Linking adhesive and structural proteins in the attachment plaque of Mytilus californianus

The byssal attachment of California mussels Mytilus californianus provides secure adhesion in the presence of moisture, a feat that still eludes most synthetic polymers. Matrix-assisted laser desorption ionization mass spectrometry was used to probe the footprints of byssal attachment plaques on glass cover slips for adhesive proteins. Besides the abundant mcfp-3 protein family (Zhao, H., Robertson, N. B., Jewhurst, S. A., and Waite, J. H. (2006) J. Biol. Chem. 281, 11090-11096), two new proteins, mcfp-5 and mcfp-6, with masses of 8.9 kDa and 11.6 kDa, respectively, were identified in footprints, partially characterized and completely sequenced from a cDNA library. mcfp-5 resembles mcfp-3 in its basic pI and abundant 3,4-dihydroxyphenyl-L-alanine (Dopa; 30 mol %), but is distinct in two respects: it is more homogeneous in primary sequence and is polyphosphorylated. mcfp-6 is basic and contains a small amount of Dopa (<5 mol %). In contrast to mcfp-3 and -5, tyrosine prevails at 20 mol %, and cysteine is present at 11 mol %, one-third of which remains thiolate. Given the oxidative instability of Dopa and cysteine at pH 8.2 (seawater), we tested the hypothesis that thiols serve to scavenge dopaquinones by adduct formation. Plaque footprints were hydrolyzed and screened for cysteine dopaquinone adducts using phenylboronate affinity chromatography. 5-S-Cysteinyldopa was detected at nearly 1 mol %. The results suggest that mcfp-6 may provide a cohesive link between the surface-coupling Dopa-rich proteins and the bulk of the plaque proteins.     

Influence of phospholipid asymmetry on fusion between large unilamellar vesicles.

The ability of lipid asymmetry to regulate Ca(2+)-stimulated fusion between large unilamellar vesicles has been investigated. It is shown that for 100-nm-diameter LUVs composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylinositol, and dioleoylphosphatidic acid (DOPC/DOPE/PI/DOPA; 25:60:5:10) rapid and essentially complete fusion is observed by fluorescent resonance energy transfer techniques when Ca2+ (8 mM) is added. Alternatively, for LUVs with the same lipid composition but when DOPA was sequestered to the inner monolayer by incubation in the presence of a pH gradient (interior basic), little or no fusion is observed on addition of Ca2+. It is shown that the extent of Ca(2+)-induced fusion correlates with the amount of exterior DOPA. Further, it is shown that LUVs containing only 2.5 mol % DOPA, but where all the DOPA is in the outer monolayer, can be induced to fuse to the same extent and with the same rate as LUVs containing 5 mol % DOPA. These results strongly support a regulatory role for lipid asymmetry in membrane fusion and indicate that the fusogenic tendencies of lipid bilayers are largely determined by the properties of the monolayers proximate to the fusion interface.     

Transmitter-Like Release of Endogenous 3,4-Dihydroxyphenylalanine from Rat Striatal Slices

Biphasic electrical field stimulation (0.5-5 Hz, 2 ms, 25 V, 3 min) and high K+ (10-30 mM, 5 min) released endogenous 3,4-dihydroxyphenylalanine (DOPA) from superfused rat striatal slices. Characteristics of the DOPA release were compared with those of 3,4-dihydroxyphenylethylamine (dopamine, DA). Electrical stimulation at 2 Hz evoked DOPA and DA over similar time courses. alpha-Methyl-p-tyrosine (0.2 mM) markedly reduced release of DOPA but not of DA. Maximal release (0.3 pmol) of DOPA was obtained at 2 Hz and at 15 mM K+. The impulse-evoked release of DOPA and DA was completely tetrodotoxin (0.3 microM) sensitive and Ca2+ dependent and the 15 mM K+-evoked release was also Ca2+ dependent. On L-[3,5-3H]tyrosine (1 microM) superfusion, high K+ (15 and 60 mM) released DOPA and DA together with concentration-dependent decreases in tyrosine 3-monooxygenase (EC 1.14.16.2) activity as indicated by [3H]H2O formation, followed by concentration-dependent increases after DOPA and DA release ended. These findings suggest that striatal DOPA is released by a Ca2+-dependent excitation-secretion coupling process similar to that involved in transmitter release.     

Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor

The effects of cholesterol (Chol) and an anionic lipid, dioleoylphosphatidic acid (DOPA) on the conformational equilibria of the nicotinic acetylcholine receptor (nAChR) have been investigated using Fourier transform infrared difference spectroscopy. The difference between spectra recorded in the presence and absence of agonist from the nAChR reconstituted into 3:1:1 egg phosphatidylcholine (EPC)/DOPA/Chol membranes exhibits positive and negative bands that serve as markers of the structural changes associated with the resting to desensitized conformational change. These markers are absent in similar difference spectra recorded from the nAChR reconstituted into EPC membranes lacking both Chol and DOPA, indicating that the nAChR cannot undergo conformational change in response to agonist binding. When low levels of either Chol or DOPA up to 25 mol % of the total lipid are included in the EPC membranes, the markers suggest the predominant stabilization of a conformation that is a structural intermediate between the resting and desensitized states. At higher levels of either Chol or DOPA, the nAChR is stabilized in a conformation that is capable of undergoing agonist-induced desensitization, although DOPA appears to be required for the nAChR to adopt a conformation fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. The ability of these two structurally diverse lipids, as well as others (Ryan, S. E., Demers, C. N., Chew, J. P., Baenziger, J. E. (1996) J. Biol. Chem. 271, 24590-24597), to modulate the functional state of the nAChR suggests that lipids act on the nAChR via an indirect effect on some physical property of the lipid bilayer. The data also suggest that anionic lipids are essential to stabilize a fully functional nAChR. We propose that membrane fluidity modulates the relative populations of nAChRs in the resting and desensitized states but that subtle structural changes in the presence of anionic lipids are essential for full activity.     

Effect of 3,4-dihydroxyphenylalanine on Cu(2+)-induced inactivation of HDL-associated paraoxonasel and oxidation of HDL; inactivation of paraoxonasel activity independent of HDL lipid oxidation

Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu²⁺-mediated inactivation of PON1 was examined. Cu²⁺-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu²⁺-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu²⁺, but not Fe²⁺. The DOPA/Cu²⁺-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu²⁺-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu²⁺. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu²⁺-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu²⁺-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu²⁺-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu²⁺ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu²⁺-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.     

Effect of electrostatic interactions on phase stability of cubic phases of membranes of monoolein/dioleoylphosphatidic acid mixtures.

To elucidate effects of electrostatic interactions resulting from surface charges on structures and phase stability of cubic phases of lipid membranes, membranes of 1-monoolein (MO) and dioleoylphosphatidic acid (DOPA) (DOPA/MO membrane) mixtures have been investigated by small-angle x-ray scattering method. As increasing DOPA concentration in the DOPA/MO membrane at 30 wt% lipid concentration, a phase transition from Q(224) to Q(229) phase occurred at 0.6 mol% DOPA, and at and above 25 mol% DOPA, DOPA/MO membranes were in the L(alpha) phase. As NaCl concentration in the bulk phase increased, for 10% DOPA/90% MO membrane in excess water, a Q(229) to Q(224) phase transition occurred at 60 mM NaCl, and then a Q(224) to H(II) phase transition occurred at 1.2 M NaCl. Similarly, for 30% DOPA/70% MO membrane in excess water, at low NaCl concentrations it was in the L(alpha) phase, but at and above 0.50 M NaCl it was in the Q(224) phase, and then at 0.65 M NaCl a Q(224) to H(II) phase transition occurred. These results indicate that the electrostatic interactions in the membrane interface make the Q(229) phase more stable than the Q(224) phase, and that, at larger electrostatic interactions, the L(alpha) phase is more stable than the cubic phases (Q(224) and Q(229)). We have found that the addition of tetradecane to the MO membrane induced a Q(224)-to-H(II) phase transition and also that to the 30% DOPA/70% MO membrane induced an L(alpha)-to-H(II) phase transition. By using these membranes, the effect of the electrostatic interactions resulting from the membrane surface charge (DOPA) on the spontaneous curvature of the monolayer membrane has been investigated. The increase in DOPA concentration in the DOPA/MO membrane reduced the absolute value of spontaneous curvature of the membrane. In the 30% DOPA/70% MO membrane, the absolute value of spontaneous curvature of the membrane increased with an increase in NaCl concentration. On the basis of these new results, the phase stability of DOPA/MO membranes can be reasonably explained by the spontaneous curvature of the monolayer membrane and a curvature elastic energy of the membrane.     

