Protective effects of (-)-epicatechin against nitrative modifications of fibrinogen

Fibrinogen appears to be particularly sensitive to toxic action of peroxynitrite; a potent oxidizing and nitrating species. An increased nitration of fibrinogen has been reported in cardiovascular diseases. The defense mechanisms against PN are crucial for complex hemostasis process. Flavonoids have antioxidative properties and could protect biomolecules against action of peroxynitrite. The aim of our studies was to establish, if (−)-epicatechin may in vitro protect fibrinogen molecule against peroxynitrite-induced nitration of tyrosines and change its thrombin-catalyzed polymerization. The exposure of purified fibrinogen (6 μM) to peroxynitrite (1-100 μM) resulted in both structural modifications and clotting ability of this glycoprotein. Peroxynitrite at the concentration of 1 μM increased maximum velocity of Fg polymerization, whereas exposure to 100 μM PN resulted in a significant decrease of Vmax. (−)-Epicatechin (1-100 μM) caused a dose-dependent inhibition of 3-nitrotyrosine formation in fibrinogen treated with peroxynitrite (100 μM) in both Western blot assays and C-ELISA assays. At the highest concentration of (−)-epicatechin (100 μM) the level of 3-NT in fibrinogen reached the control values. At lower doses (−)-epicatechin reduced tyrosine nitration by approx. 23% and 40% at the concentration of 1 μM and 10 μM, respectively. (−)-Epicatechin also abolished the pro-thrombotic effect of peroxynitrite on fibrinogen clotting. The presented in vitro results demonstrated for the first time that (−)-epicatechin might have protective effects against the impairment of structure and properties of Fg, caused by action of the strong biologic oxidant/nitration and inflammatory mediators.     

Effects of peptides derived from terminal modifications of the aβ central hydrophobic core on aβ fibrillization

Considerable research effort has focused on the discovery of mitigators that block the toxicity of the β-amyloid peptide (Aβ) by targeting a specific step involved in Aβ fibrillogenesis and subsequent aggregation. Given that aggregation intermediates are hypothesized to be responsible for Aβ toxicity, such compounds could likely prevent or mitigate aggregation, or alternatively cause further association of toxic oligomers into larger nontoxic aggregates. Herein we investigate the effect of modifications of the KLVFF hydrophobic core of Aβ by replacing N- and C-terminal groups with various polar moieties. Several of these terminal modifications were found to disrupt the formation of amyloid fibrils and in some cases induced the disassembly of preformed fibrils. Significantly, mitigators that incorporate MiniPEG polar groups were found to be effective against Aβ(1-40) fibrilligonesis. Previously, we have shown that mitigators incorporating alpha,alpha-disubstituted amino acids (ααAAs) were effective in disrupting fibril formation as well as inducing fibril disassembly. In this work, we further disclose that the number of polar residues (six) and ααAAs (three) in the original mitigator can be reduced without dramatically changing the ability to disrupt Aβ(1-40) fibrillization in vitro.     

Severe cytochrome c oxidase inhibition in vivo does not induce a pyrimidine deficiency; neuroprotective action of oral uridine prodrug PN401 requires supraphysiological levels of uridine

It has been hypothesized that mitochondrial respiratory chain dysfunction leads to a pyrimidine deficiency since the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase is coupled to the electron transport chain. The uridine prodrug triacetyluridine (PN401) is neuroprotective in several models of neurodegenerative disease involving respiratory chain toxins. Therefore, the therapeutic effects of PN401 might involve the correction of a pyrimidine deficiency secondary to respiratory chain impairment. We infused mice with the cytochrome c oxidase inhibitor azide, which inhibited brain complex IV activity. Chronic infusion of azide for 2 or 14 days induced significant toxicity and mortality but did not cause a pyrimidine deficit in the brain. In contrast, the pyrimidine synthesis inhibitor N-phosphonoacetyl-l-aspartate (PALA) produced a pyrimidine deficit with minimal mortality. Treatment with 6% PN401 decreased mortality and cerebrocortical apoptosis caused by azide. Previously, we found that optimal neuroprotection against mitochondrial complex II inhibition required 4-6% PN401. PN401 at 1, 3, 6 and 10% in chow induced nonlinear increases in plasma uridine with 6% PN401 elevating plasma uridine up to 80 muM, and these higher micromolar uridine levels were also required for neuroprotection in chemical hypoxia models in vitro. Our results indicate that severe complex IV inhibition in vivo does not lead to a pyrimidine deficiency, and therefore the protective effect of PN401 in the azide toxin model is not mediated through the correction of a pyrimidine deficiency. Furthermore, supraphysiological levels of uridine are required to produce optimal protective effects in disorders involving impairment of mitochondrial respiratory complex II or IV.     

