Audiogenic Epilepsy and Structural Features of Superior Colliculus in KM Rats

Using immunoblotting, we showed that in rats of audiogenic epilepsy (AE) prone strain (Krushinsky- Molodkina, KM) the superior colliculus tissue (SC) contains significantly less quantity of glial neurotrophic factor (GDNF), beta-tubulin and actin in comparison to the same brain region in “0” rats, nonprone to AE. This fact led to the suggestion that the histological structure of the SC in KM rats could differ significantly from that of the “0” strain. Using neuromorphologу technique, we demonstrated that the total number of SC cells, as well as the number of neurons were significantly less in KM rats than in the “0” strain rats. Particularly strong differences were found in the deep layers of SC, the area of terminals from IC. Further studies of the midbrain structures, will help to identify the novel aspects of neural networks, involved in the genesis of AE in rats of KM strain.     

Annexin A4 reduces water and proton permeability of model membranes but does not alter aquaporin 2-mediated water transport in isolated endosomes

Annexin A4 (Anx4) belongs to a ubiquitous family of Ca2+-dependent membrane-binding proteins thought to be involved in membrane trafficking and membrane organization within cells. Anx4 localizes to the apical region in epithelia; however, its physiological role is unclear. We show that Anx4 exhibited binding to liposomes (phosphatidylcholine:phosphatidylserine, 1:1) in the presence of Ca2+ and binding was reversible with EDTA. Anx4 binding resulted in liposome aggregation and a reduction in membrane water permeability of 29% (P < 0.001) at 25 degrees C. These effects were not seen in the presence of Ca2+ or Anx4 alone and were reversible with EDTA. Measurements of membrane fluidity made by monitoring fluorescence anisotropy of 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-HPC) demonstrated that Anx4 binding rigidified the outer leaflet of the bilayer (P < 0.001), thus providing a molecular explanation for the inhibition of water flux. To determine whether Anx4 would produce similar effects on physiological membranes we constructed liposomes which recapitulated the lipid composition of the inner leaflet of the MDCK apical membrane. These membranes exhibited reductions to water permeability upon Anx4 binding (19.5% at 25 degrees C, 31% at 37 degrees C; P < 0.01 and P < 0.001, respectively) and to proton permeability (15% at 25 degrees C, 19.5% at 37 degrees C; P < 0.05). Since our in vitro experiments indicated an effect on membrane permeability, we examined localization of Anx4 in the kidney collecting duct, a region of the nephron responsible for concentrating urine through water reabsorbtion. Anx4 was shown to colocalize apically with aquaporin 2 (AQP2) in collecting duct epithelia. To test for the existence of a functional interaction between Anx4 and AQP2 we isolated AQP2-containing endosomes and exposed them to Anx4/Ca2+. Water flux rates were unchanged, indicating Anx4 does not directly regulate AQP2. We conclude that Anx4 can alter the physical properties of membranes by associating with them and regulate passive membrane permeability to water and protons. These properties represent important new functions for Anx4.     

Production of wheat-rye substitution lines and identification of chromosome composition of karyotypes using C-banding, GISH, and SSR markers

Based on the cross (Triticum aestivum L. × Secale cereale L.) × T. aestivum L., wheat-rye substitution lines (2n = 42) were produced with karyotypes containing, instead of a pair of homologous wheat chromosomes, a homeologous pair of rye chromosomes. The chromosome composition of these lines was described by GISH and C-banding methods, and SSR analysis. The results of genomic in situ hybridization demonstrated that karyotype of these lines included one pair of rye chromosomes each and lacked wheat-rye translocations. C-banding and SSR markers were used to identify rye chromosomes and determine the wheat chromosomes at which the substitution occurred. The lines were designated 1R(1D), 2R(2D)₂, 2R(2D)₃, 3R(3B), 6R(6A)₂. The chromosome composition of lines 1R(1A), 2R(W)₁, 5R(W), 5R(5A), and 6R(W)₁, which were earlier obtained according to the same scheme for crossing, was characterized using methods of telocentric analysis, GISH, C-banding, and SSR analysis. These lines were identified as 1R(1A), 2R(2D)₁, 5R(5D), 5R(5A), and 6R(6A)₁, C-banding of chromosomes belonging to line 1R(1A) revealed the presence of two translocated chromosomes (3DS.3DL-del. and 4AL.W) during simultaneous amplification of SSR markers located on 3DL and 4AS arms. The “combined” long arm of the newly derived chromosome 4A is assumed to be formed from the long arm of chromosome 4AS itself and a deleted segment 3DL. All examined lines are cytologically stable, except for 3R(3B), which does not affect the stability of rye 3R chromosome transfer. Chromosome identification and classification of the lines will permit them to be models for genetic studies that can be used thereafter as promising “secondary gene pools” for the purpose of plant breeding.     

