A study on the mechanism of the vanadate-dependent NADH oxidation

The mechanism of the vanadate (V(_v))-dependent oxidation of NADH was different in phosphate buffers and in phosphate-free media. In phosphate-free media (aqueous medium or HEPES buffer) the vanadyl (V(_v)) generated by the direct V(_v)-dependent oxidation of NADH formed a complex with V(_v). In phosphate buffers V(_v) autoxidized instead of forming a complex with V(_v). The generated superoxide radical (O₂⁻) initiated, in turn, a high-rate free radical chain oxidation of NADH. Phosphate did not stimulate the V(_v)-dependent NADH oxidation catalyzed by O₂⁻-generating systems. Monovanadate proved to be a stronger catalyzer of NADH oxidation as compared to polyvanadate.     

槲皮素对人脑胶质瘤干细胞生物学行为及miR-29s家族的影响分析

目的探讨分析槲皮素对人脑胶质瘤干细胞（BGSCs）生物学行为及miR-29s家族的影响。 方法使用干细胞培养液对U87人脑胶质瘤细胞进行培养,采用CCK-8法检测槲皮素对BGSCs细胞增殖抑制率,采用流式细胞技术检测槲皮素对BGSCs细胞凋亡影响,并采用real-time PCR鉴定槲皮素对BGSCs细胞中miR-29a、miR-29b以及miR- 29c表达的影响。采用t检验以及方差分析进行统计学分析。 结果随着槲皮素浓度的增加,BGSCs细胞增殖抑制率增加24 h 0 μg/ml（0.00±0.12）%、10 μg/ml（1.36±0.38）%、20 μg/ml（15.33±3.01）%、40 μg/ ml（29.50±4.57）%、80 μg/ml（40.21±6.42）%、160 μg/ml（61.21±7.48）,F = 76.273,P < 0.05;48 h 0 μg/ml （0.09±0.05）%、10 μg/ml（9.84±2.17）%、20 μg/ml（28.57±3.84）%、40 μg/ ml（43.59±5.21）%、80 μg/ml（59.50±3.28）%、160 μg/ ml（70.21±9.48）%,F = 85.392,P < 0.05,且同浓度槲皮素作用48 h对BGSCs细胞增殖抑制率高于作用24 h（P < 0.05）。随着槲皮素浓度的升高,BGSCs细胞凋亡率升高[0 μg/ml（13.42±1.21）%、20 μg/ml（38.47±9.28）%、40 μg/ml（59.34±7.20）%、80 μg/ml（71.42±9.47）%,F = 57.493,P < 0.05]。不同浓度槲皮素处理BGSCs细胞后,可促进BGSCs细胞miR-29s家族miR- 29a/ b/ c相对表达量,且随着槲皮素浓度的增加,BGSCs细胞miR-29s家族相对表达量增加[miR-29a 0 μg/ml（1.04±0.08）、20 μg/ml（1.16±0.05）、40 μg/ml（1.30±0.10）、80 μg/ ml（1.41±0.09）,F = 19.281,P < 0.05;miR-29b 0 μg/ml（1.06±0.09）、20 μg/ml（1.13±0.05）、40 μg/ml（1.25±0.07）、80 μg/ml（1.30±0.09）,F = 13.427,P < 0.05;miR-29c 0 μg/ml（1.03±0.07）、20 μg/ml（1.15±0.03）、40 μg/ml（1.22±0.06）、80 μg/ml（1.31±0.08）,F = 14.502,P < 0.05]。 结论槲皮素可有效抑制人脑BGSCs增殖,促进人脑BGSCs凋亡,并促进人脑BGSCs中miR- 29s家族表达。     

Enkephalin-immunoreactive subpopulations in the myenteric plexus of the guinea-pig fundus project primarily to the muscle and not to the mucosa