Integrin alpha3beta1 suppresses long-term potentiation at inhibitory synapses on the cerebellar Purkinje neuron

At the GABAergic synapses between inhibitory interneurons and a Purkinje neuron (PN) in the cerebellum, the postsynaptic depolarization induces the long-term potentiation (called rebound potentiation; RP) of GABAA receptor responsiveness. Here, we show that integrins, a type of cell-adhesion molecules, are involved in the regulation of RP. Integrin activation by Mn2+ impaired the RP induction of GABA responsiveness and mIPSCs in PNs, which was abolished by the function blocking antibody against either integrin alpha3 or beta1 subunit, but not by that against alpha5 or alphaV subunit. Furthermore, overexpression of integrin alpha3 subunit in a PN by itself impaired the RP induction. We also show that Src-family of protein tyrosine kinases mediated the suppressive effect of integrin activity on the RP induction. Thus, the integrin/Src pathway negatively regulates the induction of long-term plasticity at inhibitory synapses on a cerebellar PN.     

A possible link between astrocyte activation and tau nitration in Alzheimer's disease

Alzheimer's disease (AD) pathology has been characterized, in part, by the self-assembly of the tau molecule into neurofibrillary tangles (NFT). While different post-translational modifications have been identified that accelerate tau aggregation, nitration at tyrosine residues prevents or slows tau filament formation in vitro. Of the five tyrosine residues within the molecule, nitration at the first tyrosine residue (Tyr 18) results in a profound inhibition of filament self-assembly. To determine whether nitration at Tyr 18 occurs in AD pathology, monoclonal antibodies were raised against a synthetic tau peptide nitrated at Tyr 18. A clone, termed Tau-nY18, reacts specifically with tau proteins nitrated at Tyr 18 and fails to cross-react with other nitrated tyrosine residues spanning the length of the molecule or with other proteins known to be nitrated in neurodegenerative diseases. In situ, Tau-nY18 sparsely labels the neuronal pathological hallmarks of the disease, including NFT and dystrophic neurites. Surprisingly however, Tau-nY18 robustly labels nitrated tau within activated, GFAP positive astrocytes intimately associated with amyloid plaques. Furthermore, this antibody detects nitrated tau in soluble preparations from both severe AD brains (Braak stage V, VI) and age-matched controls. Collectively, these findings suggest that nitration at Tyr 18 may be linked to astrocyte activation, an early event associated with amyloid plaque formation.     

Proteolytic cleavage of the voltage-gated Ca2+ channel alpha2delta subunit: structural and functional features

By mediating depolarization-induced Ca(2+) influx, high-voltage-activated Ca(2+) channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (alpha(1)) and three auxiliary (alpha(2)delta, beta and gamma) subunits. The alpha(2)delta subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, alpha(2)delta must undergo important post-translational modifications, including a proteolytic cleavage that separates the extracellular alpha(2) from its transmembrane delta domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature alpha(2)delta, almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of alpha(2)delta was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide-reducing agent. Interestingly, these mutations significantly decreased whole-cell patch-clamp currents without affecting the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane.     

