Effects of endokinin A/B and endokinin C/D on the antinociception properties of hemopressin in mice

The current study evaluated the effects of hemopressin (HP) on pain modulation by endokinin A/B (EKA/B) and endokinin C/D (EKC/D) at the supraspinal level in mice. Intracerebroventricular administration of HP (10 nmol) fully antagonized the hyperalgesia induced by EKA/B (10, 30, and 100 pmol), and induced a dose-dependent potent analgesic effect. HP at different concentrations (10 pmol, 100 pmol, and 1 nmol) showed varying effects on the analgesic effect of EKA/B (3 nmol). HP extended the duration of the analgesic effect of EKC/D (3 nmol). Moreover, HP at different concentrations (10 pmol, 5 pmol, 1 pmol, and 100 fmol) co-administered with EKC/D (30 pmol) induced significant analgesia at two different time points: 5 min and 50 min. To investigate the antinociceptive mechanism, we used SR140333B and SR142801. HP (1 pmol) potentiated the analgesic effect of SR140333B (100 pmol) + EKA/B (30 pmol) in 5–10 min, while HP (100 pmol) had no effect in the analgesia induced by SR140333B (3 nmol) + EKA/B (3 nmol). HP (1 nmol) fully inhibited the analgesic effect of SR140333B (3 nmol) + EKC/D (3 nmol) or SR142801 (3 nmol) + EKC/D (3 nmol). HP (1 pmol) weakened the analgesic effect of SR142801 (100 pmol) + EKA/B (30 pmol), but HP (100 pmol) strengthened the analgesic effect of SR142801 (3 nmol) + EKA/B (3 nmol). These findings may pave the way for a new strategy on investigating the interaction between tachykinins and opioids on pain modulation.     

An expedient synthesis of l-ribulose and derivatives

A significant improvement in the production of l-ribulose from inexpensive and commercially available starting materials, l-arabinose and sodium aluminate, is demonstrated. This has facilitated expeditious access to gram-scale quantities of l-ribulofuranoside derivatives.     

Intracellular l-arginine concentration does not determine NO production in endothelial cells: Implications on the “l-arginine paradox”

We examined the relative contributory roles of extracellular vs. intracellular l-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of ¹⁵N₄-ARG, ARG, or l-arginine ethyl ester (ARG-EE) for 2 h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, ¹⁵N₄-ARG, dimethylarginines, and l-citrulline by an LC–MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by¹⁵N-nitrite or estimated ¹⁵N₃-citrulline concentrations when ¹⁵N₄-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced ¹⁵N₄-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by ¹⁵N-nitrite, total nitrite and ¹⁵N₃-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the “l-arginine paradox” should not consider intracellular ARG concentration as a reference point.     

Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems

Methods with which to simply and rapidly assay l-aspartate (l-Asp) and d-aspartate (d-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of l- and d-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an l-aspartate dehydrogenase (l-AspDH) system to colorimetrically assay l-Asp and a system of three hyperthermophilic enzymes—aspartate racemase (AspR), l-AspDH, and l-aspartate oxidase (l-AO)—to assay d-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD⁺)-dependent l-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, d-Asp was measured after first removing l-Asp in the sample solution with l-AO. The remaining d-Asp was then changed to l-Asp using racemase, and the newly formed l-Asp was assayed calorimetrically using NAD⁺-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM l- and d-Asp in the assay systems. In addition, methods were applicable to the l- and d-Asp determinations in some living cells and foods.     

A facile synthesis of 5-thio-l-fucose and 5-thio-d-arabinose from d-arabinose

5-Thio-l-fucopyranose tetraacetate was synthesized in 11 steps from or d-arabinose diethyl dithioacetal by one-carbon elongation at C-5. Highly diastereo-selective addition of MeLi in ether to a derivative was achieved to give the corresponding 6-deoxy-β-d-altrofuranose isomer in good yield. A sulfur atom was introduced at C-5 of 6-deoxy-d-altrofuranose derivatives via substitution of a 5-tosylate with KSAc in HMPA with inversion of configuration, giving 5-thio-l-fucopyranose. A derivative was also prepared from 6-deoxy-β-d-altrofuranose derivatives. 5-Thio-d-arabinopyranose tetraacetate, the 5-demethyl analog of 5-thio-l-fucose, was also synthesized from in 5 steps. 5-Thio-d-arabinose showed weak inhibitory activity against α-l-fucosidase from bovine kidney (K_{i = 0.77 mM}).     