Enkephalin (ENK) immunoreactivity was localised in different neuronal subpopulations of the myenteric plexus in the guinea-pig gastric fundus using immunohistochemistry for neurone-specific enolase (NSE), ENK, choline acetyltransferase (ChAT), substance P (SP), neuropeptide Y (NPY), calretinin (CALRET), and somatostatin (SOM). NADPH-diaphorase staining was used to label nitric oxide synthase (NOS)-containing neurones. ENK was observed in 44% of the myenteric neurones. The major ENK-positive subpopulations were ChAT/ENK (35% of ENK-positive neurones), ChAT/SP/ENK (26%), NOS/NPY/ENK (22%) and ChAT/SP/ENK/CALRET (9%). The projection pathways of these ENK-positive subpopulations to the circular muscle and the mucosa were determined using retrograde labelling with DiI in organ culture followed by immunohistochemistry. Of myenteric neurones retrogradely labelled from the mucosa and the circular muscle, 13% and 48% exhibited ENK immunoreactivity, respectively. Three major ENK-positive subpopulations innervating the mucosa or circular muscle were identified: ascending ChAT/SP/ENK (7% of all mucosa neurones; 24% of all circular muscle neurones), ascending ChAT/ENK (4%; 15%) and descending NOS/NPY/ENK (1%; 8%) neurones. Only very few CALRET- or SOM-positive neurones projected to the mucosa or circular muscle. ChAT/SP/ENK and ChAT/ENK neurones might function as ascending excitatory muscle motor neurones, whereas NOS/NPY/ENK neurones are most likely descending inhibitory muscle motor neurones. The relatively few ENK-positive mucosa neurones do not favour a major involvement of ENK-positive myenteric neurones in the control of gastric mucosa activity.     

N-Ethylmaleimide inhibition of the catalytic activities of the Dunaliella salina coupling factor 1 (CF1) and the restoration of the inhibition of the CF1 ATPase activity by N-ethylmaleimide

The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF₁) to chemical modification by N-ethylmaleimide has been investigated. (i) When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF₁ ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF₁ as shown by (a) the time-course for the inhibition and (b) the maximal extent of inhibition. (ii) Treatment of the purified, latent, D. salina CF₁ with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I₅₀ ≈ 50 μM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. (iii) N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF₁ is partially reversed by either (a) prolonged incubation at low concentrations of N-ethylmaleimide or (b) short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF₁ that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original-ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF₁ are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.     

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans .
Methods and Results:  In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0·13 g l⁻¹ N) initial NaNO₃ or (NH₄)₂SO₄ levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0·78 g l⁻¹ N) initial NaNO₃ levels. EPS produced by CSTR grown cultures with high (NH₄)₂SO₄ levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO₃ levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units.
Conclusions:  While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology.
Significance and Impact of the Study:  These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.     

IL-7 Is essential for the development and the persistence of chronic colitis

Although IL-7 has recently emerged as a key cytokine involved in controlling the homeostatic turnover and the survival of peripheral resting memory CD4(+) T cells, its potential to be sustained pathogenic CD4(+) T cells in chronic immune diseases, such as inflammatory bowel diseases, still remains unclear. In this study, we demonstrate that IL-7 is essential for the development and the persistence of chronic colitis induced by adoptive transfer of normal CD4(+)CD45RB(high) T cells or colitogenic lamina propria (LP) CD4(+) memory T cells into immunodeficient IL-7(+/+) x RAG-1(-/-) and IL-7(-/-) x RAG-1(-/-) mice. Although IL-7(+/+) x RAG-1(-/-) recipients transferred with CD4(+)CD45RB(high) splenocytes developed massive inflammation of the large intestinal mucosa concurrent with massive expansion of Th1 cells, IL-7(-/-) x RAG-1(-/-) recipients did not. Furthermore, IL-7(-/-) x RAG-1(-/-), but not IL-7(+/+) x RAG-1(-/-), mice transferred with LP CD4(+)CD44(high)CD62L(-)IL-7Ralpha(high) effector-memory T cells (T(EM)) isolated from colitic CD4(+)CD45RB(high)-transferred mice did not develop colitis. Although rapid proliferation of transferred colitogenic LP CD4(+) T(EM) cells was observed in the in IL-7(-/-) x RAG-1(-/-) mice to a similar extent of those in IL-7(+/+) x RAG-1(-/-) mice, Bcl-2 expression was significantly down-modulated in the transferred CD4(+) T cells in IL-7(-/-) x RAG-1(-/-) mice compared with those in IL-7(+/+) x RAG-1(-/-) mice. Taken together, IL-7 is essential for the development and the persistence of chronic colitis as a critical survival factor for colitogenic CD4(+) T(EM) cells, suggesting that therapeutic approaches targeting IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases.     

