Sous-vide cooking as a practical strategy to improve quality attributes and shelf stability of reduced-salt chicken breast ham.

The objective of this study was to evaluate the general quality attributes and shelf stability of reduced-salt and sous-vide cooked chicken breast hams during 4 weeks of refrigerated storage (4°C). Four treatment groups of chicken breast ham were prepared using a 2 (salt level, 1.5% NaCl (regular) and 0.75% (reduced)) × 2 (cooking method, conventional and sous-vide) factorial arrangement. Based on each chicken breast weight, 20% NaCl solution was injected. Conventional cooking was done at 80°C until the core temperature reached 71°C, whereas sous-vide cooking was conducted at 60°C for 2 h. Sous-vide cooking could decrease cooking loss and shear force of reduced-salt chicken breast ham (P < 0.05). As a result, sensory scores for juiciness and tenderness of reduced-salt and sous-vide cooked chicken breast ham were similar to those of regular-salt and conventionally cooked chicken breast hams (P > 0.05). No adverse impacts on lipid oxidation and microbial safety were found in reduced-salt and sous-vide cooked chicken breast ham during 4 wk of refrigerated storage. Therefore, this study suggests that sous-vide cooking could be a practical thermal process for improving the water-holding capacity and texture of chicken breast ham without adverse impacts on shelf stability. Further studies on the combined application of sous-vide cooking with salt replacers would be warranted to improve the sensorial acceptance of saltiness of sous-vide cooked low-salt meat products.

5-HT(1B) receptors inhibit glutamate release from primary afferent terminals in rat medullary dorsal horn neurons.

BACKGROUND AND PURPOSE: Although 5-HT(1B) receptors are expressed in trigeminal sensory neurons, it is still not known whether these receptors can modulate nociceptive transmission from primary afferents onto medullary dorsal horn neurons. EXPERIMENTAL APPROACH: Primary afferent-evoked EPSCs were recorded from medullary dorsal horn neurons of rat horizontal brain stem slices using a conventional whole-cell patch clamp technique under a voltage-clamp condition. KEY RESULTS: CP93129, a selective 5-HT(1B) receptor agonist, reversibly and concentration-dependently decreased the amplitude of glutamatergic EPSCs and increased the paired-pulse ratio. In addition, CP93129 reduced the frequency of spontaneous miniature EPSCs without affecting the current amplitude. The CP93129-induced inhibition of EPSCs was significantly occluded by GR55562, a 5-HT(1B/1D) receptor antagonist, but not LY310762, a 5-HT(1D) receptor antagonist. Sumatriptan, an anti-migraine drug, also decreased EPSC amplitude, and this effect was partially blocked by either GR55562 or LY310762. On the other hand, primary afferent-evoked EPSCs were mediated by the Ca(2+) influx passing through both presynaptic N-type and P/Q-type Ca(2+) channels. The CP93129-induced inhibition of EPSCs was significantly occluded by ω-conotoxin GVIA, an N-type Ca(2+) channel blocker. CONCLUSIONS AND IMPLICATIONS: The present results suggest that the activation of presynaptic 5-HT(1B) receptors reduces glutamate release from primary afferent terminals onto medullary dorsal horn neurons, and that 5-HT(1B) receptors could be, at the very least, a potential target for the treatment of pain from orofacial tissues.

Telomeric DNA quantity, DNA damage, and heat shock protein gene expression as physiological stress markers in chickens.

