Production of tyrosine and other aromatic compounds from phenylalanine by rumen microorganisms

Summary Rumen contents from three fistulated Japanese native goats fed Lucerne hay cubes (Medicago sativa) and concentrate mixture were collected to prepare the suspensions of mixed rumen bacteria (B), mixed protozoa (P) and a combination of the two (BP). Microbial suspensions were anaerobically incubated at 39°C for 12h with or without 1 MM ofl-phenylalanine (Phe). Phe, tyrosine (Tyr) and other related compounds in both supernatant and microbial hydrolysates of the incubations were analyzed by HPLC. Tyr can be produced from Phe not only by rumen bacteria but also by rumen protozoa. The production of Tyr during 12h incubation in B (183.6 mol/g MN) was 4.3 times higher than that in P. One of the intermediate products between Phe and Tyr seems to bep-hydroxyphenylacetic acid. The rate of the net degradation of Phe incubation in B (76.O mol/g MN/h) was 2.4 times higher than in P. In the case of all rumen microorganisms, degraded Phe was mainly (>53%) converted into phenylacetic acid. The production of benzoic acid was higher in P than in B suspensions. Small amount of phenylpyruvic acid was produced from Phe by both rumen bacteria and protozoa, but phenylpropionic acid and phenyllactic acid were produced only by rumen bacteria.     

Aromatic amino acid biosynthesis and production of related compounds from p-hydroxyphenylpyruvic acid by rumen bacteria,protozoa and their mixture

Summary. Aromatic amino acid biosynthesis and production of related compounds from p-hydroxyphenylpyruvic acid (HPY) by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated. Microbial suspensions prepared from mature, fistulated goats fed Lucerne (Medicago sativa) cubes and a concentrate mixture were anaerobically incubated at 39°C for 12 h. Tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp) and other related compounds in both supernatants and hydrolyzates of all incubations were analyzed by HPLC. Large amounts of Tyr (27.0, 47.0 and 50.8% of disappeared HPY in B, P and BP, respectively) were produced from 1 mM HPY during a 12-h incubation period. The formation of Tyr in P was 1.8 and 1.6 times higher than those in B and BP, respectively. Appreciable amounts of Phe (3–12% of the disappeared HPY) and Trp (2–10% of the disappeared HPY) were also produced from HPY in B, P, and BP. Phe synthesis in B and P was almost similar but Trp synthesis in B was 1.8 times higher than that in P. The biosynthesis of both Phe and Trp from HPY in BP was higher than those in B plus P. A large amount of p-hydroxyphenylacetic acid (about 45% of the disappeared HPY) was produced from HPY in B which was 1.9 times higher than that in P. p-Hydroxybenzoic acid produced from HPY in P was 1.6 times higher than that in B. Considerable amounts of phenylpropionic acid, phenyllactic acid, and phenylpyruvic acid (2–6% of the disappeared HPY) were produced only in B. Received March 21, 2001 Accepted July 4, 2001     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

Electrogenic H<Superscript>+</Superscript> Transport and pH Gradients Generated by a V-H<Superscript>+</Superscript>-ATPase in the Isolated Perfused Larval <Emphasis Type="Italic">Drosophila</Emphasis> Midgut

A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by ≈ 17-fold and ≈ 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (V_t) of 38 to 45 mV and high resistance (R_t) of 800 to 1400 Ω • cm² measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10⁻⁴ M, ouabain 10⁻⁴ M, bumetanide 10⁻⁴ M), K⁺-free solutions, or organic solutes such as D-glucose 10 mM or DL-alanine 0.5 mM failed to affect V_t or R_t. Bafilomycin-A₁ (3 to 5 μM) decreased V_t by ≈ 40% and short-circuit current (I_sc) by ≈ 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H⁺-ATPase located in the basal membrane. Gradients of H⁺ were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO₃⁻), driven by the basal V-H⁺-ATPase.     

