HPI在肝癌组织中的表达及其对肝癌细胞特异性抑增殖效应研究

肝细胞增殖抑制因子（Ｈｅｐａｔｉｃｐｒｏｌｉｆｅｒａｔｉｏｎｉｎｈｉｂｉｔｏｒ,ＨＰＩ）粗制品、半纯品和纯品对体外培养的人肝癌细胞具有显著抑增殖作用,随样品纯度提高抑制活性逐渐增强。纯品（浓度５μｇ／ｍｌ）的抑制率达７７．７１％。正常成年大鼠肝细胞呈ＨＰＩ阳性表达。在ＤＥＮ诱发大鼠肝细胞癌的发生发展过程中,转化的癌前期细胞和肝癌细胞呈ＨＰＩ阴性表达。表明肝细胞ＨＰＩ的表达能力在其癌变过程中消失,从而失去了自身的抑癌作用。     

大鼠免疫性血小板减少模型的研究

采用注射兔抗大鼠血小板血清（ＡＰＳ）方法,建立了大鼠免疫性血小板减少模型。大鼠腹腔注射１：４稀释的ＡＰＳ（０．７ｍｌ／２００ｇ体重）,连续３ｄ,可使血小板数量显著降低,其降低率为８１±９％（ｎ＝１２）,且其骨随巨核细胞增生活跃,但注射ＡＰＳ后对血中红细胞数和白细胞数无明显影响．在注射ＡＰＳ的同时,给予大鼠灌胃强的松（１ｍｇ／２００ｇ体重）,可抑制ＡＰＳ所致的血小板减少的下降程度,并促进停止注射ＡＰＳ后血小板数的恢复。以上结果表明,该模型符合免疫性血小板减少性紫癜的病理特征。     

Inhibition of the NADH oxidase activity of plasma membranes isolated from xenografts and cell lines by the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY 181984) correlates with drug susceptibility of growth

Summary Inhibition of NADH oxidase activity of plasma membranes isolated from a series of human xenografts and cell lines by the antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl) urea (LY 181984), correlated with the ability of the sulfonylurea to inhibit cell growth. Growth of rat kidney cells either untransformed or transformed with Kirsten-ras (K-ras) were unaffected by the sulfonylurea. Similarly, the NADH oxidase activity of isolated plasma membranes from K-ras transformed cells was unaffected by LY 181984. In contrast, when transformed with Harvey-ras (H-ras), both growth and NADH oxidase activity were inhibited. With the inactive but structurally related LY 181985 (N-4-methylphenyl-sulfonyl)-N-(phenyl)urea), neither growth nor plasma membrane NADH oxidase activity of either sulfonylurea-susceptible or -resistant tissues or cell lines was inhibited. Both sulfonylureas were inactive with rat liver plasma membranes but NADH oxidase activity of plasma membranes and growth with HeLa cells was inhibited by the active (LY 181984) but not by the inactive (LY 181985) sulfonylurea. The findings suggest a possible correlation between inhibition of plasma membrane NADH oxidase activity by the antitumor sulfonylureas and their oncolytic action.     

The Second Messenger, Cyclic AMP, Is Not Sufficient for Myelin Gene Induction in the Peripheral Nervous System

Abstract: The adenylyl cyclase-cyclic AMP (cAMP) second messenger pathway has been proposed to regulate myelin gene expression; however, a clear correlation between endogenous cAMP levels and myelin-specific mRNA levels has never been demonstrated during the induction or maintenance of differentiation by the myelinating Schwann cell. Endogenous cAMP levels decreased to 8–10% of normal nerve by 3 days after crush or permanent transection injury of adult rat sciatic nerve. Whereas levels remained low after transection injury, cAMP levels reached only 27% of the normal values by 35 days after crush injury. Because P₀ mRNA levels were 60% of normal levels by 14 days and 100% by 21 days after crush injury, cAMP increased only well after P₀ gene induction. cAMP, therefore, does not appear to trigger myelin gene induction but may be involved in myelin assembly or maintenance. Forskolin, an activator of adenylyl cyclase, increased endoneurial cAMP levels only in the normal nerve, and in the crushed nerve beginning at 16 days after injury, but at no time in the transected nerve. Only by treating transected nerve with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterases, in combination with forskolin was it possible to increase cAMP levels. No induction of myelin genes, however, was observed with short- or long-term treatment with IBMX and forskolin in the transected nerve. A three-fold increase in phosphodiesterase activity was observed at 35 days after both injuries, and a nonmyelinated nerve was shown to have even higher activity. These experiments, therefore, suggest an important role for phosphodiesterase in the inactivation of this second messenger-dependent stimuli when Schwann cells are non-myelinating, such as after sciatic nerve injury or in the nonmyelinated nerve, which again implies that cAMP may be required for the maintenance of the myelin sheath.     