Cytotoxic activity of expanded coordination bis-thiosemicarbazones and copper complexes thereof

A series of bis-thiosemicarbazone agents with coordinating groups capable of multiple metal coordination modes has been generated and evaluated for potential cytotoxic effects against melanoma (MelRm) and breast adenocarcinoma (MCF-7) cell lines. The bis-thiosemicarbazones in this study generally demonstrated superior cytotoxic activity against MelRm than MCF-7 in the absence of metal ion supplementation, but in most cases could not be considered superior to the reference thiosemicarbazone Dp44mT. The key structural features for the cytotoxic activity were the central metal binding atom on the aromatic core, the thiocarbonyl residue and the nature of substitution on the N4-terminus in terms of size and lipophilicity. The cytotoxicity of bis-thiosemicarbazone ligands improved significantly with Cu(II) supplementation, particularly against MCF-7 cells. The mechanism of cytotoxicity of bis-thiosemicarbazones was proposed to be dependent on the combined effect of metal mobilisation and ROS generation which is so called a “double-punch effect”.     

Regulation of autophagy and apoptosis by Dp44mT-mediated activation of AMPK in pancreatic cancer cells

Upon activation, the 5′-adenosine monophosphate-activated protein kinase (AMPK) increases catabolism, while inhibiting anabolism. The anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), activates AMPK in multiple tumor cell-types (Biochim. Biophys Acta 2016;1863:2916–2933). This acts as an initial cell “rescue response” after iron-depletion mediated by Dp44mT. Considering Dp44mT-mediated AMPK activation, the role of AMPK on Dp44mT cytotoxicity was examined. Dp44mT increased the p-AMPK/AMPK ratio in multiple tumor cell-types over short (24 h) and longer (72 h) incubations. Notably, Dp44mT was more effective in inhibiting tumor cell proliferation after AMPK silencing, potentially due to the loss of AMPK-mediated metabolic plasticity that protects cells against Dp44mT cytotoxicity. The silencing of AMPK-increased cellular cholesterol and stabilized lysosomes against Dp44mT-mediated lysosomal membrane permeabilization. This was substantiated by studies demonstrating that the cholesterol-depleting agent, methyl-β-cyclodextrin (MβCD), restores Dp44mT-mediated lysosomal membrane permeabilization in AMPK silenced cells. The increased levels of cholesterol after AMPK silencing were independent of the ability of AMPK to inhibit the rate-limiting step of cholesterol synthesis via the inactivating phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) at Ser872. In fact, Dp44mT did not increase phosphorylation of HMGCR at (Ser872), but decreased total HMGCR expression similarly in both the presence or absence of AMPK silencing. Dp44mT was demonstrated to increase autophagic initiation after AMPK silencing via an AMPK- and Beclin-1-independent mechanism. Further, there was increased cleaved caspase 3 and cleaved PARP after incubation of AMPK silenced cells with Dp44mT. Overall, AMPK silencing promotes Dp44mT anti-proliferative activity, suggesting a role for AMPK in rescuing its cytotoxicity by inhibiting autophagy and also apoptosis.     

Rat brain monoamine levels related to behavioral assessment

Randomly selected adult, male, Sprague-Dawley rats exhibit a range of behaviors in an open field. Exploration without defecation or urination is interpreted as stable behavior. On the basis of their open field behavior we selected the five most “emotional” and five most “stable” rats from two separate groups of thirty rats. Norepinephrine (NE), dopamine (DA), and serotonin (5HT) levels were determined in brains from these ten “emotional” and ten “stable” rats. The NE levels of “emotional” rats were elevated about 60 ng/g relative to the “stable” rats. There was no difference in DA levels, but there appeared to be a trend toward elevation of 5HT levels in the “emotional” rats. These findings directly support the hypothesis that elevated central nervous system norepinephrine levels may reflect a factor which contributes to emotionality in the rat, and suggest that brain norepinephrine levels may be a biochemical mechanism which influences performance as seen with the commonly used open field behavioral test of emotionality.     

Protective effects of ascorbic acid on hepatotoxicity and oxidative stress caused by carbon tetrachloride in the liver of Wistar rats