Investigation of the mechanism of nicotine-induced relaxation on the sheep sphincter of Oddi

Possible mechanisms for nicotine-induced relaxation were investigated in the isolated sheep's sphincter of Oddi. Sheep's sphincter of Oddi rings were mounted in tissue bath with modified Krebs-Henseleit solution and aerated with 95% oxygen and 5% carbon dioxide. Tension was measured with isometric force transducers, and muscle relaxation was expressed as percent decrease of precontraction induced by carbachol. Nicotine (1 x 10(-5) to 3 x 10(-3) mol/L) produced concentration-dependent relaxation on sphincter of Oddi precontracted by carbachol (10(-6) mol/L). Nicotine-induced relaxation was 72.8 +/- 4.2% of precontraction with carbachol (10(-6) mol/L) (mean pD2 value, 3.76 +/- 0.05 mol/L). Nicotine-induced relaxation was not affected by N(w)-nitro L-arginine methyl ester (L-NAME) (3 x 10(-5) mol/L), methylene blue (10(-5) mol/L), indomethacin (10(-5) mol/L), hexamethonium (10(-5) mol/L), glibenclamide (10(-5) mol/L), 4-aminopyridine (10(-3) mol/L), tetraethylammonium (3 x 10(-4) mol/L), clotrimazole (10(-6) mol/L), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (10(-6) mol/L), and anthracene-9-carboxylate (9-AC) (10(-6) mol/L), but potentiated by bupivacain (10(-5) mol/L). A calcium-antagonizing effect of nicotine was not observed. The results suggest that nicotine-induced relaxation of the sheep's sphincter of Oddi is not mediated by the release of prostaglandins, nitric oxide (NO), or a related substance; by the activation of potassium channels or chloride channels; or by the stimulation of nicotinic cholinoceptors. Potentiation of the nicotine-induced relaxation by bupivacain indicates that blockade of sodium channels may play a role in this relaxation.     

Expression of the fibroblast growth factor-5 gene in the mouse embryo.

Fibroblast growth factors (FGFs) are structurally related mitogens that can regulate the differentiation of a wide variety of cells. As a step towards elucidating the developmental roles played by one of these factors, we have used in situ hybridization methods to examine expression of the murine F gf-5 gene during embryogenesis. F gf-5 RNA was detected at seven distinct sites in the developing mouse embryo: (1) postimplantation epiblast (embryonic day 5 1/4-7 1/2), (2) lateral splanchnic mesoderm (E9 1/2-10 1/2), (3) lateral somatic mesoderm (E10 1/2-12 1/2), (4) myotomes (E10 1/2-12 1/2), (5) mastication muscle (E11 1/2-14 1/2), (6) limb mesenchyme (E12 1/2-14 1/2), and (7) acoustic ganglion (E12 1/2-14 1/2). At several of these sites, expression is spatially restricted within the tissues. We offer several hypotheses regarding the roles of FGF-5 in murine development.     

Probing conformations of the glycerol backbones of triacyglycerols in the active site of lipase by 1,2-cyclopentane-carbamates: The meso effect for the enzyme inhibition

The aim of this study is to probe the glycerol backbone conformation of the substrate (or inhibitor) in the active site of Pseudomonas species lipase by the 1,2-cyclopentandiol analogues of the ethylene glycerol carbamate inhibitors. Cyclopentane-carbamates, cis-1,2-di-N-n- butylcarbamyl-cyclopentane (1) and trans-1,2-di-N-n-butylcarbamyl-cyclopentane (2), are the conformationally constrained analogues of 1,2-di-N-n-butylcarbamyl ethane (3). All carbamates are synthesized and characterized as the pseudo-substrate inhibitors of the enzyme. Cis-cyclopentane-di-carbamate (1) is a more potent inhibitor than both ethane-di-carbamate (3) and trans-cyclopentane-di-carbamate (2) probably because the glycerol backbone conformations of cis-cyclopentane-di-carbamate (1) are constrained by the cyclopentane ring and cis-cyclopentane-di-cabamate (1) is a meso compound but trans-cyclopentane-di-carbamate (2) is a racemate.     