N-acetyl-l-glutamate in brain: Assay,levels, and regional and subcellular distribution

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200–300 times lower than the levels of N-acetyl-l-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 g/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-l-aspartyl-l-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.Special issue dedicated to Dr. Santiago Grisolía     

Phenoloxidase activity in the reproductive system and egg masses of the pulmonate gastropod,Biomphalaria glabrata

Phenoloxidase (PO) activity in the albumen gland (AG) and egg masses (EM) ofBiomphalaria glabrata was assessed using high-performance liquid chromatography combined with electrochemical detection and colorimetric techniques. Both AG and EM extracts catalyzed the hydroxylation ofl-tyrosine (monophenol oxidase activity, MPO) and oxidation ofl-dopa (diphenol oxidase activity, DPO). However, no PO activity was found in the ovotestis. Both MPO and DPO activities in AG and EM were significantly inhibited by 1-phenyl-2-thiourea and inactivated by boiling. Approximately 35% of MPO and 44% of DPO activities were detected in the soluble fraction of homogenized EM, in contrast to that of homogenized AG, which contained about 5% and 12%, respectively, of MPO and DPO activities. N-acetyl-dopamine, a diphenolic compound, enhanced the hydroxylation of tyrosine by the PO. The presence of both MPO and DPO activities also was confirmed by the accelerated accumulation of dopachrome during incubation of EM extracts withl-tyrosine in the absence of ascorbate. Temperature and pH optima for this enzyme were 30°C and 7.5, respectively. The potential roles of PO in egg formation inB. glabrata are discussed.     

Synthesis of disaccharide fragments of the AT-III binding domain of heparin and their sulfonatomethyl analogues

d-Glucuronate and l-iduronate-containing disaccharides related to the antithrombin-binding domain of heparin were prepared. The carboxylic function of the uronic acid unit was formed on a disaccharide level in the case of the glucuronate, while on a monosaccharide level in the case of the iduronate derivatives. Synthesis of their sulfonic acid analogues was carried out analoguosly applying sulfonatomethyl-containing acceptors in the form of either salts or methyl esters. Significant difference could be observed in the methyl ether formation reactions of the sulfonatomethyl-containing uronate disaccharides and the non-sulfonic acid uronates.     

Structural studies of the O-specific polysaccharide from the lipopolysaccharide of Mesorhizobium huakuii strain S-52, the symbiotic partner of Astragalus sinicus

The O-specific polysaccharide (OPS) obtained by mild-acid degradation of the lipopolysaccharide isolated from Mesorhizobium huakuii strain S-52 was studied by sugar and ethylation analyses along with ¹H and ¹³C NMR spectroscopy. It was concluded that the OPS was composed of trisaccharide repeating units containing two residues of 6-deoxy-l-talose (6dTal) and one l-rhamnose (Rha), whose sequence in the OPS was determined by NOESY and HMBC experiments. The minor 3-O-acetylation (about 10%) of 6-deoxytalose glycosidically substituted at position-2 was judged by relative signal intensities of corresponding O-acetylated and non-acetylated 6dTal residues. Moreover, it was found that the non-reducing end of the OPS repeating unit was occupied by 3-O-methyl-d-fucose, which terminated the O-chain as a cap-residue. These data defined the structure of the OPS as:α-3-OMe-d-Fucp-(1→[2)-α-l-6dTalp-(1→3)-α-l-6dTalp-(1→2)-α-l-Rhap-(1→]_n     

A facile synthesis of (1S,5R,6S)-5-azido-6-benzyloxycyclohex-2-en-1-ol

A facile and short synthesis of (1S,5R,6S)-5-azido-6-benzyloxycyclohex-2-en-1-ol (1) has been achieved in high yield starting from 4,5-epoxycyclohex-1-ene by using a catalytic asymmetric allylic oxidation reaction.     