Degradation of tryptophan and related indolic compounds by ruminal bacteria,protozoa and their mixture in vitro

Summary.  In vitro experiments were conducted to examine the degradation of d- and l-isomers of tryptophan (Trp) and 10 related indolic compounds by mixed rumen bacteria (B), protozoa (P) and a combination of the two (BP). The analyses were carried out by HPLC. d-Trp (1.0 mM) was not degraded by rumen microorganisms during the 24-h incubation period. The net degradation of 1 mM l-Trp was 46.5%, 8.7% and 80.0% by B, P and BP suspensions, respectively. Trp was degraded into indoleacetic acid, indolelactic acid and indole by rumen bacteria and protozoa, and into skatole, p-cresol and indolepropionic acid by rumen bacteria only. Of them, indoleacetic acid was the major product of Trp found in B (15.4%) and P (3.1%), and skatole in BP (43.2%). This is the first report of the production of indolelactic acid and p-cresol from Trp by rumen microbes. Starch, d-glucose, salinomycin and monensin inhibited the production of skatole and indole from Trp, and skatole from indoleacetic acid by rumen bacteria. Received August 2, 2001 Accepted June 21, 2002 Published online November 14, 2002 Acknowledgements The authors are extremely grateful to Dr. H. Ogawa, Professor, the University of Tokyo and Dr. T. Hasegawa, Associate Professor, Miyazaki University, for inserting a permanent fistula in goats. The present study was financially supported by research grants from Kyowa Hakko Kogyo Co. Ltd. and Daiichi Seiyaku Co., Japan. Nazimuddin Mohammed thanks the Ministry of Education, Science, Sports and Culture of Japan (Monbusho) for the award of a research studentship from 1996. Authors' address: Dr. Nazimuddin Mohammed, Laboratory of Agricultural Production Technology, Faculty of Agriculture, Field Science Center, Tokyo University of Agriculture and Technology, Saiwai-cho 3-5-8, Fuchu-shi, Tokyo 183-8509, Japan     

Differential Regulation of Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Signaling and Membrane Trafficking by Multiple P2 Receptors in Brown Adipocytes

Extracellular ATP triggers changes in intracellular Ca²⁺, ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors might mediate the Ca²⁺ signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence imaging of Ca²⁺ responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology of the Ca²⁺ responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed mRNA for P2Y₂, P2Y₆, and P2Y₁₂ metabotropic receptors and P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, and P2X₇ ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2′, 3′-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca²⁺, while 100 μM suramin, pyridoxal-phosphate-6-azophenyl-2′ 4′-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca²⁺ responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely with 1 μM PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells, and that membrane trafficking is regulated by a P2 receptor showing unusual properties.     

Regulation of [<Superscript>3</Superscript>H] <Emphasis Type="SmallCaps">d</Emphasis>-Aspartate Release from Mammalian Isolated Retinae by Hydrogen Sulfide

Hydrogen sulfide (H₂S), can produce pharmacological effects on neural and non-neural tissues from several mammalian species. The present study investigates the pharmacological action of H₂S, (using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na₂S as donors) on amino acid neurotransmission (using [³H] d-aspartate as a marker for glutamate) from isolated, superfused bovine and porcine retinae. Isolated neural retinae were incubated in Krebs solution containing [³H] d-aspartate at 37°C. Release of [³H] d-aspartate was elicited by high potassium (K⁺ 50 mM) pulse. Both NaHS and Na₂S donors caused an inhibition of K⁺-evoked [³H] d-aspartate release from isolated bovine retinae without affecting basal [³H] d-aspartate efflux yielding IC₅₀ values of 0.006 and 6 μm, respectively. Furthermore, NaHS inhibited depolarization-evoked release of [³H] d-aspartate from isolated porcine retinae with an IC₅₀ value of 8 μM. The inhibitory action of NaHS on [³H] d-aspartate release from porcine retinae was blocked by propargyglycine, a selective inhibitor of cystathionine γ-lyase (CSE). Our results indicate that H₂S donors can inhibit amino acid neurotransmission from both isolated bovine and porcine retinae, an effect that is dependent, at least in part, on intramural biosynthesis of H₂S.     