Pharmacological Characterization of the Voltage-Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System

Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K⁺-induced Ca²⁺ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC₅₀ = 250 nM). This toxin at 5 µM did not block Ca²⁺ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca²⁺ uptake by rat synaptosomes (IC₅₀ = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca²⁺ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC₅₀ similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca²⁺ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca²⁺ uptake in either the rat or the chicken. In conclusion, Ca²⁺ uptake by rat synaptosomes is potently inhibited by different P-type Ca²⁺ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca²⁺ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.     

High Levels of Ascorbic Acid, Not Glutathione, in the CNS of Anoxia-Tolerant Reptiles Contrasted with Levels in Anoxia-Intolerant Species

Abstract: Ascorbic acid and glutathione (GSH) are antioxidants and free radical scavengers that provide the first line of defense against oxidative damage in the CNS. Using HPLC with electrochemical detection, we determined tissue contents of these antioxidants in brain and spinal cord in species with varying abilities to tolerate anoxia, including anoxia-tolerant pond and box turtles, moderately tolerant garter snakes, anoxia-intolerant clawed frogs (Xenopus laevis), and intolerant Long-Evans hooded rats. These data were compared with ascorbate and GSH levels in selected regions of guinea pig CNS, human cortex, and values from the literature. Ascorbate levels in turtles were typically 100% higher than those in rat. Cortex, olfactory bulb, and dorsal ventricular ridge had the highest content in turtle, 5–6 µmol g^?1 of tissue wet weight, which was twice that in rat cortex (2.82 ± 0.05 µmol g^?1) and threefold greater than in guinea pig cortex (1.71 ± 0.03 µmol g^?1). Regionally distinct levels (2–4 µmol g^?1) were found in turtle cerebellum, optic lobe, brainstem, and spinal cord, with a decreasing anterior-to-posterior gradient. Ascorbate was lowest in white matter (optic nerve) in each species. Snake cortex and brainstem had significantly higher ascorbate levels than in rat or guinea pig, although other regions had comparable or lower levels. Frog ascorbate was generally in an intermediate range between that in rat and guinea pig. In contrast to ascorbate, GSH levels in anoxia-tolerant turtles, 2–3 µmol g^?1 of tissue wet weight, were similar to those in mammalian or amphibian brain, with no consistent pattern associated with anoxia tolerance. GSH levels in pond turtle CNS were significantly higher (by 10–20%) than in rat for several regions but were generally lower than in guinea pig or frog. GSH in box turtle and snake CNS were the same or lower than in rat or guinea pig. The distribution GSH in the CNS also had a decreasing anterior-to-posterior gradient but with less variability than ascorbate; levels were similar in optic nerve, brainstem, and spinal cord. The paradoxically high levels of ascorbate in turtle brain, which has a lower rate of oxidative metabolism than mammalian, suggest that ascorbate is an essential cerebral antioxidant. High levels may have evolved to protect cells from oxidative damage when aerobic metabolism resumes after a hypoxic dive.     

Activity of amylin at CGRP1-preferring receptors coupled to positive contractile response in rat ventricular cardiomyocytes

Calcitonin gene-related peptide (CGRP) exerts a positive contractile response directly in rat ventricular cardiomyocytes. This response is mediated by receptors of the CGRP₁-subtype. Amylin is 46% homologous with CGRP and binds to receptors selective for CGRP in a range of tissues. The ability of amylin to influence ventricular contractility has been assessed using cardiomyocytes isolated from the ventricles of adult rats. Cardiomyocytes were subjected to biphasic electrical stimulation at 0.5 Hz. CGRP produced a concentration-dependent positive contractile response which became maximal 4 min after initial stimulation. CGRP increased the contractile amplitude maximally at 1 nM and to a value which was 23.3% greater than in the absence of peptide (EC₅₀ VALUE = 21 pM). Amylin increased the contractile amplitude maximally at 20 nM and to a value which was 17.3% greater than in the absence of peptide (EC₅₀ VALUE = 216 pM). In the presence of amylin (20 nM), the concentration-dependence of the contractile response to CGRP was shifted to the left, so that the response became maximal when CGRP was present at 50 pM. In the presence of CGRP_8–37 (100 nM), a selective antagonist at CGRP₁-preferring receptors, the concentration-dependence of the contractile response to CGRP was shifted to the right (dose RATIO = 54). Similarly, in the presence of CGRP_8–37 (100 nM), the contractile response to amylin was inhibited significantly (P ≤ 0.01). Amylin_8–37 (100 nM) did not inhibit the concentration-dependence of the contractile responses to CGRP and amylin significantly (dose RATIOS = 4.2 and 2.4, respectively). In conclusion, these data indicate that amylin exerts a contractile response directly in rat ventricular cardiomyocytes via CGRP₁-preferring receptors. This effect could assume greater significance in non-insulin-dependent diabetes mellitus and in hypertensive states, in which the concentration of amylin is elevated in plasma.     