In this longitudinal study with Single Comb White Leghorn chickens, we investigated the effects of stress conditions in birds that were subjected to a high stocking density with feed restrictions on the quantity of telomeric DNA, the rate of DNA damage, and the expression levels of heat shock proteins (HSP) and hydroxyl-3-methyl-glutaryl coenzyme A reductase (HMGCR) genes. The telomere length and telomere-shortening rates were analyzed by quantitative fluorescence in situ hybridization on the nuclei of lymphocytes. The DNA damage rate of lymphocytes was quantified by the comet assay. The expression levels of HSP70, HSP90, and HMGCR genes were measured by quantitative real-time PCR in lymphocytes. The telomere-shortening rate of the lymphocytes was significantly higher in the stress group than in the control. The DNA damage also increased in birds raised under stress conditions, as compared with the control group. The stress conditions had a significant effect on the expressions of HMGCR and HSP90α in lymphocytes but had no significance on HSP70 and HSP90ß in blood. We conclude that the telomere length, especially the telomere-shortening rates, the quantification of total DNA damage, and the expression levels of the HMGCR and HSP90α genes can be used as sensitive physiological stress markers in chickens.

Modulation of presynaptic GABA(A) receptors by endogenous neurosteroids.

BACKGROUND AND PURPOSE: Although 3α-hydroxy, 5α-reduced pregnane steroids, such as allopregnanolone (AlloP) and tetrahydrodeoxycorticosterone, are endogenous positive modulators of postsynaptic GABA(A) receptors, the functional roles of endogenous neurosteroids in synaptic transmission are still largely unknown. EXPERIMENTAL APPROACH: In this study, the effect of AlloP on spontaneous glutamate release was examined in mechanically isolated dentate gyrus hilar neurons by use of the conventional whole-cell patch-clamp technique. KEY RESULTS: AlloP increased the frequency of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in a dose-dependent manner. The AlloP-induced increase in sEPSC frequency was completely blocked by a non-competitive GABA(A) receptor blocker, tetrodotoxin or Cd(2+) , suggesting that AlloP acts on presynaptic GABA(A) receptors to depolarize presynaptic nerve terminals to increase the probability of spontaneous glutamate release. On the other hand, γ-cyclodextrin (γ-CD) significantly decreased the basal frequency of sEPSCs. However, γ-CD failed to decrease the basal frequency of sEPSCs in the presence of a non-competitive GABA(A) receptor antagonist or tetrodotoxin. In addition, γ-CD failed to decrease the basal frequency of sEPSCs after blocking the synthesis of endogenous 5α-reduced pregnane steroids. Furthermore, γ-CD decreased the extent of muscimol-induced increase in sEPSC frequency, suggesting that endogenous neurosteroids can directly activate and/or potentiate presynaptic GABA(A) receptors to affect spontaneous glutamate release onto hilar neurons. CONCLUSIONS AND IMPLICATIONS: The modulation of presynaptic GABA(A) receptors by endogenous neurosteroids might affect the excitability of the dentate gyrus-hilus-CA3 network, and thus contribute, at least in part, to some pathological conditions, such as catamenial epilepsy and premenstrual dysphoric disorder.

Pregnenolone sulfate enhances spontaneous glutamate release by inducing presynaptic Ca2+-induced Ca2+ release.

Pregnenolone sulfate (PS) acts as an excitatory neuromodulator and has a variety of neuropharmacological actions, such as memory enhancement and convulsant effects. In the present study, we investigated the effect of PS on glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in acutely isolated dentate gyrus (DG) hilar neurons by use of a conventional whole-cell patch-clamp technique. PS significantly increased sEPSC frequency in a concentration-dependent manner without affecting the current amplitude, suggesting that PS acts presynaptically to increase the probability of spontaneous glutamate release. However, known molecular targets of PS, such as α7 nicotinic ACh, NMDA, σ1 receptors and voltage-dependent Ca(2+) channels, were not responsible for the PS-induced increase in sEPSC frequency. In contrast, the PS-induced increase in sEPSC frequency was completely occluded in a Ca(2+)-free external solution, and was significantly reduced by either the depletion of presynaptic Ca(2+) stores or the blockade of ryanodine receptors, suggesting that PS elicits Ca(2+)-induced Ca(2+) release (CICR) within glutamatergic nerve terminals. In addition, the PS-induced increase in sEPSC frequency was completely occluded by transient receptor potential (TRP) channel blockers. These data suggest that PS increases spontaneous glutamate release onto acutely isolated hilar neurons via presynaptic CICR, which was triggered by the influx of Ca(2+) through presynaptic TRP channels. The PS-induced modulation of excitatory transmission onto hilar neurons could have a broad impact on the excitability of hilar neurons and affect the pathophysiological functions mediated by the hippocampus.