The role of prostaglandins in the excitatory innervation of the rat urinary bladder

The possible role of PGs in hyoscine-resistant nerve mediated responses of the rat urinary bladder was investigated. Responses to electrical stimulation were inhibited by cinchocaine (30 μmol/l) but were only partially inhibited by a high concentration of hyoscine (25 μmol/l) or by the choline uptake inhibitors, hemicholinium-3 (500 μmol/l) and troxypyrrolidinium (500 μmol/l). Indomethacin (50 μmol/l) produced partial blockade (30%) of responses to electrical stimulation without markedly affecting responses to acetylcholine and the degree of blockade was of a similar order in the presence of hyoscine or troxypyrrolidinium. PGE₂ (0.028 – 2.8 μmol/l) or F_2α (0.029 – 2.9 μmol/l) produced a slowly developing increase in tone and spontaneous activity. Responses to electrical stimulation were at most only slightly increased in the presence of either PG. However, the PGs always increased the responses to electrical stimulation after indomethacin, indomethacin plus hyoscine or indomethacin plus troxypyrrolidinium. Responses to acetylcholine in the presence of indomethacin were not increased by PGE₂. It is concluded that PGE₂ and F_2α do not function as transmitters responsible for resistance to anti-muscarinic drugs in the bladder but may exert a modulating effect on nervous transmission.     

Adhesion of mussel foot protein mefp-5 to mica: an underwater superglue

Mussels have a remarkable ability to attach their holdfast, or byssus, opportunistically to a variety of substrata that are wet, saline, corroded, and/or fouled by biofilms. Mytilus edulis foot protein-5 (Mefp-5) is one of several proteins in the byssal adhesive plaque of the mussel M. edulis. The high content of 3,4-dihydroxyphenylalanine (Dopa) (～30 mol %) and its localization near the plaque-substrate interface have often prompted speculation that Mefp-5 plays a key role in adhesion. Using the surface forces apparatus, we show that on mica surfaces Mefp-5 achieves an adhesion energy approaching E(ad) = ～-14 mJ/m(2). This exceeds the adhesion energy of another interfacial protein, Mefp-3, by a factor of 4-5 and is greater than the adhesion between highly oriented monolayers of biotin and streptavidin. The adhesion to mica is notable for its dependence on Dopa, which is most stable under reducing conditions and acidic pH. Mefp-5 also exhibits strong protein-protein interactions with itself as well as with Mefp-3 from M. edulis.     

Role of tyrosine, DOPA and decarboxylase enzymes in the synthesis of monoamines in the brain of the locust

The metabolic transformation of tyrosine (TYR) by the decarboxylase and hydroxylase enzymes was investigated in the central nervous system of the locust, Locusta migratoria. It has been demonstrated that the key amino acids, 3,4-dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan (5HTP) and tyrosine are decarboxylated in all part of central nervous system. DOPA and 5HTP decarboxylase activities show parallel changes in the different ganglia, but the rank order of the activity of TYR decarboxylase is different. Enzyme purification has revealed that the molecular weights of TYR decarboxylase and DOPA/5HTP decarboxylase are 370,000 and 112,000, respectively. The decarboxylation of DOPA by DOPA/5HTP decarboxylase is stimulated, whereas the decarboxylation of DOPA by TYR decarboxylase is inhibited in the presence of the cofactor pyridoxal-5'-phosphate. TYR hydroxylase could not be detected and 3H-TYR is found to be metabolised to tyramine (TA), but not to DOPA. The haemolymph contains a significant concentration of DOPA (120 pmol/100 microl haemolymph), and the ganglia incorporates DOPA from the haemolymph by a high affinity uptake process (K(M)=12 microM and V(max)=24 pmol per ganglion/10 min). Our results suggest that no tyrosine hydroxylase is present in the locust CNS and the DOPA uptake into the ganglia by a high affinity uptake process as well as the DOPA decarboxylase enzyme may be responsible for the regulation of the ganglionic dopamine (DA) level. Two types of decarboxylases exist, one of them decarboxylating DOPA and 5HTP (DOPA/5HTP decarboxylase), other decarboxylating TYR (TYR decarboxylase). The DOPA/5HTP decarboxylase enzyme present in the insect brain may correspond to the 5HTP/DOPA decarboxylase in vertebrate brain, whereas TYR decarboxylase is characteristic only for the insect brain.     