Characterization of NADP<Superscript>+</Superscript>-specific <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose dehydrogenase from the thermoacidophilic Archaeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Thermoplasma acidophilum utilizes l-rhamnose as a sole carbon source. To determine the metabolic pathway of l-rhamnose in Archaea, we identified and characterized l-rhamnose dehydrogenase (RhaD) in T. acidophilum. Ta0747P gene, which encodes the putative T. acidophilum RhaD (Ta_RhaD) enzyme belonging to the short-chain dehydrogenase/reductase family, was expressed in E. coli as an active enzyme catalyzing the oxidation of l-rhamnose to l-rhamnono-1,4-lactone. Analysis of catalytic properties revealed that Ta_RhaD oxidized l-rhamnose, l-lyxose, and l-mannose using only NADP⁺ as a cofactor, which is different from NAD⁺/NADP⁺-specific bacterial RhaDs and NAD⁺-specific eukaryal RhaDs. Ta_RhaD showed the highest activity toward l-rhamnose at 60 °C and pH 7. The K _m and k _cat values were 0.46 mM, 1,341.3 min⁻¹ for l-rhamnose and 0.1 mM, 1,027.2 min⁻¹ for NADP⁺, respectively. Phylogenetic analysis indicated that branched lineages of archaeal RhaD are quite distinct from those of Bacteria and Eukarya. This is the first report on the identification and characterization of NADP⁺-specific RhaD.     

Block of a Ca<Superscript>2+</Superscript>-activated Potassium Channel by Cocaine

The primary target for cocaine is believed to be monoamine transporters because of cocaine’s high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K⁺ channel in hippocampal neurons grown in culture (IC₅₀ = ∼30 μM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca²⁺-activated K⁺ channels are 261 ± 37 μM⁻¹s⁻¹ and 11451 ± 1467 s⁻¹. The equilibrium dissociation constant (K_B) for cocaine, determined from single-channel parameters, is 43 μM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca²⁺-dependent K⁺ channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.     

C-terminal residues of ferredoxin-NAD(P)<Superscript>+</Superscript> reductase from <Emphasis Type="Italic">Chlorobaculum tepidum</Emphasis> are responsible for reaction dynamics in the hydride transfer and redox equilibria with NADP<Superscript>+</Superscript>/NADPH

Ferredoxin-NAD(P)⁺ reductase ([EC 1.18.1.2], [EC 1.18.1.3]) from Chlorobaculum tepidum (CtFNR) is structurally homologous to the bacterial NADPH-thioredoxin reductase (TrxR), but possesses a unique C-terminal extension relative to TrxR that interacts with the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group. In this study, we introduce truncations to the C-terminal residues to examine their role in the reactions of CtFNR with NADP⁺ and NADPH by spectroscopic and kinetic analyses. The truncation of the residues from Tyr326 to Glu360 (the whole C-terminal extension region), from Phe337 to Glu360 (omitting Phe337 on the re-face of the isoalloxazine ring) and from Ser338 to Glu360 (leaving Phe337 intact) resulted in a blue-shift of the flavin absorption bands. The truncations caused a slight increase in the dissociation constant toward NADP⁺ and a slight decrease in the Michaelis constant toward NADPH in steady-state assays. Pre-steady-state studies of the redox reaction with NADPH demonstrated that deletions of Tyr326–Glu360 decreased the hydride transfer rate, and the amount of reduced enzyme increased at equilibrium relative to wild-type CtFNR. In contrast, the deletions of Phe337–Glu360 and Ser338–Glu360 resulted in only slight changes in the reaction kinetics and redox equilibrium. These results suggest that the C-terminal region of CtFNR is responsible for the formation and stability of charge-transfer complexes, leading to changes in redox properties and reactivity toward NADP⁺/NADPH.     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

Studies on the utilization of methionine sulfoxide and methionine sulfone by rumen microorganisms in vitro