Simplified recombinant plasmin: production and functional comparison of a novel thrombolytic molecule with plasma-derived plasmin

A simplified and fully functional deletion mutant of plasminogen was created in which the middle portion of the molecule was removed, resulting in kringle 1 attachment to the serine protease domain. This recombinant plasminogen deletion mutant, Delta(K2-K5)Pg, was produced in the form of inclusion bodies at the yield of up to 200 mg/l in an Escherichia coli T7 expression system. Following protein refolding and purification on lysine-Sepharose, the conversion of the recombinant molecule Delta(K2-K5)Pg to the active enzyme mutant Delta(K2-K5)Pm by plasminogen activators was evaluated, and functional characteristics of the simplified plasmin were studied. Properties of Delta(K2-K5)Pg were similar to native, human plasma-derived plasminogen. Delta(K2-K5)Pg effectively bound epsilon-aminocaproic acid (K(d) = 11.3 +/- 2.3 muM) and fibrin (C(50) approximately 0.3 muM). The plasminogen activators streptokinase, urokinase, and tissue plasminogen activator effectively converted the recombinant zymogen Delta(K2-K5)Pg to the active recombinant enzyme, Delta(K2-K5)Pm. Additionally, Delta[K2-K5]Pm was rapidly inhibited by alpha(2)-antiplasmin (1.1 +/- 0.1 x 10(7) M(-1)s(-1)) and alpha(2)-macroglobulin (7.6 +/- 0.6 x 10(5) M(-1)s(-1)). In an in-vitro model, Delta(K2-K5)Pm demonstrated fibrinolytic potency comparable to human plasma-derived plasmin. Because of their unique biochemistry, including fibrin-binding properties and rapid inhibition by alpha(2)-antiplasmin, both native plasmin and a simplified deletion mutant of plasmin are potentially safe and effective direct thrombolytic agents for various thrombotic conditions. Further studies evaluating the in-vivo pharmacologic safety and clinical efficacy of this simplified plasmin (i.e. Delta[K2-K5]Pm) are warranted.     

A sulphated polysaccharide - the effects in vivo and in vitro on the haemostatic system in healthy volunteers

Fischer rats were immunized with fragment D (Fg-D) of human fibrinogen (Fg) to obtain antibody specific for neoantigens unique to this molecule. Absorption of serum with whole Fg indicated that some of the antibody produced reacted preferentially with Fg-D. Hybridoma cultures were prepared by fusion of immune rat spleen cells with mouse myeloma P3-X63-Ag8. Monoclonal antibodies obtained from these cultures fell into two classes: (a) Those reacting equally well with Fg and Fg-D. (b) Those reacting preferentially but not absolutely with Fg-D. Antibody from hybridoma 104-14, a member of the first group had an affinity for Fg-D of 1.5 × 10⁹ M^?1 while antibodies from 106-59 and 106-71 (group 2) demonstrated much lower affinities of 1.0 × 10⁷ and 4.7 × 10⁶ M^?1, respectively. The cross reactivity of antibodies in the second group indicated that they react with protein conformations that are altered during production of Fg-D from Fg.     

Characterization of neurons of the nucleus tractus solitarius pars centralis

Esophageal sensory afferent inputs terminate principally in the central subnucleus of the tractus solitarius (cNTS). Neurons of the cNTS comprise two major neurochemical subpopulations. One contains neurons that are nitric oxide synthase (NOS) immunoreactive (-IR) while the other comprises neurons that are tyrosine hydroxylase (TH)-IR. We have shown recently that TH-IR neurons are involved in esophageal-distention induced gastric relaxation. We used whole cell patch clamp techniques in rat brainstem slices combined with immunohistochemical and morphological reconstructions to characterize cNTS neurons. Postrecording reconstruction of cNTS neurons revealed two morphological neuronal subtypes; one group of cells (41 out of 131 neurons, i.e., 31%) had a multipolar soma, while the other group (87 out of 131 neurons, i.e., 66%) had a bipolar soma. Of the 43 cells in which we conducted a neurochemical examination, 15 displayed TH-IR (9 with bipolar morphology, 6 with multipolar morphology) while the remaining 28 neurons did not display TH-IR (18 with bipolar morphology, 10 with multipolar morphology). Even though the range of electrophysiological properties varied significantly, morphological or neurochemical distinctions did not reveal characteristics peculiar to the subgroups. Spontaneous excitatory postsynaptic currents (sEPSC) recorded in cNTS neurons had a frequency of 1.5 +/- 0.15 events s(-1) and an amplitude of 27 +/- 1.2 pA (Vh = -50 mV) and were abolished by pretreatment with 30 muM AP-5 and 10 muM CNQX, indicating the involvement of both NMDA and non-NMDA receptors. Some cNTS neurons also received a GABAergic input that was abolished by perfusion with 30-50 muM bicuculline. In conclusion, our data show that despite the heterogeneity of morphological and neurochemical membrane properties, the electrophysiological characteristics of cNTS neurons are not a distinguishing feature.     