An organic solvent resistant tyrosinase from Streptomyces sp. REN-21: purification and characterization

We found a tyrosinase, which has high activity in the presence of organic solvents, in the culture filtrate of Streptomyces sp. REN-21. The organic solvent resistant tyrosinase (OSRT) was purified from the culture filtrate by three column chromatographies. About 1.2 mg of purified OSRT was obtained from 5.6 liters of the culture filtrate with a yield of 26.0%. The purified enzyme had a single polypeptide chain with a molecular mass of about 32,000 Da. The optimum pH and temperature of OSRT were pH 7.0 and 35 degrees C using L-beta-(3,4-dihydroxyphenyl)alanine (L-DOPA) as substrate. OSRT showed stereospecificity toward L-, DL-, and D-enantiomers of DOPA or tyrosine. OSRT had 44% of the activity of the control even in the presence of 50% ethanol, while a mushroom tyrosinase showed only 6% activity under the same conditions. Moreover, OSRT retained its original activity even after 20 h of incubation at 30 degrees C in the presence of 30% ethanol.     

Polyphosphoprotein from the adhesive pads of Mytilus edulis

Achieving a satisfactory biochemical explanation for the opportunistic underwater adhesion of marine invertebrates such as mussels and barnacles requires a detailed characterization of proteins extracted from holdfast structures produced by these organisms. Mefp-5 is an adhesive protein derived from the foot of the common mussel, Mytilus edulis, and deposited into the byssal attachment pads. Purification and primary structure of mefp-5 was determined by peptide mapping and cDNA sequencing. The protein is 74 residues long and has a mass of about 9500 Da. Mefp-5 composition shows a strong amino acid bias: aromatic amino acids, lysine, and glycine represent 65 mol % of the composition. More than a third of all the residues in the protein are posttranslationally modified by hydroxylation or phosphorylation. The conversion of tyrosine to 3, 4-dihydroxyphenyl-L-alanine (DOPA) and serine to O-phosphoserine accounts for the hydroxylation and phosphorylation, respectively. Neither modification is complete since variations in the extent of phosphorylation and hydroxylation can be detected by mass spectrometry. More than 75% of the DOPA is adjacent to basic residues, e.g., Lys-DOPA and DOPA-Lys. Phosphoserine occurs in sequences strikingly reminiscent of acidic mineral-binding motifs that appear in statherin, osteopontin, and others. This may be an adaptation for adhesion to the most common substrata for mussels, i.e., calcareous materials.     

Increased tyrosine availability increases brain regional DOPA levels in vivo

Tyrosine hydroxylation is considered to be the rate-limiting step in catecholamine synthesis. It is also assumed that under usual conditions, tyrosine 3-monooxygenase (EC 1.14.16.2) (tyrosine hydroxylase - TH) is close to full saturation with its l-tyrosine substrate and hence that raising the availability of l-tyrosine does not substantially increase 3,4-dihydroxyphenylalanine (DOPA) synthesis. We evaluated this in vivo by reverse dialysis of the aromatic-l-amino-acid decarboxylase (EC 4.1.1.28) inhibitor NSD-1015 (20 μM) and selected concentrations of l- or d-tyrosine. In striatum, extracellular DOPA levels increased linearly (maximum 250% control) as l-tyrosine concentrations were raised from 0–1000 μM. In medial prefrontal cortex, DOPA levels reached a maximum (300% control) at l-tyrosine 62.5–125 μM but still remained significantly elevated (200% control) at higher l-tyrosine concentrations (250–500 μM). At the l-tyrosine concentrations tested, DOPA levels were never below those of controls. d-tyrosine (62.5 μM) did not affect DOPA levels.     