Sympathetic Sprouting: Time Course of Changes of Noradrenergic, Cholinergic, and Serotonergic Markers in the Denervated Rat Hippocampus

Abstract: As a first step for experiments investigating the presynaptic characteristics of sympathetic fibers grown into the denervated hippocampus, we studied the time course of changes of neurochemical markers in the rat hippocampus, subsequent to aspiration lesions of the fimbria-fornix and the overlying callosal and cortical structures. At various postsurgical delays (1, 2, 8, 24, and 40 weeks), the activity of choline acetyltransferase, the high-affinity synaptosomal uptake of choline and noradrenaline, and the concentrations of noradrenaline, serotonin, and 5-hydroxyindoleacetic acid were measured in a dorsal, an intermediate, and a ventral part of the hippocampus. Levels of all markers were significantly reduced shortly (1–2 weeks) after the lesions. However, whereas the cholinergic (choline uptake and choline acetyltransferase activity) and the serotonergic (concentrations of serotonin and 5-hydroxyindoleacetic acid) markers remained significantly reduced for up to 40 weeks, both noradrenergic markers recovered to near-normal (noradrenaline uptake) or even supranormal (noradrenaline concentration) levels, although with clear-cut differences in the time course and the regional characteristics. The noradrenaline content reached control levels already 8 weeks after lesion surgery and was about two to three times higher 40 weeks later, with the most dramatic effects in the ventral hippocampus. In contrast, high-affinity noradrenaline uptake reached control values only 24 weeks after lesion and exceeded them only in the ventral hippocampus 40 weeks after surgery. It is concluded (a) that hippocampal noradrenaline concentration is a more sensitive marker for sympathetic sprouting than high-affinity noradrenaline uptake and (b) that functional in vitro studies on hippocampal sympathetic ingrowth appear to fit optimal conditions in the ventral hippocampus at a delay of at least 40 weeks after surgery.     

Specific Activity of Brain Palmitoyl-CoA Pool Provides Rates of Incorporation of Palmitate in Brain Phospholipids in Awake Rats

Abstract: In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-³H]palmitate to maintain constant plasma specific activity. Animals were killed after 2–10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-³H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with >50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6–1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by ∼60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.     

Characterization of a Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Comparison with a 6S-Form

Abstract: 6R-l -erythro-Tetrahydrobiopterin (6R-BH₄) is a cofactor for aromatic l -amino acid hydroxylases and nitric oxide synthase. Recently, we have reported that independently of its cofactor activities, 6R-BH₄ acts from the outside of neurons in the brain to enhance the release of monoamine neurotransmitters such as dopamine. To characterize the pharmacological properties of the action, we examined the effects of 6S-BH₄, a diastereoisomer of 6R-BH₄, on dopamine release in the rat striatum by using brain microdialysis and compared its effects with those of 6R-BH₄. Perfusion of 6S-BH₄ or 6R-BH₄ through the dialysis probe increased extracellular dopamine levels (an index of in vivo dopamine release) concentration dependently; the maximal increase by 6S-BH₄, was one-sixth of that by 6R-BH₄. 6S-BH₄ increased extracellular DOPA levels in the presence of NSD 1015, an inhibitor of aromatic l -amino acid decarboxylase (an index of in vivo tyrosine hydroxylase activity), to an extent similar to the increase induced by 6R-BH₄. The increase in the DOPA levels induced by either of the pteridines was abolished after pretreatment of rats with α-methyl-p-tyrosine (an inhibitor of tyrosine hydroxylase). Under the same conditions, the 6S-BH₄-induced dopamine release was abolished, but most of the 6R-BH₄-induced increase persisted. Coadministration of 6S-BH₄ with 6R-BH₄ inhibited the increase in dopamine release induced by 6R-BH₄ alone. These results show that 6R-BH₄ stimulates dopamine release by acting at the specific recognition site on the neuronal membrane, and that 6S-BH₄ acts as an antagonist of 6R-BH₄ at this site, although it has cofactor activities.