Narcolepsy: regional cerebral blood flow during sleep and wakefulness.

Serial measurements of regional cerebral blood flow were made by the 135Xe inhalation method during the early stages of sleep and wakefulness in eight normal volunteers and 12 patients with narcolepsy. Electroencephalogram, electro-oculogram, and submental electromyogram were recorded simultaneously. In normals, mean hemispheric gray matter blood flow (Fg) during stages I and II sleep was significantly less (-9.2 percent) than waking values (84.3 +/- 13 ml per 100 gm brain per minute). Maximum regional blood flow decreases during sleep occurred in the brainstem-cerebellar (-25.1 percent), right inferior temporal (-23.1 percent) and bilateral frontal (-18.9 percent) regions (p less than 0.05). In patients with narcolepsy, mean hemispheric Fg while awake was 80.5 +/- 13 ml per 100 gm brain per minute. During REM sleep (n = 2), mean hemispheric Fg increased by 9.8 percent concurrently with large increases (+34.6 percent) in brainstem-cerebellar region flow. During stages I and II sleep without REM (n = 6), there were significant increases in mean hemispheric Fg of +/-20.2 percent (p less than 0.01) and brainstem-cerebellar Fg of 38.0 percent (p less than 0.01), just the opposite of changes in normals. In narcolepsy, there appears to be a reversal of normal cerebral deactivation patterns, particularly involving the brainstem, during stages I and II sleep.     

Sex differences in GABAAergic system in rat substantia nigra pars reticulata

The substantia nigra pars reticulata (SNR) is involved in the control of movement disorders including seizures through its GABAergic neurons. Microinfusions of muscimol (a GABA(A) receptor agonist) produce specific effects on seizures depending on sex, infusion site (SNR(anterior) or SNR(posterior)) and age. To assess whether these effects are due to sex differences in GABAergic indices within the SNR we analyzed the expression of alpha(1) subunit mRNA of the GABA(A) receptor and the levels of GABA immunoreactivity (IR) of male and female rats at postnatal day 15 (PN15) and PN30. In each age, within the same SNR region, expression of alpha(1) subunit mRNA and intensity of GABA IR per neuron was higher in females compared to males. At PN15, in both sexes, there were no regional differences in expression of alpha(1) subunit mRNA and intensity of GABA IR. However, at PN30 in both sexes, expression of alpha(1) subunit mRNA and intensity of GABA IR per cell was higher in SNR(anterior) than in SNR(posterior). These results demonstrate that expression of alpha(1) subunit mRNA for GABA(A) receptor and levels of GABA IR in the SNR are sex- and site-specific, which may contribute to sex-, regional- and age-related differences in the expression of movement disorders and seizures.     