青藏高原东部地区田鼠物种的分子鉴定

以线粒体细胞色素b基因序列（Cyt b）为分子标记,结合系统发育分析（最大似然法和贝叶斯法）和遗传距离统计（JC遗传距离模型）等方法,分析青藏高原东部地区田鼠类动物的物种组成和地理分布特征。从16个样点共采集到189个田鼠样品,成功获得所有个体的Cyt b全序列,长度为1 143 bp,共检测到248个变异位点和65个单倍型。系统发育分析显示,最大似然树和贝叶斯树的结构基本一致,将65个单倍型分为三组（G1-G3）,分别与已知的柴达木根田鼠（Microtus limnophilus）、青海松田鼠（Neodon fuscus）和高原松田鼠（N. irene）聚为单系群,支持率都为100%。遗传距离统计显示,G1、G2、G3组内两两单倍型之间的遗传距离范围分别为0.09%~3.04%、0.09%~0.70%、0.18%~1.95%;同时,G1与柴达木根田鼠参考序列之间、G2与青海松田鼠参考序列之间、G3与高原松田鼠参考序列之间遗传距离范围分别为0.61%-2.49%、0.53%-0.97%、1.77%-2.22%。结合系统发育和遗传距离分析结果,本研究中采集到的田鼠个体可以鉴定为3个物种：柴达木根田鼠（n = 135）、青海松田鼠（n = 30）和高原松田鼠（n = 24）。其中,柴达木根田鼠分布最广（10个地点）,青海松田鼠（4个地点）和高原松田鼠（3个地点）的分布区则相对狭小;3种动物在分布区上重叠度很小,仅河南县同时发现有柴达木根田鼠和青海松田鼠分布。研究表明,青藏高原东部地区至少有3种田鼠分布,种间遗传界限清晰,空间分布具有一定规律性。研究结果为了解青藏高原东部地区田鼠类动物的物种分布提供可靠的基础资料,同时,为这一地区的田鼠类动物的分子鉴定方法提供参考。     

The beta G156C substitution in the F1-ATPase from the thermophilic Bacillus PS3 affects catalytic site cooperativity by destabilizing the closed conformation of the catalytic site

Fluorescence titrations of the alpha(3)(betaG(156)C/Y(345)W)(3)gamma, alpha(3)(betaE(199)V/Y(345)W)(3)gamma, and alpha(3)(betaY(345)W)(3)gamma subcomplexes of TF(1) with nucleotides show that the betaG(156)C substitution substantially lowers the affinity of catalytic sites for ATP and ADP with or without Mg(2+), whereas the betaE(199)V substitution increases the affinity of catalytic sites for nucleotides. Whereas the alpha(3)(betaG(156)C)(3)gamma and alpha(3)(betaE(199)V)(3)gamma subcomplexes hydrolyze 2 mM ATP at 2% and 0.7%, respectively, of the rate exhibited by the wild-type enzyme, the alpha(3)(betaG(156)C/E(199)V)(3)gamma hydrolyzes 2 mM ATP at 9% the rate exhibited by the wild-type enzyme. The alpha(3)(betaG(156)C)(3)gamma, alpha(3)(betaG(156)C/E(199)V)(3)gamma, and alpha(3)(betaG(156)C/E(199)V/Y(345)W)(3)gamma subcomplexes resist entrapment of inhibitory MgADP in a catalytic site during turnover. Product [(3)H]ADP remains tightly bound to a single catalytic site when the wild-type, betaE(199)V, betaY(345)W, and betaE(199)V/Y(345)W subcomplexes hydrolyze substoichiometric [(3)H]ATP, whereas it is not retained by the betaG(156)C and betaG(156)C/Y(345)W subcomplexes. Less firmly bound, product [(3)H]ADP is retained when the betaG(156)C/E(199)V and betaG(156)C/E(199)V/Y(345)W mutants hydrolyze substoichiometric [(3)H]ATP. The Lineweaver-Burk plot obtained with the betaG(156)C mutant is curved downward in a manner indicating that its catalytic sites act independently during ATP hydrolysis. In contrast, the betaG(156)C/E(199)V and betaG(156)C/E(199)V/Y(345)W mutants hydrolyze ATP with linear Lineweaver-Burk plots, indicating cooperative trisite catalysis. It appears that the betaG(156)C substitution destabilizes the closed conformation of a catalytic site hydrolyzing MgATP in a manner that allows release of products in the absence of catalytic site cooperativity. Insertion of the betaE(199)V substitution into the betaG(156)C mutant restores cooperativity by restricting opening of the catalytic site hydrolyzing MgATP for product release until an open catalytic site binds MgATP.     