Summary.  An in vitro experiment was conducted to test the ability of mixed rumen bacteria (B), protozoa (P), and their mixture (BP) to utilize the oxidized forms of methionine (Met) e.g., methionine sulfoxide (MSO), methionine sulfone (MSO₂). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39°C for 12 h with or without MSO (1 mM) or MSO₂ (1 mM) as a substrate. Met and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by HPLC. During 6- and 12-h incubation periods, MSO disappeared by 28.3 and 42.0%, 0.0 and 0.0%, and 40.6 and 62.4% in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were capable to reduce MSO to Met, and the production of Met from MSO in BP (156.6 and 196.1 μmol/g MN) was about 17.3 and 14.1% higher than that in B alone (133.5 and 171.9 μmol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to utilize MSO. Other metabolites produced from MSO were found to be MSO₂ and 2-aminobutyric acid (2AB) in B and BP. MSO₂ as a substrate remained without diminution in all-microbial suspensions. It was concluded that B, P, and BP cannot utilize MSO₂; but MSO can be utilized by B and BP for producing Met. Received December 28, 2001 Accepted May 21, 2002 Published online October 14, 2002 Acknowledgements The authors are extremely grateful to Professor H. Ogawa, the University of Tokyo, Japan and Dr. Takashi Hasegawa, Miyazaki University, Japan for inserting permanent rumen fistulae in goats. We would like to thank MONBUSHO for the award of a research scholarship to Mamun M. Or-Rashid since 1996–2001. Authors' address: Shaila Wadud, Laboratory of Animal Nutrition and Biochemistry, Division of Animal Science, Miyazaki University, Miyazaki 889-2192, Japan, Fax. +81-985-58-7201, E-mail: rafatkun@hotmail.com     

Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.     

Copper (II) complex of 1,10-phenanthroline and <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine with DNA oxidative cleavage activity in the gallic acid

Copper (II) complex of formulation [Cu–Phen–Tyr](H₂O)](ClO₄) (Phen = 1,10-phenanthroline, l-Tyr = l-tyrosine), has been prepared, and their induced DNA oxidative cleavage activity studied. The complex binds to DNA by intercalation, as deduced from the absorption and fluorescence spectral data. Scatchard plots constructed from the absorption titration data gave binding constant 2.44 × 10⁴ M⁻¹ of base pairs. Extensive hypochromism, broadening, and red shifts in the absorption spectra were observed. Upon binding to DNA, the fluorescence from the DNA–ethidium bromide system was efficiently quenched by the copper (II) complex. Stern–Volmer quenching constant 0.61 × 10³ M⁻¹ obtained from the linear quenching plots. [Cu–Phen–Tyr] complex efficiently cleave the supercoiled DNA to its nicked circular form with gallic acid as biological reductant at appropriate complex concentration. The gallic acid as reductant could observably improve copper (II) complex to DNA damage. The pseudo-Michaelis–Menten kinetic parameters (k _cat, K _M) were calculated to be 1.32 h⁻¹ and 5.46 × 10⁻⁵ M for [Cu–Phen–Tyr] complex. Mechanistic studies reveal the involvement of superoxide anions and hydroxyl radical (HO^·) as the reactive species under an aerobic medium.     

Ca<Superscript>2+</Superscript>-activated nonselective cation channels in rat neonatal atrial myocytes

A nonselective cation channel activated by intracellular Ca²⁺ was identified in inside-out membrane patches taken from cultured rat atrial myocytes. Ca²⁺ (0.01–1.00 mM) reversibly activated the channel in a concentration-dependent manner. The channel often showed a quick and irreversible rundown within a few minutes after patch excision. The I-V relationship of the channel was linear between –100 and +100 mV. The single channel conductance was 26.0 ± 0.5 pS and its open probability was weakly voltage-dependent. Ion-substitution experiments showed that the channel was permeable to monovalent cations (P_x/P_Cs: Li⁺ (1.5) = K⁺ (1.5)> Na⁺ (1.2) > Rb⁺ (1.1) > Cs⁺ (1.0)) but not to Cl^– (P_Cl/P_Cs < 0.01) and Ca²⁺ (P_Ca/P_Cs =0.02 ± 0.01). Present address: A.B. Zhainazarov UF Center for Smell and Taste, McKnight Brain Institute, University of Florida, 100 S Newell Dr., L1-131, P.O. Box 100015, Gainesville, FL 32610-0015, USA     

Antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine bearing B cell,CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cell epitopes