Differences in properties between aromatic amino acid: Aromatic keto acid aminotransferases and aromatic amino acid: α-ketoglutarate aminotransferases

Homogenates of rat liver transaminate phenylpyruvate (PP), as well as α-ketoglutarate (α-KG), in the presence of L-tyrosine, 3,4-dihydroxyphenylalanine (L-DOPA) or L-tryptophan. Aminotransferase activity with phenylpyruvate and DOPA, but not with tyrosine, was inhibited by excess phenylpyruvate. Tyrosine and DOPA aminotransferase activities with phenylpyruvate were more heat stable than the corresponding activities with α-ketoglutarate. Aminotransferase activities with phenylpyruvate were not significantly induced following intraperitoneal injections of cortisol, glucagon or serotonin, compared with a 3 to 7-fold increase in the aminotransferase activities with α-ketoglutarate. Tyrosine:phenylpyruvate aminotransferase activity rose 40% at night, compared with a 300% increase in tyrosine:α-ketoglutarate aminotransferase activity. The results suggest that aminotransferases catalysing transfers between aromatic keto acids and aromatic amino acids are separate enzymes from those utilizing α-ketoglutarate as the acceptor keto acid.     

Effect of 3,4-dihydroxyphenylalanine on Cu2+-induced Inactivation of HDL-associated Paraoxonase1 and Oxidation of HDL; Inactivation of Paraoxonase1 Activity Independent of HDL Lipid Oxidation

Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu²⁺-mediated inactivation of PON1 was examined. Cu²⁺-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu²⁺-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu²⁺, but not Fe²⁺. The DOPA/Cu²⁺-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu²⁺-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu²⁺. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu²⁺-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu²⁺-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu²⁺-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu²⁺ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu²⁺-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.     

Phenylalanine residues in the active site of tyrosine hydroxylase: mutagenesis of Phe300 and Phe309 to alanine and metal ion-catalyzed hydroxylation of Phe300.

Residues Phe300 and Phe309 of tyrosine hydroxylase are located in the active site in the recently described three-dimensional structure of the enzyme, where they have been proposed to play roles in substrate binding. Also based on the structure, Phe300 has been reported to be hydroxylated due to a naturally occurring posttranslational modification [Goodwill, K. E., Sabatier, C., and Stevens, R. C. (1998) Biochemistry 37, 13437-13445]. Mutants of tyrosine hydroxylase with alanine substituted for Phe300 or Phe309 have now been purified and characterized. The F309A protein possesses 40% less activity than wild-type tyrosine hydroxylase in the production of DOPA, but full activity in the production of dihydropterin. The F300A protein shows a 2.5-fold decrease in activity in the production of both DOPA and dihydropterin. The K(6-MPH4) value for F300A tyrosine hydroxylase is twice the wild-type value. These results are consistent with Phe309 having a role in maintaining the integrity of the active site, while Phe300 contributes less than 1 kcal/mol to binding tetrahydropterin. Characterization of Phe300 by MALDI-TOF mass spectrometry and amino acid sequencing showed that hydroxylation only occurs in the isolated catalytic domain after incubation with a large excess of 7, 8-dihydropterin, DTT, and Fe(2+). The modification is not observed in the untreated catalytic domain or in the full-length protein, even in the presence of excess iron. These results establish that hydroxylation of Phe300 is an artifact of the crystallography conditions and is not relevant to catalysis.     

Reaction of pyruvate kinase with the new nucleotide affinity labels 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate

Two new reactive nucleotides have been synthesized and characterized: 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate (8-BDB-TADP and 8-BDB-TATP). ADP or ATP was converted to 8-thio-ADP (-ATP) via 8-bromo-ADP (-ATP), followed by condensation with 1,4-dibromobutanedione. Rabbit muscle pyruvate kinase is inactivated by both reagents in a biphasic manner with an initial rapid loss of 75% activity, followed by a slow total inactivation. The initial fast reaction with both compounds exhibits nonlinear dependence on reagent concentration, indicating formation of a reversible enzyme-reagent complex prior to covalent attachment. The presence of the gamma-phosphoryl group improves the performance of the affinity label: KI values for the fast phase are similar (about 100 microM), whereas kmax for 8-BDB-TATP is about three times greater than that of 8-BDB-TADP (0.286 min-1 vs 0.0835 min-1). After an 80-min incubation with 175 microM of either reagent, about 2 mol/mol of subunit is incorporated with 76% inactivation caused by 8-BDB-TADP and 97% inactivation by 8-BDB-TATP. Loss of activity is prevented by substrates, with the best protection afforded by a combination of ATP, Mn2+, K+, and phosphoenolpyruvate. Reaction of pyruvate kinase with either compound in the presence of protecting ligands leads to incorporation of about 1 mol of reagent/mol of subunit with only about 15% loss of activity. These results suggest that 8-BDB-TADP and 8-BDB-TATP react with two groups on the enzyme, one of which is at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurotensin interacts with dopaminergic neurons in rat brain