A new definition for the consensus sequence of the peroxisome targeting signal type 2

All organisms except the nematode Caenorhabditis elegans have been shown to possess an import system for peroxisomal proteins containing a peroxisome targeting signal type 2 (PTS2). The currently accepted consensus sequence for this amino-terminal nonapeptide is -(R/K)(L/V/I)X(5)(H/Q)(L/A)-. Some C.elegans proteins contain putative PTS2 motifs, including the ortholog (CeMeK) of human mevalonate kinase, an enzyme known to be targeted by PTS2 to mammalian peroxisomes. We cloned the gene for CeMeK (open reading frame Y42G9A.4) and examined the subcellular localization of CeMeK and of two other proteins with putative PTS2s at their amino termini encoded by the open reading frames D1053.2 and W10G11.11. All three proteins localized to the cytosol, confirming and extending the finding that C.elegans lacks PTS2-dependent peroxisomal protein import. The putative PTS2s of the proteins encoded by D1053.2 and W10G11.11 did not function in targeting to peroxisomes in yeast or mammalian cells, suggesting that the current PTS2 consensus sequence is too broad. Analysis of available experimental data on both functional and nonfunctional PTS2s led to two re-evaluated PTS2 consensus sequences: -R(L/V/I/Q)XX(L/V/I/H)(L/S/G/A)X(H/Q)(L/A)-, describes the most common variants of PTS2, while -(R/K)(L/V/I/Q)XX(L/V/I/H/Q)(L/S/G/A/K)X(H/Q)(L/A/F)-, describes essentially all variants of PTS2. These redefined PTS2 consensus sequences will facilitate the identification of proteins of unknown cellular localization as possible peroxisomal proteins.     

Destabilization of the duplex and the high-salt Z-form of poly(dG-methyldC) by substitution of ethyl for the 5-methyl group

The B-to-Z conformational transition of poly(dG-dC) is highly promoted by 5-methyl substitution of the dC moiety, i.e. in poly(dG-methyl⁵dC). By the synthesis of a new poly(dG-dC) analogue, poly(dG-ethyl⁵dC), the effect of a longer alkyl-chain substituent of dC on structure and conformation has been studied with ultraviolet absorption melting profiles and circular dichroism spectroscopy. The 5-ethyl substituent in poly(dG-ethyl⁵dC) destabilizes the duplex structure against thermal denaturation compared with both poly(dG-methyl⁵dC) and poly(dG-dC). C.d. studies also reveal that for the high-salt B-Z transition of poly(dG-ethyl⁵dC) a higher NaCl concentration is required than for that of poly(dG-methyl⁵dC), although much lower than for poly(dG-dC). However low-salt Z-DNA in poly(dG-ethyl⁵dC) shows unique features, e.g. it needs no divalent cations to be stable. The low-salt B-Z transition of poly(dG-ethyl⁵dC) can also be observed by the absorption-temperature melting profile, in constrast to both poly(dG-methyl⁵dC) and poly(dG-dC). The effects of MgCl₂ concentration, temperature, acid pH and trifluorethanol on the conformation of poly(dG-ethyl⁵dC) have also been determined.     

Roles for TAB1 in regulating the IL-1-dependent phosphorylation of the TAB3 regulatory subunit and activity of the TAK1 complex

The protein kinase TAK1 (transforming growth factor-beta-activated kinase 1), which has been implicated in the activation of MAPK (mitogen-activated protein kinase) cascades and the production of inflammatory mediators by LPS (lipopolysaccharide), IL-1 (interleukin 1) and TNF (tumour necrosis factor), comprises the catalytic subunit complexed to the regulatory subunits, termed TAB (TAK1-binding subunit) 1 and either TAB2 or TAB3. We have previously identified a feedback-control mechanism by which p38alpha MAPK down-regulates TAK1 and showed that p38alpha MAPK phosphorylates TAB1 at Ser(423) and Thr(431). In the present study, we identified two IL-1-stimulated phosphorylation sites on TAB2 (Ser(372) and Ser(524)) and three on TAB3 (Ser(60), Thr(404) and Ser(506)) in human IL-1R cells [HEK-293 (human embryonic kidney) cells that stably express the IL-1 receptor] and MEFs (mouse embryonic fibroblasts). Ser(372) and Ser(524) of TAB2 are not phosphorylated by pathways dependent on p38alpha/beta MAPKs, ERK1/2 (extracellular-signal-regulated kinase 1/2) and JNK1/2 (c-Jun N-terminal kinase 1/2). In contrast, Ser(60) and Thr(404) of TAB3 appear to be phosphorylated directly by p38alpha MAPK, whereas Ser(506) is phosphorylated by MAPKAP-K2/MAPKAP-K3 (MAPK-activated protein kinase 2 and 3), which are protein kinases activated by p38alpha MAPK. Studies using TAB1(-/-) MEFs indicate important roles for TAB1 in recruiting p38alpha MAPK to the TAK1 complex for the phosphorylation of TAB3 at Ser(60) and Thr(404) and in inhibiting the dephosphorylation of TAB3 at Ser(506). TAB1 is also required to induce TAK1 catalytic activity, since neither IL-1 nor TNFalpha was able to stimulate detectable TAK1 activity in TAB1(-/-) MEFs. Surprisingly, the IL-1 and TNFalpha-stimulated activation of MAPK cascades and IkappaB (inhibitor of nuclear factor kappaB) kinases were similar in TAB1(-/-), MEKK3(-/-) [MAPK/ERK (extracellular-signal-regulated kinase) kinase kinase 3] and wild-type MEFs, suggesting that another MAP3K (MAPK kinase kinase) may mediate the IL-1/TNFalpha-induced activation of these signalling pathways in TAB1(-/-) and MEKK3(-/-) MEFs.     