This study was planned to investigate the protective effect of l (+)‐ascorbic acid (Vit C) on CCl₄‐induced hepatotoxicity and oxidative stress in the liver of Wistar rats (Rattus Norvegicus, strain Wistar). Twenty‐four adult male Wistar rats were fed with standard rat chow diet for 10 days and randomly were divided into four groups of six each as follows: (1) control, (2) CCl₄, (3) “CCl₄ + Vit C”, (4) Vit C groups. CCl₄ was applied to rats belonging to CCl₄ and “CCl₄ + Vit C” groups subcutaneously at 1 mg kg^?1 dose CCl₄ for 3 days. Vit C applied to “CCl₄ + Vit C” and “Vit C” group rats intraperitoneally at 300 mg kg^?1 dose for 3 days. All rats were sacrificed and livers were quickly removed on the fourth day of the experiment. MDA, total glutathione (T.GSH) levels and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH‐PX) activities were measured in the liver of all groups of rats and also serum alanine amino transferase (ALT) and aspartate amino transferase (AST) activities were detected to determine liver functions in all groups of rats. Histopathological changes were evaluated by light and transmission electron microscopes. In “CCl₄ + Vit C” group, MDA level was significantly decreased (p < 0.05) and SOD, CAT, GSH‐PX activities were significantly increased (p < 0.005, 0.01, 0.05) respectively, T.GSH level was significantly increased (p < 0.005) and serum ALT and AST activities were significantly decreased (p < 0.01, 0.05), respectively, when compared with CCl₄ group. These results show that Vit C has a highly protective effect on hepatotoxicity and oxidative stress caused by CCl₄. Copyright © 2009 John Wiley & Sons, Ltd.     

Audiogenic kindling changes the subunit composition of BK-channels in dentate gyrus of Krushinskii-Molodkina rats

Subunit composition of voltage- and Ca²⁺-sensitive high-conductance K⁺ channels (BK channels) in dentate gyrus (DG) of Krushinskii-Molodkina (KM) rats, genetically prone to audiogenic seizures, was compared with that of normal Wistar rats, resistant to sound effects. Additionally, long-lasting changes in protein expression of α- and β4-subunits in DG of KM rats after audiogenic kindling (model of temporal lobe epilepsy) was investigated. Western blot analysis revealed no differences between the levels of the pore-forming α-subunit expression in DG of KM and Wistar rats. In contrast, the level of brain-specific auxiliary β4-subunit in DG of KM rats was increased twofold in comparison to that in Wistar rats. It is likely that the observed changes in the BK channel α/β4 subunits ratio can prevent the development of excessive neuronal exitability in DG of KM rats. The results obtained on the model of audiogenic kindling (20 convulsion fits) confirmed this assumption. Thus, α-subunit expression levels in DG of KM rats on day 3 and 14 after the last seizure were increased 2.5 times in comparison with intact KM rats. The expression level of β4 in DG of KM rats 3 days after kindling was reduced to 30% of the control level. On day 14 after finishing audiogenic kindling, a further reduction of β4 protein expression level occurred. We suggest that the changes in the subunit composition of BK channels in DG following chronic seizures can alter functional properties of DG as a physiological filter, which normally prevents the propagation of epileptiform activity in the hippocampus.     

Annexin 2 Is Not Required for Human Immunodeficiency Virus Type 1 Particle Production but Plays a Cell Type-Dependent Role in Regulating Infectivity