Neurotensin (NT) injected intracerebroventricularly in rat increases dopamine (DA) turnover in the corpus striatum and nucleus accumbens. Significant increases in 3,4-dihydroxyphenylacetic acid (DOPAC) levels occurred within 15 minutes after injection with peak levels at 60 minutes. The effect on NT on DOPAC and homovanillic acid (HVA) accumulation was dose-dependent at 3–100 μg. NT, like haloperidol, stimulated 3,4-dihydroxyphenylalanine (DOPA) accumulation in striatal neurons, in the presence of DOPA decarboxylase inhibitor, after injection of gamma-butyrolactone (GBL). NT had a similar stimulatory effect on DOPA levels in the accumbens while haloperidol (0.25 mg·kg^?1) had no significant effect in this brain region. NT did not block the inhibitory effect of apomorphine on DOPA accumulation in both the striatum and accumbens, while haloperidol inhibited apomorphine effect in both regions. NT also failed to displace ³H-spiperone from DA receptors and the presence of NT in the binding assay did not alter the ability of DA to displace ³H-spiperone in either brain region. These experiments demonstrate that NT increases DA turnover in both the nigrostriatal and mesolimbic pathways.     

Modified FGF4 signal peptide inhibits entry of herpes simplex virus type 1

Entry of herpes simplex virus type 1 (HSV-1) into host cells occurs through fusion of the viral envelope with the plasma membrane and involves complex and poorly understood interactions between several viral and cellular proteins. One strategy for dissecting the function of this fusion machine is through the use of specific inhibitors. We identified a peptide with antiviral activity that blocks HSV-1 infection at the entry stage and during cell-to-cell spreading. This peptide (called EB for "entry blocker") consists of the FGF4 signal sequence with an RRKK tetramer at the amino terminus to improve solubility. The activity of EB depends exclusively but not canonically on the signal sequence. Inhibition of virus entry (hrR3) and plaque formation (KOS) strongly depend on virus concentrations and serum addition, with 50% inhibitory concentrations typically ranging from 1 to 10 microM. Blocking preadsorbed virus requires higher EB concentrations. Cytotoxic effects (trypan blue exclusion) are first noted at 50 microM EB in serum-free medium and at > or = 200 microM in the presence of serum. EB does not affect gC-dependent mechanisms of virus attachment and does not block virus attachment at 4 degrees C. Instead, EB directly interacts with virions and inactivates them irreversibly without, however, disrupting their physical integrity as judged by electron microscopy. At subvirucidal concentrations, EB changes the adhesive properties of virions, causing aggregation at high virus concentrations. This peptide may be a useful tool for studying viral entry mechanisms.     

Interference of some tryptophan metabolites in the formation of melanin in vitro

Melanin (eumelanin) is commonly produced in mammals starting from tyrosine and/or 3,4-dioxyphenylalanine (DOPA) under the action of tyrosinase. 3-Hydroxyanthranilic acid and 3-hydroxykynurenine are intermediates occurring in the kynurenine pathway of tryptophan catabolism. In this paper, we show that these substances can interfere in melanin formation in vitro when tyrosine or DOPA is oxidized by molecular oxygen under catalysis by tyrosinase. In particular, when 3-hydroxyanthranilic acid is present, a brown and apparently water-soluble pigment is formed, whereas the typical eumelanin granules seem to become more and more rare as the concentration of 3-hydroxyanthranilic acid increases. Also in the presence of the latter, the rate of tyrosine and/or DOPA consumption decreases. A very complicated (13)C-NMR spectrum indicates the high complexity of the reaction. This involves both the true melanin precursor(s) and the tryptophan metabolite, even if with peculiar mechanism and kinetics. When 3-hydroxykynurenine is substituted for 3-hydroxyanthranilic acid the reaction leads to reddish pigments whereas xanthommatins (the typical oxidation products of 3-hydroxykynurenine) are absent. A possible relationship between some dischromic pathologies and tryptophan metabolic disorders is suggested.