Novel anti-apoptotic mechanism of A20 through targeting ASK1 to suppress TNF-induced JNK activation.

The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of nuclear factor-κB (NF-κB) activation and apoptosis in tumor necrosis factor (TNF) receptor 1 signaling pathway. Although the molecular basis for the anti-NF-κB function of A20 has been well elucidated, the anti-apoptotic function of A20 is largely unknown. Here, we report a novel mechanism underlying the anti-apoptotic function of A20: A20 blocks TNF-induced apoptosis through suppression of c-jun N-terminal kinase (JNK) by targeting apoptosis signal-regulating kinase1 (ASK1). First, the ectopic expression of A20 drastically inhibits TNF-induced JNK activation and apoptosis in multiple cell types including those deficient of NF-κB activation. Unexpectedly, the blunting effect of A20 on TNF-induced JNK activation is not mediated by affecting the TNFR1 signaling complex formation. Instead, A20 interacts with ASK1, an important MAPKK kinase in the JNK signaling cascade. More importantly, overexpression of wild-type A20, but not of mutant A20 (ZnF4; C624A, C627A), promotes degradation of the ASK1 through the ubiquitin-proteasome system. Taken together, the results from this study reveal a novel anti-apoptotic mechanism of A20 in TNF signaling pathway: A20 binds to ASK1 and mediates ASK1 degradation, leading to suppression of JNK activation and eventually blockage of apoptosis.

High potassium-induced facilitation of glycine release from presynaptic terminals on mechanically dissociated rat spinal dorsal horn neurons in the absence of extracellular calcium.

The high potassium-induced potentiation of spontaneous glycine release in extracellular Ca2+-free conditions was studied in mechanically dissociated rat spinal dorsal horn neurons using whole-cell patch-clamp technique. Elevating extracellular K+ concentration reversibly increased the frequency of spontaneous glycinergic inhibitory postsynaptic currents (IPSCs) in the absence of extracellular Ca2+. Blocking voltage-dependent Na+ channels (tetrodotoxin) and Ca2+ channels (nifedipine and omega-grammotoxin-SIA) had no effect on this potassium-induced potentiation of glycine-release. The high potassium-induced increase in IPSC frequency was also observed in the absence of extracellular Na+, although the recovery back to baseline levels of release was prolonged under these conditions. The action of high potassium solution on glycine release was prevented by BAPTA-AM, by depletion of intracellular Ca2+ stores by thapsigargin and by the phospholipase C inhibitor U-73122. The results suggest that the elevated extracellular K+ concentration causes Ca2+ release from internal stores which is independent of extracellular Na+- and Ca2+-influx, and may reveal a novel mechanism by which the potassium-induced depolarization of presynaptic nerve terminals can regulate intracellular Ca2+ concentration and exocytosis.

beta(2)-Adrenoceptor-mediated facilitation of glutamatergic transmission in rat ventromedial hypothalamic neurons.