Bi- and trivalent glycopeptide mannopyranosides as inhibitors of type 1 fimbriae-mediated bacterial adhesion: variation of valency, aglycon and scaffolding

In order to test relevant structural parameters for effective inhibition of mannose-specific bacterial adhesion, bi- and trivalent glycopeptide α-d-mannopyranosides were synthesized that differ in their conformational properties as well as in the spatial arrangement of attached mannosyl residues. They were tested in an inhibition adhesion assay with fluorescent Escherichia coli bacteria and testing results were referenced to the inhibitory potency of methyl α-d-mannopyranoside. It was shown, that besides the nature of the mannoside aglycon moiety, scaffolding of α-d-mannopyranosides on a peptide backbone was important for the performance of the synthesized glycopeptides as inhibitors of bacterial adhesion.     

Prolonged inhibition of brain nitric oxide synthase by short-term systemic administration of nitro-l-arginine methyl ester

We studied the dose-response characteristics and the temporal profile of inhibition of brain nitric oxide (NO) synthase (NOS) elicited by i.v. administration of the NOS inhibitor nitro-l-arginine methyl ester (L-NAME). L-NAME was administered i.v. in awake rats equipped with a venous cannula. L-NAME was injected in cumulative doses of 5, 10, 20 and 40 mg/kg and rats were sacrificed 30 min after the last dose. NOS catalytic activity was assayed in forebrain cytosol as the conversion of [³H]l-arginine into [³H]l-citrulline. L-NAME attenuated brain NOS activity in a dose-dependent manner but enzyme activity could not be inhibited by more than 50%. After a single 20 mg/kg injection of L-NAME the inhibition of brain NOS activity was time dependent and reached a stable level at 2 hrs (52% of vehicle). Inhibition after a single injection was still present at 96 hrs, albeit to a lower magnitude. We conclude that intravenous administration of L-NAME in rats at concentrations commonly used in physiological experiments leads to a dose and time-dependent but partial inhibition of brain NOS catalytic activity. The finding that the inhibition persists for several days after a single administration is consistent with the hypothesis that nitro-L-arginine, the active principle of L-NAME, binds to NOS irreversibly.     

Facile syntheses of the disaccharide repeating unit of the O-antigenic polysaccharide of Burkholderia pseudomallei strain 304b and its dimer and trimer

A highly efficient strategy for the preparation of a disaccharide-repeating unit of the O-antigenic polysaccharide of Burkholderia pseudomallei strain 304b, and its dimer and trimer, has been developed through a regio- and stereoselective manner using p-methoxylphenyl 2,4,6-tri-O-benzoyl-α-d-glucopyranoside and 3-O-allyloxycarbonyl-2,4-di-O-benzoyl-6-deoxy-α-l-talopyranosyl trichloroacetimidate as the key synthons. The target molecules were equipped with a p-methoxylphenyl handle at the reducing terminus to allow for their further functionalization and attachment to a carrier protein.     

Modulation of striatal dopamine release in cerebral ischemia byL-arginine

The effect ofL-arginine, the precursor of nitric oxide, on ischemic dopamine release from the striatum was investigated in Mongolian gerbils subjected to bilateral carotid artery occlusion (15 min) alone or with reflow (2 h). Dopamine and its metabolites were measured in the striatal extracellular space dialysate after continuous perfusion (2 l/min) of artificial extracellular fluid in the presence or absence of 15 mmol/literL- orD-arginine or 1 mmol/liter nitro-L-arginine.L-Arginine but notD-arginine increased the striatal content of dopamine in pre- and postischemia whereas it lowered the levels of dopamine and 3-methoxytyramine induced by ischemia. In contrast, nitro-L-arginine reduced the preischemic levels of dopamine and 3,4-dihydroxyphenyl-acetic acid, and had no effect on the ischemic release of dopamine. These findings indicate thatL-arginine stereospecifically modified the ischemic release and metabolism of dopamine. The data also suggest that the basal level of nitric oxide is not involved in dopamine release during ischemia but may participate in regulating dopamine release under physiological conditions.Presented in part at the 19th International Joint Conference on Stroke and Cerebral Circulation, San Diego, California, February 17–19, 1994.     