Molecularly defined synthetic vaccines capable of inducing both antibodies and cellular anti-tumor immune responses, in a manner compatible with human delivery, are limited. Few molecules achieve this target without utilizing external immuno-adjuvants. In this study, we explored a self-adjuvanting glyco-lipopeptide (GLP) as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. A prototype B and T cell epitope-based GLP molecule was constructed by synthesizing a chimeric peptide made of a CD8⁺ T cell epitope, from ovalbumin (OVA_257–264) and an universal CD4⁺ T helper (Th) epitope (PADRE). The resulting CTL–Th peptide backbones was coupled to a carbohydrate B cell epitope based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules at C-terminal. The N terminus of the resulting glycopeptides (GP) was then linked to a palmitic acid moiety (PAM), obviating the need for potentially toxic external immuno-adjuvants. The final prototype OVA-GLP molecule, delivered in adjuvant-free PBS, in mice induced: (1) robust RAFT-specific IgG/IgM that recognized tumor cell lines; (2) local and systemic OVA_257–264-specific IFN-γ producing CD8⁺ T cells; (3) PADRE-specific CD4⁺ T cells; (4) OVA-GLP vaccination elicited a reduction of tumor size in mice inoculated with syngeneic murine MO5 carcinoma cells and a protection from lethal carcinoma cell challenge; (5) finally, OVA-GLP immunization significantly inhibited the growth of pre-established MO5 tumors. Our results suggest self-adjuvanting glyco-lipopeptide molecules as a platform for B Cell, CD4⁺, and CD8⁺ T cell epitopes-based immunotherapeutic cancer vaccines. Both I. Bettahi and G. Dasgupta have contributed equally to this work.     

Differential Contribution of L-, N-, and P/Q-type Calcium Channels to [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> Changes Evoked by Kainate in Hippocampal Neurons

We investigated the contribution of L-, N- and P/Q-type Ca²⁺ channels to the [Ca²⁺]_i changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca²⁺ imaging. Selective Ca²⁺ channel blockers, namely nitrendipine, ω-Conotoxin GVIA (ω-GVIA) and ω-Agatoxin IVA (ω-AgaIVA) were used. The [Ca²⁺]_i changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but the N-methyl-d-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca²⁺ channel blocker caused differential inhibitory effects on [Ca²⁺]_i responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above 90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by ω-GVIA or ω-AgaIVA. Thus, in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case of ω-GVIA or ω-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what concerns their L-, N- and P/Q- type Ca²⁺ channels activated by stimulation of the AMPA/kainate receptors. Special issue article in honor of Dr. Ricardo Tapia.     

Modulation of [<Superscript>3</Superscript>H]Dopamine Release by Glutathione in Mouse Striatal Slices

Glutathione (γ-glutamylcysteinylglycine, GSH and oxidized glutathione, GSSG), may function as a neuromodulator at the glutamate receptors and as a neurotransmitter at its own receptors. We studied now the effects of GSH, GSSG, glutathione derivatives and thiol redox agents on the spontaneous, K⁺- and glutamate-agonist-evoked releases of [³H]dopamine from mouse striatal slices. The release evoked by 25 mM K⁺ was inhibited by GSH, S-ethyl-, -propyl-, -butyl- and pentylglutathione and glutathione sulfonate. 5,5′-Dithio-bis-2-nitrobenzoate (DTNB) and l-cystine were also inhibitory, while dithiothreitol (DTT) and l-cysteine enhanced the K⁺-evoked release. Ten min preperfusion with 50 μM ZnCl₂ enhanced the basal unstimulated release but prevented the activation of K⁺-evoked release by DTT. Kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) evoked dopamine release but the other glutamate receptor agonists N-methyl-d-aspartate (NMDA), glycine (1 mM) and trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD, 0.5 mM), and the modulators GSH, GSSG, glutathione sulfonate, S-alkyl-derivatives of glutathione, DTNB, cystine, cysteine and DTT (all 1 mM) were without effect. The release evoked by 1 mM glutamate was enhanced by 1 mM GSH, while GSSG, glutathionesulfonate and S-alkyl derivatives of glutathione were generally without effect or inhibitory. NMDA (1 mM) evoked release only in the presence of 1 mM GSH but not with GSSG, other peptides or thiol modulators. l-Cysteine (1 mM) enhanced the glutamate-evoked release similarly to GSH. The activation by 1 mM kainate was inhibited by S-ethyl-, -propyl-, and -butylglutathione and the activation by 0.5 mM AMPA was inhibited by S-ethylglutathione but enhanced by GSSG. Glutathione alone does not directly evoke dopamine release but may inhibit the depolarization-evoked release by preventing the toxic effects of high glutamate, and by modulating the cysteine–cystine redox state in Ca²⁺ channels. GSH also seems to enhance the glutamate-agonist-evoked release via both non-NMDA and NMDA receptors. In this action, the γ-glutamyl and cysteinyl moieties of glutathione are involved.     