Adrenergic modulation of glutamatergic spontaneous miniature excitatory postsynaptic currents (mEPSCs) was investigated in mechanically dissociated rat ventromedial hypothalamic (VMH) neurons using a conventional whole-cell patch clamp technique. Noradrenaline (NA) reversibly increased mEPSC frequency without affecting the current amplitude in a concentration-dependent manner, indicating that NA acts presynaptically to facilitate the probability of spontaneous glutamate release. NA (10 microM) action on glutamatergic mEPSC frequency was completely blocked by 1 microM ICI-188551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methyl-ethyl)amino]-2-butanol], a selective beta(2)-adrenoceptor antagonist, and mimicked by 1 microM formoterol, a selective beta(2)-adrenoceptor agonist. Neither alpha-adrenoceptor nor beta(1)-adrenoceptor blockers affected the NA-induced increase in mEPSC frequency. NA action on glutamatergic mEPSC frequency was completely occluded in the presence of either 10 microM forskolin, an adenylyl cyclase (AC) activator, or blocked by 1 microM SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine], a selective AC inhibitor. Furthermore, the NA-induced increase in mEPSC frequency was completely attenuated by either 1 muM KT5720 or 1 microM H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), specific PKA inhibitors. However, NA still could increase mEPSC frequency either in the Ca(2+)-free external solution or in the presence of 1 microM thapsigargin. The results suggest that activation of presynaptic beta(2)-adrenoceptors facilitates spontaneous glutamate release to VMH neurons via cAMP/PKA signal transduction pathway. beta(2)-Adrenoceptor-mediated presynaptic modulation of excitatory glutamatergic transmission would therefore be expected to play a pivotal role in the regulation of a variety of behavioral functions, which are mediated by the VMH.

Presynaptic GABAA receptors facilitate spontaneous glutamate release from presynaptic terminals on mechanically dissociated rat CA3 pyramidal neurons.

Mossy fiber-derived giant spontaneous miniature excitatory postsynaptic currents have been suggested to be large enough to generate action potentials in postsynaptic CA3 pyramidal neurons. Here we report on the functional roles of presynaptic GABA(A) receptors on excitatory terminals in contributing to spontaneous glutamatergic transmission to CA3 neurons. In mechanically dissociated rat hippocampal CA3 neurons with adherent presynaptic nerve terminals, spontaneous excitatory postsynaptic currents were recorded using conventional whole-cell patch clamp recordings. In most recordings, unusually large spontaneous excitatory postsynaptic currents up to 500 pA were observed. These large spontaneous excitatory postsynaptic currents were highly sensitive to group II metabotropic glutamate receptor activation, and were still observed even after the blockade of voltage-dependent Na(+) or Ca(2+) channels. Exogenously applied muscimol (0.1-3 microM) significantly increased the frequency of spontaneous excitatory postsynaptic currents including the large ones. This facilitatory effect of muscimol was completely inhibited in the presence of 10 microM 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid HBr, a specific GABA(A) receptor antagonist. Pharmacological data suggest that activation of presynaptic GABA(A) receptors directly depolarizes glutamatergic terminals resulting in the facilitation of spontaneous glutamate release. In the current-clamp condition, a subset of large spontaneous excitatory postsynaptic potentials triggered action potentials, and muscimol greatly increased the frequency of spontaneous excitatory postsynaptic potential-triggered action potentials in postsynaptic CA3 pyramidal neurons. The results suggest that presynaptic GABA(A) receptors on glutamatergic terminals play an important role in the excitability of CA3 neurons as well as in the presynaptic modulation of glutamatergic transmission onto hippocampal CA3 neurons.

Feed-forward facilitation of glutamate release by presynaptic GABA(A) receptors.

Disynaptic GABAergic inputs from Schaffer collateral (SC) afferents on to the soma of glutamatergic CA1 pyramidal neurons are involved in feed-forward inhibition in the hippocampal neural circuits. Here we report the functional roles of presynaptic GABA(A) receptors on SC afferents projecting to CA1 pyramidal neurons. Muscimol (0.5 microM), a selective GABA(A) receptor agonist, increased SC-evoked EPSC amplitude and decreased paired-pulse ratio in the slice preparation, in addition, it facilitated spontaneous glutamate release on to mechanically dissociated CA1 pyramidal neurons in an external Ca2+-dependent manner. In field recordings, muscimol at low concentrations (< or = 0.5 microM) increased not only the excitability of SC afferents but glutamate release, however, it at high concentrations (> or = 1 microM) changed bidirectionally. These results suggest that the moderate activation of presynaptic GABA(A) receptors depolarizes SC afferents and enhances SC-mediated glutamatergic transmission. When endogenous GABA was disynaptically released by brief trains of stimulation of SC afferents, the axonal excitability in addition to glutamate release was increased. The effects of endogenous GABA on the excitability of SC afferents were blocked by either SR95531 or AMPA receptor blockers, which would be expected to block disynaptic feed-forward neural circuits. The present results provide a novel form of presynaptic modulation (feed-forward facilitation) of glutamatergic transmission by presynaptic GABA(A) receptors within the intrinsic hippocampal neural circuits.