Tyrosine phosphorylation of L1 family adhesion molecules: Implication of the Eph kinase Cek5

The L1 family comprises transmembrane cell adhesion molecules of the immunoglobulin superfamily that play an important role in neuronal migration and axon outgrowth, fasciculation, and myelination. Consistent with a crucial role in developmental processes, mutations in L1 cause severe brain malformations. Although L1 activates intracellular signaling pathways, little is known about the membrane proximal events of L1 signaling. The cytoplasmic domains of L1 family proteins contain several conserved tyrosine residues that are potential targets for receptor tyrosine kinases. Here, we report that the L1 family protein Ng-CAM is phosphorylated on tyrosine in embryonic day 13 chicken retina. This is the first demonstration of in (vivo) tyrosine phosphorylation of an L1-like molecule. Because chicken embryo kinase 5 (Cek5) is a receptor tyrosine kinase expressed in neuronal processes and activated in the chicken embryonic retina, we have analyzed the possible role of Cek5 in L1 phosphorylation. The rat glioblastoma cell line B28 was stably transfected with human L1. Additional transient transfection with Cek5 cDNA led to expression of Cek5 in its tyrosine-phosphorylated, activated form. Biochemical analysis revealed that L1 is phosphorylated on tyrosine in Cek5-transfected cells but not in control transfectants. Furthermore, direct phosphorylation of the L1 cytoplasmic domain by Cek5 was demonstrated in an in vitro kinase assay. Tyrosine phosphorylation may represent a novel mechanism of signal cascade initiation through L1. J. Neurosci. Res. 47:655–665, 1997. © 1997 Wiley-Liss, Inc.     

Beneficial effects of carnosic acid on dieldrin-induced dopaminergic neuronal cell death

Carnosic acid (CA) is one of the bioactive polyphenols present in extracts of the herb rosemary (Rosmarinus officinalis). In this study, we examined possible protective effects of CA on neurotoxicity induced by dieldrin, an organochlorine pesticide implicated in sporadic Parkinson's disease, in cultured dopaminergic cells (SN4741). CA (5-10 muM) pretreatment showed potent protective effects in a concentration-related manner and prevented dieldrin (10 muM)-induced caspase-3 activation, Jun N-terminal kinase phosphorylation, and caspase-12 activation. Furthermore, dieldrin-induced downregulation of brain-derived neurotrophic factor production was significantly attenuated by CA. These results suggest that CA may safeguard dopaminergic neuronal cells from environmental neurotoxins by enhancing brain-derived neurotrophic factor and repressing apoptotic molecules.     

Oxidation of human alpha-thrombin by the myeloperoxidase-H2O2-chloride system: structural and functional effects

The myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This, study investigated the biochemical and functional effects of alpha-thrombin oxidation by MPOS. This system, in the presence of 100 microM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCI, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human alpha-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5-fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2-fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin.     

Identification of residues of functional importance within the central turn motifs present in the cytoplasmic tails of integrin alphaIIb and alphaV subunits

INTRODUCTION: Previous studies demonstrated that cell-permeable alpha(IIb) cytoplasmic peptides can modulate the activation of alpha(IIb)beta(3). An integrin activation motif was mapped to its membrane proximal region and a double proline mutant peptide and receptor indicated that its central turn motif had inhibitory capacity. However, the residues critical for inhibition of alpha(IIb)beta(3) activation were not identified. Using central turn peptides derived from alpha(IIb) and alpha(V), residues critical for suppression of integrin activation were identified and the importance of these residues in protein-protein interactions was assessed. MATERIALS AND METHODS: Cell-permeable peptides were used to determine the capacity of the central turn peptides to suppress alpha(IIb)beta(3) and alpha(V)beta(3) activation. Far Western analysis was used to characterize the capacity of the peptides to interact with CIB1 and surface plasmon resonance was used to characterize the binding of an antibody to the cytoplasmic tails of alpha(IIb) and alpha(V). RESULTS AND CONCLUSIONS: The central turn peptide from alpha(V), alpha(V)(993-1001), has full inhibitory capacity while that derived from alpha(IIb) requires additional residues located adjacent to alpha(IIb)(995-1003). Within these two sequences there is a switch in the position of an asparaginine and leucine residue for a valine and glutamine (alpha(IIb), RNRPPLEED; alpha(V), RVRPPQEEQ). This switch had a dramatic effect on their inhibitory capacity and on protein-protein interactions. The two arginine and glutamic residues, juxtapositioned at identical locations in both subunits, appeared to be important in specifying the orientation by which proteins can dock to this region in alpha(IIb) and alpha(V).     

