Potassium-evoked efflux of transmitter amino acids and purines from rat cerebral cortex

Repeated applications of elevated K+ (50 or 75 mM) in cerebral cortical cup superfusates was used to evoke an efflux of gamma-aminobutyric acid (GABA), glutamate, aspartate, glycine, adenosine, and inosine from the in vivo rat cerebral cortex. K+ (50 mM) significantly elevated GABA levels in cup superfusates but had little effect on the efflux of glutamate, aspartate, glycine, adenosine, or inosine. K+ (75 mM) significantly enhanced the efflux of GABA, aspartate, adenosine, and inosine and caused nonsignificant increases in glutamate and glycine efflux. The adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA), applied in cup superfusates at a concentration of 10(-10) M had no effect on either basal or K(+)-evoked release of any of the amino acids or purines measured. At 10(-6) M CPA significantly enhanced aspartate release, and depressed GABA efflux. The selective A2 adenosine receptor agonist 2-p(2-carboxyethyl) phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS 21680) (10(-8) M) was without effect on either basal, or K(+)-evoked, efflux of amino acids or purines. The enhancement of aspartate (an excitotoxic amino acid) efflux by higher concentrations of CPA is likely due to activation of adenosine A2b receptors. This observation may be of relevance when selecting adenosinergic agents to treat ischemic or traumatic brain injuries. Overall, the results suggest that effects of adenosine receptor agonists on K(+)-evoked efflux of transmitter amino acids from the in vivo rat cerebral cortex may not be comparable to those observed with in vitro preparations.     

Differential regulation of mouse embryo development and viability by amino acids

The amino acid requirements of the preimplantation mouse embryo in culture changes as development proceeds from the zygote to the blastocyst stage. Eagle's non-essential amino acids and glutamine significantly increased cleavage rates during the first four cell cycles, while Eagle's essential amino acids without glutamine did not confer any benefit to embryo development before the eight-cell stage. After the eight-cell stage, non-essential amino acids and glutamine no longer stimulated cleavage rates but significantly increased blastocoel development and blastocyst hatching. In contrast, after the eight-cell stage essential amino acids increased cleavage rates as well as stimulating development of the inner cell mass of the resultant blastocysts. Fetal development after transfer of blastocysts was also significantly increased by culture with essential amino acids from the eight-cell stage. Consequently highest rates of development in vitro and viability after transfer were achieved when embryos were cultured with non-essential amino acids and glutamine to the eight-cell stage followed by development to the blastocyst stage in the presence of all 20 amino acids. Analysis of the parameters measured revealed a significant relationship between number of blastocyst cells and inner cell mass development with viability after transfer. Blastocyst formation and hatching could not be used to assess subsequent developmental potential.     

Intrinsic inhibitory pathways in mouse barrel cortex

The organization of intrinsic connections in the mouse somatosensory cortex was studied by combining tract-tracing and immunocytochemical techniques. Injections of HRP or fluorescent beads were made into the supragranular or infragranular layers of the posteriomedial barrel subfield. Numerous cells were retrogradely labeled within a vertical column centered on each injection site. Retrogradely labeled cells were also found > 3 mm horizontal to the injection. Dual-label immunocytochemistry identified the population of inhibitory, GABAergic cells forming intrinsic horizontal connections. Double-labeled cells were found predominantly within a 200 microns radius horizontal to an injection site. These findings indicate that intrinsic inhibitory pathways in the barrel cortex do not provide a substrate for direct GABAergic interactions among barrel columns, and that inter-columnar interactions are primarily excitatory.     

Isolation of hydrophobic proteins biding amino acids: L-aspartic acid-binding protein from the rat cerebral cortex

The interaction of hashish compounds, delta 1-tetrahydrocannabinol and cannabidiol, with dipalmitoyl phosphatidylcholine was investigated using differential scanning calorimetry. Both drugs affect the transition of dipalmitoyl phosphatidylcholine from the gel to liquid crystalline state, decreasing both the melting temperature and the enthalpy of melting. At a drug to dipalmitoyl phosphatidylcholine ratio of approx. 1:5, two peaks appear in the transition profile, suggesting a phase separation in the drug dipalmitoyl phosphatidylcholine mixture.     