Involvement of the protein kinase C pathway in thyrotropin-induced STAT3 activation in FRTL-5 thyroid cells.

The binding of thyrotropin (TSH) to the TSH receptor (TSHR) activates two signaling pathways: the cAMP-protein kinase A (PKA) and the protein kinase C (PKC) systems. We have recently demonstrated that TSH activates the Janus kinases (JAK)/signal transducer and activator of transcription (STAT) pathway via TSHR. This study aimed to investigate whether the cAMP/PKA or the PKC system is involved in STAT3 activation in response to TSH. Treatment with TSH activated STAT3 phosphorylation in FRTL-5 thyrocytes and human TSHR-expressing Chinese hamster ovary cells. TSH-induced STAT3 activation was inhibited by a blocking antibody directed against TSHR that was isolated from patients with primary myxoedema. Increased intracellular cAMP activated STAT3 but inhibition of PKA did not affect STAT3 activation. On the other hand, the PKC stimulant PMA induced STAT3 phosphorylation and the PKC inhibitors inhibited it. Moreover, inhibition of PKC blocked STAT3 activation induced by a stimulator of cAMP. Our data suggest that TSH activates STAT3 via TSHR and cAMP- and PKC-dependent pathways, and provide evidence that PKC may be involved in the pathway downstream from cAMP.

Isoform-specific changes of adenylate cyclase mRNA expression in rat brains following chronic electroconvulsive shock.

1. Electroconvulsive shock (ECS) has been reported to regulate the cAMP signaling system at various levels, suggesting that the cAMP system is involved in the therapeutic mechanism. 2. Chronic ECS has been suggested to change the expressions of adenylate cyclase (AC) genes, which constitute at least 9 families. However, little is known about its effect on the expression of AC. Therefore, to understand how chronic ECS alters the expression of AC genes in the brain, the authors analyzed the expression of 9 AC isoforms at the transcriptional level in rat hippocampus and cerebellum by quantitative RT-PCR following chronic ECS treatment. 3. Chronic ECS treatment was found to induce differential changes in the expression of AC isoforms in an isoform- and brain region-specific manner in the rat hippocampus and cerebellum. 4. Thus, it is concluded that chronic ECS induces differential changes in the expression of AC isoform mRNA in an isoform- and brain region-specific manner in the rat hippocampus and cerebellum. This suggests that the differential expression of AC isoforms might be an important mechanism by which chronic ECS treatment regulates the cAMP signaling system in rat brains.

Xenobiotic response in humanized double transgenic mice expressing tetracycline-controlled transactivator and human CYP1B1.

The cytochrome P450 enzymes (P450s or CYPs) are a superfamily of hemeproteins that catalyze the monooxygenation of a wide range of endobiotic and xenobiotic substrates. A typical strategy in toxicological research and testing involves applying a toxicant at high doses for a short period to homogeneous animals under controlled conditions. However, the conditions of this approach have very little in common with actual human exposure. Transgenic (Tg) mice carrying human genes encoding a drug-metabolizing enzyme (CYP) offer a solution to many of the difficulties in the evaluation of chemical toxicity. It has been demonstrated that the expression of human CYP transgenes under the control of mammalian-inducible promoters exhibits relatively poor fold increases after induction. In this study, we used the tetracycline-regulated (tet) promoter system to increase the expression of the human CYP1B1 (hCYP1B1) gene in the tissues of transgenic mice. By mating two lineages of transgenic mice, double transgenic (dTg) mice expressing both tTA and hCYP1B1 genes under the control of the tet promoter were successfully produced, into which the two transgenes were introduced in an embryo. The expression pattern of tTA-driven hCYP1B1 transgene featured a fold induction of more than 3 to 12 in the brain, heart, and lung and 2- to 4-fold induction in the liver, kidney, and intestine upon doxycycline removal. Immunohistochemical staining with hCYP1B1 antibody was also increased by the removal of doxycycline. In addition, the activities of CYP liver microsomes in the dTg mice without doxycycline showed an increase compared to that in the dTg mice treated with doxycycline. The level of activities correspond to the levels of human CYP1B1 protein expression in the Tg mice (-dox) that was increased by 2-fold induction as compared to that of the dTg mice with doxycycline. Thus, overproduction in Tg can be purified and the activity of purified human CYP1B1 can be characterized by alterations to the coding sequence in order to solve the physiological function of this enzyme in a humanized in vivo system. It is also possible to examine the activity of purified human CYP1B1 using several environmental toxicants such as procarcinogens.

