The total folate content of various tissues was: liver, 8.9 μg/g; kidney, 2.6; adrenal, 2.6; bone marrow, 2.4; spleen, 0.9; erythrocytes, 0.8; small intestinal mucosa, 0.7; small intestinal smooth muscle, 0.8; heart, 0.6; brain, 0.4, and skeletal muscle, 0.1 μg/g tissue. For most tissues, with the exception of muscle and kidney, approximately 80% of the total folates assayed as longer chain length folylpolyglutamates.When liver folates were analyzed from rats fed folate-supplemented, control and folate-deficient diets, a relationship was found between folate nutrition and distribution of folylpolyglutamates. The proportion of total folates in the form of longer chain length folylpolyglutamates was greatest in the livers of folate-deficient rats and least in the livers of folate-supplemented rats.     

Distribution of prostaglandin E2 binding sites in the porcine gastrointestinal tract

Binding of biologically active ³H-PGE₂ to particulate fractions of porcine gastrointestinal mucosa and muscle was investigated. Specific binding activity was detected in the 2500 xg and 30,000 xg sedimentation fractions of mucosa from esophagus, fundus, antrum, duodenum, ileum and colon, as well as in serosal muscle taken from the antrum, ileum, and colon. Optimal binding (> 40 fmol/mg protein) was observed in the 30,000 xg fraction of fundic mucosa incubated at pH 5.0. The characteristics of ³H-PGE₂ binding were variable in the remainder of the gastrointestinal tract although binding in these tissues was significantly less (0.2 to 15 fmol/mg protein) than that observed in the fundic mucosa. These data suggest that the cellular and/or subcellular site of PG binding is not uniform throughout the gastrointestinal tract. In fundic mucosa removal of the surface epithelial layer by scraping did not significantly alter the total binding activity for PGE. This result suggests that in gastric secretory mucosa optimal binding activity for PGE₂ occurs within the gastric pits deep to the surface epithelium.     

Substance P and substance K receptor binding sites in the human gastrointestinal tract: Localization by autoradiography

Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for ¹²⁵I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: 1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; 2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; 3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and 4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells.     

Mucosal nerves and smooth muscle relationships with gastric glands of the opossum: an ultrastructural and three-dimensional reconstruction study

The precise anatomical relation by which autonomic nerve endings contact gastric epithelial cells to enhance the rate of gastric secretions is not fully understood. The aim of the present study was to clarify this issue by using the technique of serial section reconstruction of areas of the gastric mucosa. The work also explored the possibility of a functional role for a system of smooth muscle strands in the gastric mucosa that emanate from the muscularis mucosa, run in the lamina propria, and are associated in a unique manner with the gastric glands. Electron microscopic serial sections of the gastric mucosa were performed to visualize the entire limiting membrane of gastric epithelial cells to determine any nerve associations (especially varicose endings) with these cells. Evaluation of serial sections of five separate parietal cells showed that their basal membrane did not come in close contact (nearest distance 500 nm) with any nerve axon or varicosity. Moreover, the axons passing in the area of these cells ultimately showed varicose endings associated with smooth muscle cells in the adjacent connective tissue (often separated by only 20 nm), with mast cells or with vascular elements. Additionally, the lateral membrane of these five parietal cells did not contact any endocrine cell in the epithelium, although other parietal cells in the area were adjacent to endocrine cells. Chief cells in the immediate area also did not form any close associations with nerve varicosities. Random analysis of 5,000 additional epithelial cells in these sections showed no close associations to nerve elements with significant accumulations of neurosecretory vesicles (varicosities). Because of the observed existence of innervation to the smooth muscle strands in the area of the gastric glands, serial 1-micron epoxy sections of the gastric mucosa were prepared, and profiles of smooth muscle and gastric glands were entered into a computer-assisted reconstruction system. Three-dimensional reconstruction techniques were employed to reveal the existence of a unique association between the mucosal smooth muscle strands and the gastric glands. The muscle strands arose from the muscularis mucosa at regular intervals and became branched to form an intricate wrap around a series of gastric glands that empty into one gastric pit.(ABSTRACT TRUNCATED AT 400 WORDS)     

New insights on the role of free d-aspartate in the mammalian brain

Free d-aspartate (d-Asp) occurs in substantial amounts in the brain at the embryonic phase and in the first few postnatal days, and strongly decreases in adulthood. Temporal reduction of d-Asp levels depends on the postnatal onset of d-aspartate oxidase (DDO) activity, the only enzyme able to selectively degrade this d-amino acid. Several results indicate that d-Asp binds and activates N-methyl-d-aspartate receptors (NMDARs). Accordingly, recent studies have demonstrated that deregulated, higher levels of d-Asp, in knockout mice for Ddo gene and in d-Asp-treated mice, modulate hippocampal NMDAR-dependent long-term potentiation (LTP) and spatial memory. Moreover, similarly to d-serine, administration of d-Asp to old mice is able to rescue the physiological age-related decay of hippocampal LTP. In agreement with a neuromodulatory action of d-Asp on NMDARs, increased levels of this d-amino acid completely suppress long-term depression at corticostriatal synapses and attenuate the prepulse inhibition deficits produced in mice by the psychotomimetic drugs, amphetamine and MK-801. Based on the evidence which points to the ability of d-Asp to act as an endogenous agonist on NMDARs and considering the abundance of d-Asp during prenatal and early life, future studies will be crucial to address the effect of this molecule in the developmental processes of the brain controlled by the activation of NMDARs.