The interaction of von Willebrand factor (vWf) with collagen: investigation of vWf-binding sites in the collagen molecule

Following fragmentation of the collagen molecule, we have examined the ability of the isolated fragments to bind vWf. In view of the importance of collagen tertiary and quaternary structure for binding, fragments were first renatured to restore triple-helical conformation and then polymerized. Results indicate the presence of specific vWf-binding sites in both the alpha 1(I)- and alpha 2(I)-chains of type I collagen. Cleavage of the alpha 1(I)-chain with cyanogen bromide suggests the presence of at least four (conceivably several more) binding sites implying a wide distribution of sites along the length of the collagen type I molecule. Collagen type III appears to possess a similar wide distribution of sites. Chemical modification of specific amino acid residues indicates that interaction involves arginyl residues in collagen and carboxyl groups in vWf. Although interaction between fibronectin and collagen fibres also involves collagen arginyl residues and carboxyl groups in fibronectin (authors' unpublished results), fibronectin does not compete with vWf in the binding to collagen fibres.     

The onset of dopaminergic innervation during ontogeny decreases melanotrope proliferation in the intermediate lobe of the rat pituitary

The onset of dopaminergic innervation and its effects on melanotrope proliferation were investigated in the rat pituitary intermediate lobe. Dopamine, and its synthetic rate-limiting enzyme tyrosine hydroxylase, were first detected immunohistochemically on late post-natal day 3 or early postnatal day 4. Axon density was highest at the neural lobe/intermediate lobe border, and decreased toward the pituitary cleft. By postnatal day 10, the adult pattern of tyrosine hydroxylase immunoreactivity was established and remained through post-natal day 14. Neurointermediate lobe dopamine levels, measured by HPLC, correlated well with the increased axon density observed in the immunohistochemical studies. Dopamine could not be measured by our assay (100 fg limit) until post-natal day 3 (439.32 fg/NIL). Dopamine concentration increased to 2.09 +/- 0.425 ng at PN 4, 86.31 +/- 20.42 ng at PN 7, 168.72 +/- 18.37 ng at PN 10. Melanotrope proliferation was determined by [3H]thymidine incorporation before and after innervation. Concomitant with the onset of innervation, the proliferation index dropped from 13.4 +/- 0.01% to 6.5 +/- 0.002% at PN 4, and continued to decrease until a level of 3 +/- 0.003% was established by PN 10. To confirm the inhibitory action of dopaminergic innervation on melanotrope proliferation, rat neonates were injected intracisternally with 150 mg 6-hydroxydopamine to destroy dopaminergic axons within the intermediate lobe. Measurement of dopamine concentrations in neurointermediate lobes of injected animals showed a decrease in dopamine levels as compared to controls. From PN 4 (0.88 +/- 0.165 ng), DA levels gradually increased during development: at PN 5, [DA] = 0.689 +/- 0.104 ng; PN 6 [DA] = 11.60 +/- 2.24 ng; PN 7 [DA] = 20.93 +/- 3.80 ng; and PN 10 [DA] = 27.95 +/- 3.46 ng. Melanotrope proliferation also increased in 6-hydroxydopamine-treated animals. At PN 4, the onset of innervation reduced the pre-innervation proliferation index to 8.75 +/- 0.002%, only a 30% reduction in contrast to the greater than 50% decrease observed in control animals. A stable proliferation level of approximately 7.5% persisted in all subsequent stages with 6-OHDA administration. Our results demonstrated the time of dopamine innervation onset and a characteristic developmental pattern for axons within the rat intermediate lobe. The onset of innervation and increased dopamine concentration suggests increased dopaminergic control of the melanotropes, illustrated specifically by a decrease in their level of proliferation. This is the first presentation of evidence showing that dopaminergic innervation within the intermediate lobe of the rat pituitary regulates melanotrope proliferation.     

Uptake of 3,4-dimethoxyphenylethylamine-1-14C (14C-DMPEA) by rat tissues in vitro.