Developmental changes in P2X purinoceptors on glycinergic presynaptic nerve terminals projecting to rat substantia gelatinosa neurones.

1. In mechanically dissociated rat spinal cord substantia gelatinosa (SG) neurones attached with native presynaptic nerve endings, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using nystatin perforated patch recording mode under voltage-clamp conditions. Under these conditions, it was tested whether the changes in P2X receptor subtype on the glycinergic presynaptic nerve terminals occur during postnatal development. 2. ATP facilitated glycinergic mIPSC frequency in a concentration-dependent manner through all developmental stages tested, whereas alphabeta-methylene-ATP (alphabeta-me-ATP) was only effective at later developmental stages. 3. alphabeta-me-ATP-elicited mIPSC frequency facilitation was completely occluded in the Ca2+-free external solution, but it was not affected by adding 10(-4) M Cd2+. 4. alphabeta-me-ATP still facilitated mIPSC frequency even in the presence of 10(-6) M thapsigargin, a Ca2+ pump blocker. 5. In later developmental stages, ATP-elicited presynaptic or postsynaptic responses were reversibly blocked by 10(-5) M pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), but only partially blocked by 10(-7) M 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP). However, alphabeta-me-ATP-elicited presynaptic or postsynaptic responses were completely and reversibly blocked by either 10(-5) M PPADS or 10(-7) M TNP-ATP. 6. alphabeta-me-ATP significantly reduced the evoked glycinergic IPSC amplitude in postnatal 28-30 day neurones, whereas it had no effect in 10-12 day neurones. 7. It was concluded that alphabeta-me-ATP-sensitive P2X receptors were functionally expressed on the glycinergic presynaptic nerve terminals projecting to SG neurones in later developmental stages. Such developmental changes of presynaptic P2X receptor subtypes might contribute to synaptic plasticity such as the regulation of neuronal excitability and the fine controlling of the pain signal in spinal dorsal horn neurones.

Thymidine-dependent attenuation of the mitochondrial apoptotic pathway in adenosine-induced apoptosis of HL-60 cells.

OBJECTIVE: We previously reported that adenosine-induced apoptosis in HL-60 cells was attenuated by cotreating the cells with pyrimidine nucleosides. The mechanism involved in this adenosine-induced apoptosis by the differential supply of nucleosides is studied here with a particular focus on the regulation of apoptosis-associated mitochondrial events. METHODS: Time-dependent changes in the mitochondrial membrane potential (MMP) after treatment with adenosine and/or thymidine were monitored. RESULTS: The cells did not show any decrease of MMP level up to 2.5 h after 1 mM adenosine exposure, whereas cytochrome c release, caspase-9 and caspase-3 activity, and DNA fragmentation were already activated, suggesting that mitochondrial depolarization is not a prerequisite of other apoptosis-related mitochondrial events. In contrast, the translocation of Bax to mitochondria and the release of cytochrome c began within the first hour of adenosine treatment. CONCLUSION: Thus, it is believed that adenosine-induced apoptosis is mediated by the activation of the caspase cascade by cytochrome c release with concomitant increase of Bax in the mitochondria, which implies that the translocation of Bax might be a leading event in the adenosine-induced apoptosis. Moreover, we found that most of the apoptotic parameters in adenosine-induced cellular changes, such as translocation of Bax, the release of cytochrome c, and the consequent activation of caspase-9 and caspase-3, were attenuated by thymidine supplement, thus indicating that the sensing of a nucleoside or nucleotide balance might be an upstream event of cytochrome c release. Therefore, it can be concluded that thymidine can attenuate adenosine-induced apoptosis by modulating the earliest stage of the mitochondrial apoptotic pathway.