Species differences in the behavioral effects of cerulein--an agonist of the receptors of the octapeptide cholecystokinin--in white mice and rats

It has been shown in the behavioural experiments that combined pretreatment with haloperidol (0.25 mg/kg) and caerulein (40 micrograms/kg), and to a lesser extent pretreatment with caerulein alone caused long-term reversal of amphetamine (2 mg/kg) induced hyperexcitability in rats. Administration of proglumide (50 mg/kg), an antagonist of CCK-8 receptors, did not reverse long-term antiamphetamine effect of caerulein. In mice pretreatment with caerulein (50 and 100 micrograms/kg) alone or in combination with haloperidol (0.25 mg/kg) caused hypersensitivity to the behavioural effect of amphetamine (3 mg/kg). Intraventricular (I ng), but not systemic (100-500 micrograms/kg) administration of caerulein selectively antagonized seizures in mice induced by intraventricular administration of quinolinic acid (5 micrograms) and N-methyl-D-aspartate (0.2 microgram). Pretreatment with proglumide (50 mg/kg) reversed the anticonvulsive effect of caerulein in mice. In rats, caerulein failed to affect the seizures caused by intraventricular administration of quinolinic acid. The results of the present study demonstrate the existence of obvious interspecies differences in the behavioural effects of caerulein, the agonist of CCK-8 receptors, in mice and rats.     

Chronic systemic treatment with epidermal growth factor in the rat increases the mucosal surface of the small intestine

We examined the effects of treatment with human recombinant epidermal growth factor (EGF) on the functioning small intestine in the rat. Male Wistar rats, 7–8 weeks old, were treated with EGF administered subcutaneously in doses of 0 (n = 7) or 150 μg/kg/day (n = 8) for 4 weeks. The histological composition and mucosal surface area of the perfusion-fixed small intestine was quantified with stereological principles. The length of the gut remained unchanged. The amount of tissue and surface area per length of gut (median (ranges)) were increased from 117 (101–131) mg/cm and 2.6 (2.1–3.5) cm²/cm in the controls to 146 (138–152) mg/cm and 3.5 (2.5–3.8) cm²/cm for the complete small intestine (both comparisons p < 0.02). The weight increase was due to mucosal growth in all parts of the intestine, whereas the surface area was only increased in proximal and middle parts. It is concluded that EGF treatment in rats increases the mucosal weight and surface area of the functioning small intestine.     