Laminar development of the mouse barrel cortex: effects of neurotoxins against monoamines

To investigate the inductive activities of the vertebrate organizer, we transplanted the chicken organizer (Hensen's node) into zebrafish gastrula and analyzed resulting secondary axes. Grafted Hensen's node did not differentiate or participate in the secondary axis. It also did not induce a secondary notochord or expression of the genes normally expressed by the fish organizer including no tail, axial, goosecoid. Nevertheless, it recruited fish cells to organize a variety of tissues: the dorsal portion of the central nervous system including Rohon-Beard sensory neurons, otic vesicles, dorsal pigment stripe, dorsal fin, somites, heart, and pronephric ducts. Enlarged neural plate induced by the organizer was shown by the expression pattern of dlx3 and msxB genes, which demarcates the early presumptive neural tissue. In addition, Hensen's node of an earlier stage chicken embryo displayed differential movement in zebrafish from that of a later stage. This might reflect unknown differences in properties between the organizer at two different developmental stages related to its normal organizer activity. To create a model system to study the molecular mechanisms of the organizer, we next transplanted genetically modified mouse cells into zebrafish embryos. We found that Wnt3A-transfected NIH3T3 cells are much more potent in inducing a secondary axis than NIH3T3 cells alone. These results suggest that formation of a variety of tissues are controlled by signalling from the organizer itself with no requirement of participation of the organizer-derived tissues. Additionally, the activities of the organizer may involve a function of Wnt-family genes.     

Inhibition by anion channel blockers of ischemia-evoked release of excitotoxic and other amino acids from rat cerebral cortex

In febrile neutropenic patients with high-grade hematologic malignancies, empirical antimicrobial intervention is mandatory. Large randomized clinical trials have elucidated the benefit of broad-spectrum beta lactam antibiotics used as single drugs or in combination with aminoglycosides in order to provide activity against gram-negative aerobes as well as against streptococci and Staphylococcus aureus. As a result, infection-related mortality was reduced to less than 10% also in patients undergoing intensified remission induction or consolidation therapy for acute leukemias. Distinct subgroups of patients have been identified who need an empirical modification of antimicrobial treatment i.e., patients with catheter-related infections, patients with pulmonary infiltrates, and patients with unexplained fever not responding to first-line antibiotics. In two consecutive, prospectively randomized trials conducted by the Paul Ehrlich Society it was demonstrated that empirical antifungal therapy is beneficial for second-line treatment in patients with persistent unexplained fever and should be part of the first-line approach in patients with lung infiltrates. The empirical addition of glycopeptides, however, should be restricted to patients with catheter-related infections due to coagulase-negative staphylococci.     

Effects of amino acids and gastric inhibitory polypeptide on insulin release in dogs

Insulin release following intravenous administration of an amino acid solution with and without a simultaneous infusion of varying amounts of porcine gastric inhibitory polypeptide (GIP) was studied in dogs. Group I received a 10-amino acid mixture (300 mosmol/kg iv) at 16.6 ml/min for 1 h; group II, amino acid mixture plus 0.5 micrograms.kg-1.h-1 porcine GIP; group III, amino acid mixture plus 1.0 micrograms.kg-1.h-1 of GIP; group IV (a and b) received either 0.5 or 1.0 micrograms.kg-1.h-1 of GIP alone. Compared to group I, groups II and III had a greater insulin response during the first 30 min of the infusion. Group] IV (a and b) showed no insulin release. Glucose concentrations showed no significant change in all groups. From these results, it is concluded that insulin release after intravenous infusion of an amino acid mixture plus GIP is greater than after amino acids or GIP alone. It appears that this effect is more pronounced in the early phase of insulin release.     