Contribution of the Na-K-Cl cotransporter on GABA(A) receptor-mediated presynaptic depolarization in excitatory nerve terminals.

GABA(A) receptor-mediated responses manifest as either hyperpolarization or depolarization according to the intracellular Cl(-) concentration ([Cl(-)](i)). Here, we report a novel functional interaction between the Na-K-Cl cotransporter (NKCC) and GABA(A) receptor actions on glutamatergic presynaptic nerve terminals projecting to ventromedial hypothalamic (VMH) neurons. The activation of presynaptic GABA(A) receptors depolarizes the presynaptic nerve terminals and facilitates spontaneous glutamate release by activating TTX-sensitive Na(+) channels and high-threshold Ca(2+) channels. This depolarizing action of GABA was caused by an outwardly directed Cl(-) driving force for GABA(A) receptors; that is, the [Cl(-)](i) of glutamatergic nerve terminals was higher than that predicted for a passive distribution. The higher [Cl(-)](i) was generated by bumetanide-sensitive NKCCs and was responsible for the GABA-induced presynaptic depolarization. Thus, GABA(A) receptor-mediated modulation of spontaneous glutamatergic transmission may contribute to the development and regulation of VMH function as well as to the excitability of VMH neurons themselves.

Histaminergic modulation of GABAergic transmission in rat ventromedial hypothalamic neurones.

1. The ventromedial nucleus of the hypothalamus (VMH) is a key nucleus in the homeostatic regulation of neuroendocrine and behavioural functions. In mechanically dissociated rat VMH neurones with attached native presynaptic nerve endings, GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded using the nystatin perforated patch recording mode under voltage-clamp conditions. 2. Histamine reversibly inhibited the sIPSC frequency in a concentration-dependent manner without affecting the mean current amplitude. The selective histamine receptor type 3 (H(3)) agonist imetit (100 nM) mimicked this effect and it was completely abolished by the selective H(3) receptor antagonists clobenpropit (3 microM) and thioperamide (10 microM). 3. The GTP-binding protein inhibitor N-ethylmaleimide (10 microM) removed the histaminergic inhibition of GABAergic sIPSCs. 4. Elimination of external Ca(2+) reduced the GABAergic sIPSC frequency without affecting the distribution of current amplitudes. In this condition, the inhibitory effect of imetit on the sIPSC frequency completely disappeared, suggesting that the histaminergic inhibition requires extracellular Ca(2+). 5. The P/Q-type Ca(2+) channel blocker omega-agatoxin IVA (300 nM) attenuated the histaminergic inhibition of the GABAergic sIPSC frequency, but neither the N-type Ca(2+) channel blocker omega-conotoxin GVIA (3 microM) nor the L-type Ca(2+) channel blocker nicardipine (3 microM) was effective. 6. Activation of adenylyl cyclase with forskolin (10 microM) had no effect on histaminergic inhibition of the sIPSCs. 7. In conclusion, histamine inhibits spontaneous GABA release from presynaptic nerve terminals projecting to VMH neurones by inhibiting presynaptic P/Q-type Ca(2+) channels via a G-protein coupled to H(3) receptors and this may modulate the excitability of VMH neurones.

Adaptation of cAMP signaling system in SH-SY5Y neuroblastoma cells following expression of a constitutively active stimulatory G protein alpha, Q227L Gsalpha.