Rat heart and spleen slices were incubated with 3,4-dimethoxyphenylethylamine-1-14C(14C-DMPEA) in Krebs medium at 37 C. At the end of 5-20 min of incubation, the heart did not take up the radioactivity while the spleen did. The Km and Vmax values of uptake in the spleen were 1 x 10(-4) M and 20 nmole/g per min, respectively, and the uptake was reduced to 16.0-35.1% in the cold (4 C) and to 40.3-64.0% in Na+-free medium. Thus, the uptake was an energy-dependent active process but was only partially Na+-dependent. Spleen slices incubated with 14C-DMPEA-free medium for 15 min following incubation with 14C-DMPEA retained 41.0-74.8% of radioactivity. The uptake was insensitive to norepinephrine (0.313 and 0.939 muM), dopamine (9.98 muM), 5-hydroxytryptamine (5 muM), cocaine (14.8 muM), 1-amphetamine (0.3 and 300 muM), d-amphetamine (300 muM), and normetanephrine (45.7 muM). 6-Hydroxydopamine treatment of rats, which produced 93% reduction in the splenic norepinephrine content, did not significantly reduce uptake. Thus, the uptake of DMPEA into the spleen is not by adrenergic neurones.     

Peroxynitrite inactivation of tyrosine hydroxylase: mediation by sulfhydryl oxidation, not tyrosine nitration.

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the biosynthesis of dopamine (DA). TH activity is significantly diminished in Parkinson's disease (PD) and by the neurotoxic amphetamines, thereby accentuating the reductions in DA associated with these conditions. Reactive oxygen and nitrogen species have been implicated in the damage to DA neurons seen in PD and in reaction to amphetamine drugs of abuse, so we investigated the hypothesis that peroxynitrite (ONOO(-)) could interfere with TH catalytic function. ONOO(-) caused a concentration-dependent inactivation of TH. The inactivation was associated with tyrosine nitration (maximum of four tyrosine residues nitrated per TH monomer) and extensive sulfhydryl oxidation. Tetranitromethane, which causes sulfhydryl oxidation at pH 6 and 8 but which nitrates tyrosines only at pH 8, inactivated TH equally at either pH. Bicarbonate protected TH from ONOO(-)-induced inactivation and sulfhydryl oxidation but increased significantly tyrosine nitration. PNU-101033 blocked ONOO(-)-induced tyrosine nitration in TH but could not prevent enzyme inactivation or sulfhydryl oxidation. Together, these results indicate that the inactivation of TH by ONOO(-) is mediated by sulfhydryl oxidation. The coincident nitration of tyrosine residues appears to exert little influence over TH catalytic function.     

Structural Analysis of the Murine Cell Adhesion Molecule L1 by Electron Microscopy and Computer-assisted Modelling

In the present study we have analysed the morphology of two fragments with apparent molecular weights of 180 and 140 kDa (L1-180 and L1-140) derived from the extracellular region of the murine neural cell adhesion molecule L1. The fragment L1-180 consists of almost the entire extracellular part of the molecule, and is built up of six immunoglobulin-like and five fibronectin type III-like domains. Fragment L1-140 lacks one-half of the third, the fourth and the fifth fibronectin type III-like domains. By electron microscopic analysis of rotary-shadowed molecules, L1-140 and L1-180 revealed fibrillar structures 31-43 nm long and 7-12 nm wide with one pronounced globular terminal domain. As determined by complex formation with an L1 antibody, this terminal part of the molecule is formed by the fibronectin type III-like domains. The individual structures showed variation and complexity, and four distinct aspects were identified. These different forms probably represent two-dimensional projections of the same three-dimensional helical structure. Computer-assisted modelling of the L1 molecule, i.e. the protein backbone, showed no strong intramolecular interaction between the different fibronectin type III- or Ig-like domains, suggesting that the formation of the globular part of the molecule is probably achieved by protein-carbohydrate and/or carbohydrate-carbohydrate rather than protein-protein interactions. In addition, our model proposes that interactions occur within the interfaces between the different domains. The highly conserved amino acid residues in these regions point to the necessity of maintaining the orientation between the different domains.