Muscarinic cholinergic components in the carp brain

The activity of the muscarinic cholinergic system (acetylcholine, ACh; acetylcholinesterase, AChE; choline acetyltransferase, ChAT; muscarinic acetylcholine receptors) was studied in the carp brain. The ACh content (13.9 ± 1.1 nmol/g wet tissue) was estimated by gas chromatography after microwave irradiation focused to the head. The AChE and ChAT activities were 153 ± 13 nmol/min/mg protein and 817 ± 50 pmol/min/mg protein, respectively. The characteristics of [³H](−)quinuclidinyl benzilate ([³H](−)QNB) and [³H]pirenzepine ([³H]PZ) binding were also studied in brain membranes. Their specific binding was linearly dependent on the protein content and they appeared to bind with high affinity to a single, saturable binding site. A dissociation constant (K_d) of 47 ± 6.3 pM and a maximum number of binding sites (B_max) of 627 ± 65 fmol/mg protein were obtained for [³H](−)QNB, with a K_d value of 3.85 ± 0.67 nM and a B_max value of 95.3 ± 6.25 fmol/mg protein for [³H]PZ binding. The [³H]PZ binding amounted to only 15% of the [³H](−)QNB-labeled sites, as estimated from the ratio of the B_max values of [³H](−)QNB and [³H]PZ, suggesting a low density of M₁ subtype. Atropine sulfate, atropine methylnitrate and PZ inhibited the binding of both radioligands with Hill slopes (n_H) close to unity. The n_H value of AF-DX 116 was close to 1 against [³H](−)QNB binding, while it was 0.75 against [³H]PZ binding. The displacement curves of oxotremorine and carbachol were shallow for the binding of both radioligands. The rank order of potency of muscarinic ligands against [³H](−)QNB binding (K_i nM) was atropine sulfate (0.55) > atropine methylnitrate (1.61) > PZ (61.19) > oxotremorine (156.3) > AF-DX 116 (307) > carbachol (1301), while in the case of [³H]PZ binding it was atropine sulfate (0.24) > atropine methylnitrate (0.34) > PZ (10.38) > AF-DX 116 (55.87) > oxotremorine (62.79) > carbachol (1696). The results indicate the presence of a well-developed muscarinic cholinergic system with predominantly M₂ receptors in the carp brain.     

Association found between the promoter region polymorphism in the apolipoprotein A-V gene and the serum triglyceride level in Japanese schoolchildren

The purpose of this study was to assess the influence of single nucleotide polymorphism 3 (SNP3) of the apolipoprotein A-V ( APOA5) gene on the serum triglyceride (TG) level in Japanese schoolchildren. To determine the frequency of the genotype, we analyzed 552 schoolchildren. The frequencies of the T/T, T/C and C/C genotypes of the APOA5 gene were 225 (40.8%), 263 (47.6%) and 64 (11.6%), respectively. The serum TG level was significantly different among the genotypic groups after adjustments for age, gender and obesity index (T/T 71.6+/-34.8 mg/dl, T/C 80.7+/-36.1 mg/dl, C/C 94.4+/-69.4 mg/dl, P<0.0001). The odds ratio (95% confidence interval) for hypertriglyceridemia of the C allele was 2.4 (1.0-6.2). Our data suggested that the T/C promoter region polymorphism of the APOA5 gene appears to be a genetic risk factor for hypertriglyceridemia in Japanese children.     

Synthesis of the tetrasaccharide repeating-unit of the lipopolysaccharide isolated from pseudomonas maltophilia

The title tetrasacharide having the structure 3-O-Me-β- -Xylp-(1→4)-- -Rhap-(1→4)-- -Rhap-(1→2)- -Rhap was obtained by reaction of the -acetobromo derivative of 4-O-(3-O-methyl-β- -xylopyranosyl)- -rhamnopyranose and benzyl 3,4-di-O-benzyl-2-O-(2,3-O-isopropylidene-- -rhamnopyranosyl)-- -rhamnopyranoside, followed by removal of the protecting groups. The synthesised compounds were characterised on the basis of n.m.r. data.     

Participation of m- and n-cholinergic mechanisms in the development of the kindling phenomenon of the amygdala in cats

The kindling phenomenon was produced after chronic electrostimulation of the cat amygdala. The duration and intensity of petit mal and grand mal were recorded. The M-cholinomimetic arecoline (0.3 mg/kg), the acetylcholinesterase inhibitor galanthamine (1-3 mg/kg) and the N-cholinergic blockers eterofen (5-10 mg/kg) and ganglerone (035-3.5 mg/kg) intraperitoneally decreased or abolished the kindling phenomenon. Combination of M-cholinomimetics with N-blockers facilitated the anticonvulsant effects. Nicotine (0.5 mg/kg) and the M-cholinergic blocker methylbenactyzine (0.5-1 mg/kg) as well as combination of methylbenactyzine with galanthamine, on the contrary, facilitated and aggravated seizures in cats. In the authors' opinion, the M- and N-cholinergic mechanisms are involved in formation of the kindling phenomenon. It is suggested that N-cholinergic blockers or their combinations with M-cholinomimetics may be used as anticonvulsants.