Effects of inhibitory amino acids on the frequency components in sympathetic nerve discharge

The effects of the GABA antagonist picrotoxin and the glycine antagonist strychnine on the frequency components in sympathetic inferior cardiac nerve activity were observed. Picrotoxin (0.03-1.0 mg/kg) increased power in the 10-Hz component of sympathetic activity and produced a dramatic shift in the rhythm to higher frequencies. Only small changes were noted in the 2- to 6-Hz component. Strychnine produced a small generalized increase in power in both frequency bands in sympathetic activity. These data suggest that GABA may play an important role in the generation and maintenance of the 10-Hz rhythm in sympathetic activity while glycine likely inhibits activity at a site of convergence of the two rhythms in sympathetic activity.     

Keto acids and free amino acids during leaf growth in Bauhinia purpurea L

The biosynthesis of keto acids and free amino acids was studied during the growth of Bauhinia purpurea leaves. Alpha-KGA, OAA, pyruvic acid and PEP are the important keto acids observed at various stages. The first 2 metabolites show a progressive increase and PEP leads to OAA pathway is very active during the process of growth.     

A set of genes expressed in response to light in the adult cerebral cortex and regulated during development

Activity-dependent plasticity is thought to underlie both formation of appropriate synaptic connections during development and reorganization of adult cortical topography. We have recently cloned many candidate plasticity-related genes (CPGs) induced by glutamate-receptor activation in the hippocampus. Screening the CPG pool for genes that may contribute to neocortical plasticity resulted in the identification of six genes that are induced in adult visual cortical areas in response to light. These genes are also naturally induced during postnatal cortical development. CPG induction by visual stimulation occurs primarily in neurons located in cortical layers II-III and VI and persists for at least 48 hr. Four of the visually responsive CPGs (cpg2, cpg15, cpg22, cpg29) are previously unreported genes, one of which (cpg2) predicts a mini-dystrophin-like structural protein. These results lend molecular genetic support to physiological and anatomical studies showing activity-dependent structural reorganization in adult cortex. In addition, these results provide candidate genes the function of which may underlie mechanisms of adult cortical reorganization.     

Importance of specific amino acids in protein binding sites for heparin and heparan sulfate

The present study tests whether lesions small enough to allow the rapid reestablishment of a normally aligned tract glial framework would provide a permissive environment for the regeneration of cut adult CNS axons. We made penetrating microlesions which cut a narrow beam of axons in the adult rat cingulum, but caused minimal damage to the tract glial framework and no cavitation. The proximal tips of cut axons were identified by enhanced immunoreactivity for low affinity neurotrophin receptor, p75. From 1 day they became expanded into large growth-cone-like structures. At later times some axons turned back and extended in the reverse direction. Up to 14 days (after which time p75 could no longer be used as a marker), no axons advanced beyond the line of the lesion. From 1 to 2 days, OX42 immunostaining and electron microscopy showed that the lesion site was densely infiltrated by macrophages, which disappeared by 3 to 4 days. This was followed by a local hypertrophy of the OX42 immunoreactive resident tract microglial cells and an increase in both GFAP and vimentin immunoreactivity of the tract astrocytes. These responses were greatly reduced by 8 days, when the longitudinal alignment of glial processes across the lesion site was similar to that of an undamaged tract. The large growth-cone-like structures formed at the ends of the cut axons resemble those of developing axons exposed to chemorepulsive factors. This suggests that cellular elements in adult tract lesions may also exert chemorepulsive influences blocking regeneration of axons even in an apparently "open" tract framework.     