Backbone and sidechain 1H, 15N and 13C assignments of the human G-actin binding protein profilin IIa

The resonance assignment of the human profilin IIa have been determined, based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. These assignments facilitate further studies of interactions between profilin IIa and its poly-l-proline rich ligands.     

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.     

A simple assay for 3-deoxy-d-manno-octulosonate cytidylyltransferase and its use as a pathway screen

This article describes the adaptation of a simple colorimetric assay for inorganic pyrophosphate to the enzyme 3-deoxy-d-manno-octulosonate cytidylyltransferase (CMP–KDO synthetase, KdsB, EC 2.7.7.38), a key enzyme in the biosynthesis of lipopolysaccharide (LPS) in Gram-negative organisms. This assay is particularly useful because it can be combined with the malachite green (MG) assay for inorganic phosphate to form an assay system capable of determining inorganic phosphate and inorganic pyrophosphate in the same solution (the MG/EK (eikonogen reagent) assay). This assay system has the potential for simultaneous screening of the 3-deoxy-d-manno-octulosonate (KDO) biosynthesis pathway. We tested this potential using two enzymes, KdsB and KdsC, involved in the biosynthesis and use of the key bacterial 8-carbon sugar, KDO.     

Human lymphokine-activated killer (LAK) cells: III. Effect ofl-phenylalanine methyl ester on LAK cell activation from human peripheral blood mononuclear cells: Possible protease involvement of monocytes,natural killer cells and LAK cells

Summary We have shown that depletion of monocytes from human peripheral blood mononuclear cells (PBMC) byl-phenylalanine methyl ester (PheOMe) enhanced lymphokine-activated killer cell (LAK) generation by recombinant interleukin-2 (rIL-2) at high cell density. In this study, we have investigated the mechanism of action of PheOMe on LAK activation by using trypsin, chymotrypsin, tosylphenylalaninechloromethanol (TPCK, a chymotrypsin inhibitor), tosyl-l-lysinechloromethane (TLCK, a trypsin inhibitor), phenylalaninol (PheOH), and benzamidine. PBMC were treated with 1–5 mM PheOMe for 40 min at room temperature in combination with the various agents, washed and assessed for their effects on natural killer (NK) activity against K562 cells and monocyte depletion. The treated cells were then cultured with or without rIL-2 for 3 days. LAK cytotoxicity was assayed against⁵¹Cr-labeled K562 and Raji tumor target cells. TPCK at 10 µg/ml partially inhibited depletion of monocytes by PheOMe. TLCK did not prevent depletion of monocytes nor inhibition of NK activity induced by PheOMe. TPCK and TLCK inhibited NK activity by themselves. TPCK but not TLCK inhibited rIL-2 induction of LAK cells. On the other hand, PheOH and benzamidine (analogs of PheOMe) lacked any effect on monocyte depletion but abrogated the inhibitory effect of PheOMe on NK activity. They had no effect on rIL-2 activation of LAK activity enhanced by PheOMe. Trypsin potentiated the inhibitory effect of PheOMe on NK activity and monocyte depletion. Trypsin partially inhibited IL-2 activation of LAK activity enhanced by PheOMe. Chymotrypsin had little effect on NK activity but prevented the inhibitory effect of PheOMe on NK activity. It had little effect on monocyte depletion induced by PheOMe. PheOMe was hydrolysed by monocytes and chymotrypsin to Phe and methanol as determined by HPLC. TPCK inhibited hydrolysis of PheOMe by monocytes. Our data suggest that the effects of PheOMe on monocytes, NK cells and LAK activation involve protease activities of monocytes.     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.