Isovaleric Acid Reduces Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity in Synaptic Membranes from Cerebral Cortex of Young Rats

1. Patients affected by isovaleric acidemia (IVAcidemia) suffer from acute episodes of encephalopathy. However, the mechanisms underlying the neuropathology of this disease are poorly known. The objective of the present study was to investigate the in vitro effects of the metabolites that predominantly accumulate in IVAcidemia, namely isovaleric acid (IVA), 3-hydroxyisovaleric acid (3-OHIVA) and isovalerylglycine (IVG), on important parameters of energy metabolism, such as ¹⁴CO₂ production from acetate and the activities of the respiratory chain complexes I–IV, creatine kinase and Na⁺, K⁺-ATPase in synaptic plasma membranes from cerebral cortex homogenates of 30-day-old rats. 2. We observed that 3-OHIVA acid and IVG did not affect all the parameters analyzed. Similarly, ¹⁴CO₂ production from acetate (Krebs cycle activity), the activities of creatine kinase, and of the respiratory chain complexes was not modified by IVA. In contrast, IVA exposition to cortical homogenates provoked a marked inhibition of Na⁺, K⁺-ATPase activity. However, this activity was not changed when IVA was directly exposed to purified synaptic plasma membranes, suggesting an indirect effect of this organic acid on the enzyme. Furthermore, pretreatment of cortical homogenates with α-tocopherol and creatine totally prevented IVA-induced inhibition on Na⁺, K⁺-ATPase activity from synaptic plasma membranes, whereas glutathione (GSH) and the NO synthase inhibitor N^ω-nitro-l-arginine methyl ester (L-NAME) did not alter this inhibition. 3. These data indicate that peroxide radicals were probably involved in this inhibitory effect. Since Na⁺, K⁺-ATPase is a critical enzyme for normal brain development and functioning and necessary to maintain neuronal excitability, it is presumed that the inhibitory effect of IVA on this activity may be involved in the pathophysiology of the neurological dysfunction of isovaleric acidemic patients.     

Kinetics of the Reverse Mode of the Na<Superscript>+</Superscript>/Glucose Cotransporter

This study investigates the reverse mode of the Na⁺/glucose cotransporter (SGLT1). In giant excised inside-out membrane patches from Xenopus laevis oocytes expressing rabbit SGLT1, application of α-methyl-D-glucopyranoside (αMDG) to the cytoplasmic solution induced an outward current from cytosolic to external membrane surface. The outward current was Na⁺- and sugar-dependent, and was blocked by phlorizin, a specific inhibitor of SGLT1. The current-voltage relationship saturated at positive membrane voltages (30–50 mV), and approached zero at −150 mV. The half-maximal concentration for αMDG-evoked outward current (K_0.5^αMDG) was 35 mM (at 0 mV). In comparison, K_0.5^αMDG for forward sugar transport was 0.15 mM (at 0 mV). K_0.5^Na was similar for forward and reverse transport (≈35 mM at 0 mV). Specificity of SGLT1 for reverse transport was: αMDG (1.0) > D-galactose (0.84) > 3-O-methyl-glucose (0.55) > D-glucose (0.38), whereas for forward transport, specificity was: αMDG ≈ D-glucose ≈ D-galactose > 3-O-methyl-glucose. Thus there is an asymmetry in sugar kinetics and specificity between forward and reverse modes. Computer simulations showed that a 6-state kinetic model for SGLT1 can account for Na⁺/sugar cotransport and its voltage dependence in both the forward and reverse modes at saturating sodium concentrations. Our data indicate that under physiological conditions, the transporter is poised to accumulate sugar efficiently in the enterocyte.