Telomere length regulation during postnatal development and ageing in Mus spretus

Telomere shortening has been causally implicated in replicative senescence in humans. To examine the relationship between telomere length and ageing in mice, we have utilized Mus spretus as a model species because it has telomere lengths of approximately the same length as humans. Telomere length and telomerase were analyzed from liver, kidney, spleen, brain and testis from >180 M.spretus male and female mice of different ages. Although telomere lengths for each tissue were heterogeneous, significant changes in telomere lengths were found in spleen and brain, but not in liver, testis or kidney. Telomerase activity was abundant in liver and testis, but weak to non-detectable in spleen, kidney and brain. Gender differences in mean terminal restriction fragment length were discovered in tissues from M.spretus and from M.spretus xC57BL/6 F1 mice, in which a M. spretus -sized telomeric smear could be measured. The comparison of the rank order of tissue telomere lengths within individual M. spretus showed that certain tissues tended to be longer than the others, and this ranking also extended to tissues of the M.spretus xC57BL/6 F1 mice. These data suggest that telomere lengths within individual tissues are regulated independently and are genetically controlled.     

The in vivo inactivation of GABA and other inhibitory amino acids in the cat nervous system

In cats anaesthetised with pentobarbitone, the effect of inhibitors of the in vitro cellular uptake of GABA were tested on the responses of single central neurones to GABA and other depressant amino acids. (4)- AND (-)-nepecotic acid, (4)-2,4-diaminobutyric acid (DABA) and 2,2-dimethyl-beta-alanine, enhanced the action of GABA on spinal, cerebellar and cerebral cortical neurones. In the spinal cord DABA, and to a less estent (-)-nipecotic acid, enhanced the action of beta-alanine, whereas the actions of glycine and taurine were unaffected by DABA and reduced by (-)-nipecotic acid. In the cerebellum and cerebral cortex, these two substances enhanced the action of GABA, usually to a greater extent than that of beta-alanine and taurine, although this specificity was not marked. The GABA-mediated basket cell inhibition of Purkinje cells in the cerebellum was unaffected by DABA and (-)-nipecotic acid, and neither substance appears suitable for determining the role of uptake processes in the inactivation of synapitcally released GABA. Quantitatively these in vivo results agree more closely with recent vitro uptake studies in cat tissue than the previously published data on rat cerebral cortex and dorsal root ganglia, and the observations provide further evidence for the importance of cellular uptake in maintaining low extraneuronal concentrations of inhibitory amino acid transmitters.     

Increases in strychnine-insensitive glycine binding sites in cerebral cortex of chronic schizophrenics: evidence for glutamate hypothesis

Strychnine-insensitive glycine binding sites, an absolute requirement of the responses mediated by N-methyl-D-aspartate (NMDA) receptors, were measured in the postmortem brains of 13 chronic schizophrenics and 10 controls, using a radiolabeled receptor assay. Specific [3H]glycine binding was significantly increased in six of the 16 areas of the cerebral cortex that were investigated. Scatchard analysis performed in these areas showed a significant increase in the maximum number of binding sites, with no change in the affinity of binding. Multiple regression analysis confirmed that the increase was not due to age at death or interval from death to freezing. The increase was also observed in the off-drug cases of schizophrenics who had not taken antipsychotics for more than 40 days before death. These results suggest that the increases in NMDA-associated glycine binding sites, possibly ascribed to the postsynaptic compensation for impaired glutamatergic neurotransmission, might be implicated in the pathophysiology of schizophrenia.     

Trigeminal inputs to reticulospinal neurones in lampreys are mediated by excitatory and inhibitory amino acids

Using stable isotope dilution/gas chromatography-mass spectrometry (ID/GC-MS), a physicochemical method, we have profiled the plasma steroids 17 alpha-hydroxyprogesterone, 4-androstenedione, and testosterone in normal children of various age groups. Comparison of our values with those obtained by direct immunologic assays and those using an extraction or purification step showed that immunoassays in general overestimate steroid concentrations. This was especially true for plasma samples in the neonatal period and was most expressed for the concentrations of 17 alpha-hydroxyprogesterone. Our study demonstrated the applicability of ID/GC-MS to routine clinical steroid analysis. The application of ID/GC-MS is recommended whenever problems from matrix effects or cross-reactivity are likely to arise or suspicious results by immunoassays need to be rechecked.     