Heterotrimeric GTP-binding proteins (G protein) are known to participate in the transduction of signals from ligand activated receptors to effector molecules to elicit cellular responses. Sustained activation of cAMP-G protein signaling system by agonist results in desensitization of the pathway at receptor levels, however it is not clear whether such receptor responses induce other changes in post-receptor signaling path that are associated with maintenance of AMP levels, i.e. cAMP-forming adenylate cyclase (AC), cAMP-degrading cyclic nucleotide phosphodiesterase (PDE) and cAMP-dependent protein kinase (PKA). Experiments were performed to determine the expression of AC, PDE, and PKA isoforms in SH-SY5Y neuroblastoma cells, in which cAMP system was activated by expressing a constitutively activated mutant of stimulatory G protein (Q227L Gsalpha). Expression of ACI mRNA was increased, but levels of ACVIII and ACIX mRNA were decreased. All of the 4 expressed isoforms of PDE (PDE1C, PDE2, PDE 4A, and PDE4B) were increased in mRNA expression; the levels of PKA RIalpha, RIbeta, and RIIbeta were increased moderately, however, those of RIIalpha and Calpha were increased remarkably. The activities of AC, PDE and PKA were also increased in the SH-SY5Y cells expressing Q227L Gsalpha. The similar changes in expression and activity of AC, PDE and PKA were observed in the SH-SY5Y cells treated with dbcAMP for 6 days. Consequently, it is concluded that the cAMP system adapts at the post-receptor level to a sustained activation of the system by differential expression of the isoforms of AC, PDE, and PKA in SH-SY5Y neuroblastoma. We also showed that an increase in cellular cAMP concentration might mediate the observed changes in the cAMP system.

Mammary gland tumor in transgenic mice expressing targeted beta-casein/HPV16E6 fusion gene.

The human papillomaviruses (HPV)-16 and HPV-18 referred to as high-risk HPVs are strongly associated with anogenital malignancies as well as benign epithelial cysts. It has been demonstrated that transgenic mice carrying HPV-16 E6-E7 under the control of the MMTV LTR developed malignant tumors including salivary gland carcinoma, lymphoma, skin histiocytomas and testicular tumors in a non-mammary gland specific manner. Another regulatory unit of rat beta-casein gene can confer the expression of fusion gene preferentially in the mammary glands of transgenic mice in a developmentally regulated manner. In order to generate mammary tumor formation in transgenic mice directing HPV16E6 gene alone into the mammary gland, this regulatory unit was fused to the E6 gene of HPV-16 type to constructing fusion gene. By screening 51 newborn founder transgenic mice, three mice carrying transgenes were identified. One line termed TG32 developed in a mammary gland tumor with large subcutaneous mass in the left rib region at 17 months of age. The levels of E6 transcript in the mass-tumor of TG32 line were lower than those in non-tumor mammary gland of identical TG32 and of TG250. In each tissue of TG32 line, high expression of E6 transcript was detected both in the mammary gland and brain. Histological analysis showed that cells from mammary gland tumor of the TG32 line had also hyperplasia appearance, with irregular or increased total number of mitotic rate. These observations suggest that developing phenotype and the level of E6 transcripts in the process of malignant transformation may have different mechanisms involving the capacity to bind and destabilize p53, although for confirmation it is necessary to investigate many more transgenic mice.

Beta-glucuronidase-inhibitory activity and hepatoprotective effect of Ganoderma lucidum.

To prove the relationship between the fluctuation in serum beta-glucuronidase level and hepatotoxicity, an inhibitor of beta-glucuronidase from G. lucidum was isolated and its hepatoprotective activity was investigated. The ether fraction of G. lucidum, which had potent beta-glucuronidase-inhibitory activity, protected against CCl4-induced liver injury. From this ether fraction, ganoderenic acid A, was isolated as the potent inhibitor of beta-glucuronidase. It had a potent hepatoprotective effect against CCl4-induced liver injury. These results suggest that the beta-glucuronidase seems to be closely related to liver injury, which could be prevented by beta-glucuronidase inhibitors.