Isolation of hydrophobic proteins binding amino acids. Stereoselectivity of the binding of L-[14C]glutamic acid in cerebral cortex

Forty-one patients with rheumatoid arthritis, including 6 with acute vasculitis, 13 with chronic vasculitis, and 22 without vasculitis, were studied for evidence of intravascular coagulation and fibrinolysis (ICF). The mean plasma fibrinogen levels were elevated in all groups. The fibrinogen, platelet count, and fibrin split products were usually elevated in acute vasculitis. Fewer patients on corticosteroids had abnormal coagulation tests. Active plasmin was detected in 12 patients primarily with chronic vasculitis. Plasminogen activator activity was not diminished in vascular endothelium of normal appearing skin of those patients with or without vasculitis. None of the patients demonstrated decompensated intravascular coagulation and fibrinolysis. The results suggest overcompensated ICF occurring in rheumatoid arthritis, but rheumatoid patients with vasculitis cannot be clearly distinguished from those without vasculitis on the basis of the usual tests performed for coagulation and fibrinolysis abnormalities.     

Amino acids and ammonium regulate mouse embryo development in culture

The conformation of the bulge formed between the hairpin ribozyme R derived from (-)sTRSV and noncleavable all-deoxy-substrate analogues dS was studied by photoaffinity labelling. The photolabel deoxy-6-thioinosine was inserted in place of residue G+1 or A-1, located immediately 3' and 5' to the cleavage site, respectively. Upon 335 nm irradiation both substrate analogues were linked to ribozyme at multiple sites. Formation of the R-dS complex is absolutely required for the generation of the crosslinks, since they were detected neither in the absence of Mg2+ nor upon using a ds6I containing 14-mer, unable to interact with the ribozyme. The fraction of ribozyme crosslinked at completion of the reaction increased with increasing analogue concentrations, yielding apparent KD values for the R-dS complex in the range of 5 +/- 2 microM. Multiple crosslinks between ribozyme and each one of the substrate analogues provide clear evidence for a large flexibility of the bulge region.     

Effects of excitatory amino acids on cerebral oxygen consumption and blood flow in rat

The oncogene Tpr-Met is a constitutively active form of the hepatocyte growth factor/scatter factor (HGF/SF) receptor Met. It comprises the intracellular moiety of Met linked to the dimerization domain of the nuclear envelope protein Tpr, thus functioning as a constitutively activated Met. HGF/SF is responsible for various biological processes including angiogenesis and wound healing, in which secreted serine protease urokinase-type plasminogen activator (uPA) is implicated. The action of HGF/SF on cells is mediated by the autophosphorylation of Met on two carboxyterminal tyrosine residues, Y1349VHVNATVY1356VNV. The two tyrosine residues provide docking sites for various effector molecules, suggesting that multiple signaling pathways are activated to exert biological effects of HGF/SF [Ponzetto et al., Cell (1994) 77: 261]. We found that Tpr-Met efficiently activates the uPA gene via a SOS/Ras/extracellular signal regulated kinase (ERK)-dependent signaling pathway. Mutation of Y1356, which abrogates GRB2 binding, reduced the induction to half of the control level, while mutation of Y1349 showed little effect on uPA induction, suggesting an important but partly replaceable role for GRB2 in Met-dependent uPA gene induction. Mutation of both Y1349VHV and Y1356VNV into optimal PI 3-kinase sites resulted in a residual induction of about one quarter of the control level, suggesting a potential role for PI 3-kinase. Dose-response analysis of the Tpr-Met showed a biphasic curve. These results suggest that the interplay among different signaling molecules on the receptor is important for full induction of the pathway leading to the activation of the uPA gene.