During assembly and budding of retroviruses, host cell proteins are incorporated into viral particles. Identification of virion-associated proteins may help pinpoint key cellular components required for virus production and function. The cellular protein annexin 2 (Anx2) is incorporated into HIV-1 particles, and knockdown of Anx2 has been reported to cause defects in Gag processing and infectivity of HIV-1 particles in macrophages. Here, we tested whether Anx2 was required for HIV-1 production in other cell types capable of producing HIV-1 virions. Endogenous Anx2 levels were knocked down by ∼98% using lentivirus encoding short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting Anx2. Under these conditions, there was no reduction in HIV-1 virus-like particle (VLP) production in either COS-1, 293T, or Jurkat T cells or primary human monocyte-derived macrophages (MDMs). Murine embryonic fibroblasts derived from Anx2^−/− mice produced the same levels of VLPs as matched cells from wild-type mice. The calcium-mediated spike in VLP production still occurred in Anx2-depleted COS-1 cells, and there was no apparent alteration in the intracellular Gag localization. Overexpression of Anx2 in trans had no effect on Gag processing or VLP production. Neither Anx2 depletion nor Anx2 overexpression altered the infectivity of HIV-1 particles produced by COS-1 or 293T cells. However, supernatants containing virus from Anx2 siRNA-treated primary human MDMs exhibited decreased infectivity. These data indicate that Anx2 is not required for HIV-1 assembly or Gag processing but rather plays a cell type-dependent role in regulating production of infectious HIV-1 by macrophages.The Gag polyprotein generates the key structural proteins for all retroviruses. Gag is necessary and sufficient for the formation of virus-like particles (VLPs), which are morphologically similar to immature virions. Following its synthesis in the cytoplasm, HIV-1 Gag is trafficked to sites of particle production on membranes. Viral particle production depends on Gag-membrane interactions mediated by the myristoylated MA domain of Gag (18, 22, 31) and Gag-Gag interactions mediated by the CA and NC domains. Budding and release of the new virion are mediated by the Gag p6 domain. For successful particle production to occur, HIV-1 Gag must also interact with numerous host cell proteins and protein complexes. Identification of these interactions provides a crucial window into determining Gag trafficking intermediates as well as clues to the mechanism of virion production.The host cell protein annexin 2 (Anx2) has recently attracted attention for its potential to regulate key processes in both cells and viruses (9, 14, 17, 24). Anx2 belongs to a family of conserved calcium-regulated proteins and interacts with actin, membranes, and negatively charged phospholipids. The major protein binding partner for Anx2 is p11, also known as S100A10. Two populations of Anx2 have been identified: a heterotetrameric complex with two molecules of Anx2 and two molecules of p11 (found predominantly at the plasma membrane) and a monomeric form found mainly in the cytoplasm. Anx2 performs multiple functions in the cell, including regulation of actin-based dynamics, fibrinolysis, calcium-mediated exocytosis, and transport of intermediates from early to late endosomes (10, 14-16) Anx2 also enhances binding and fusion of cytomegalovirus with phospholipid membranes (21). In addition, Anx2 can be detected within influenza virus particles (28), where it has been shown to aid in virus replication (9).Several lines of evidence suggest that Anx2 may play a role in HIV-1 biogenesis. Both Anx2 and its binding partner p11 are incorporated in HIV-1 particles produced by macrophages (2). Anx2 interacts with Gag in macrophages, and annexin 2 knockdown has been reported to cause defective Gag processing and reduced infectivity of the released particles (24). Blockade of Anx2 function, with either anti-Anx2 antibody or small interfering RNA (siRNA)-mediated knockdown, results in suppression of HIV-1 infection in macrophages (11). Anx2 also binds to Gag in 293T cells, and expression of Anx2 in trans in these cells has been reported to lead to increased Gag processing and HIV-1 production (7). Taken together, these findings suggest that Anx2 might play a universal role in Gag trafficking and particle production. To test this hypothesis, we exploited methods to efficiently knock down Anx2 expression and determined the effect of Anx2 knockdown in a variety of cell lines capable of producing HIV-1 virions. Here we show that, in the absence of Anx2 expression, HIV-1 Gag is expressed, trafficked, and capable of mediating viral particle formation in a manner similar to that of control cells expressing Anx2. However, a cell type-dependent effect of Anx2 depletion on HIV-1 infectivity was detected in primary human monocyte-derived macrophages (MDMs). These findings suggest that Anx2 might be a macrophage-specific host cell factor that regulates HIV-1 infectivity.     

Plant annexins form calcium-independent oligomers in solution

The oligomeric state in solution of four plant annexins, namely Anx23(Ca38), Anx24(Ca32), Anx(Gh1), and Anx(Gh2), was characterized by sedimentation equilibrium analysis and gel filtration. All proteins were expressed and purified as amino-terminal His(n) fusions. Sequencing of the Anx(Gh1) construct revealed distinct differences with the published sequence. Sedimentation equilibrium analysis of Anx23(Ca38), Anx24(Ca32), and Anx(Gh1) suggests monomer-trimer equilibria for each protein with association constants in the range of 0.9 x 10(10)-1.7 x 10(11) M(-2). All four proteins were subjected to analytical gel filtration under different buffer conditions. Observations from this experiment series agree quantitatively with the ultracentrifugation results, and strongly suggest calcium independence of the annexin oligomerization behavior; moreover, binding of calcium ions to the proteins seems to require disassembly of the oligomers. Anx(Gh2) showed a different elution profile than the other plant annexins; while having only a very small trimer content, this annexin seems to exist in a monomer-dimer equilibrium in solution.     

The Anx7(+/-) knockout mutation alters electrical and secretory responses to Ca(2+)-mobilizing agents in pancreatic β-cells

Insulin secretion from the pancreatic β-cell is controlled by changes in membrane potential and intracellular Ca(2+). The contribution of intracellular Ca(2+) stores to this process is poorly understood. We have previously shown that β-cells of mice lacking one copy of the Annexin 7 gene (Anx7(+/-)) express reduced levels of IP(3) receptors and defects in IP(3)-dependent Ca(2+) signaling. To further elucidate the effect of the Anx7(+/-) mutation on signaling related to intracellular Ca(2+) stores in the β-cell, we measured the effects of Ca(2+) mobilizing agents on electrical activity, intracellular Ca(2+) and insulin secretion in control and mutant β-cells. We found that the muscarinic agonist carbachol and the ryanodine receptor agonists caffeine and 4-chloro-m-cresol had more potent depolarizing effects on Anx7(+/-) β-cells compared to controls. Accordingly, glucose-induced insulin secretion was augmented to a greater extent by caffeine in mutant islets. Surprisingly, ryanodine receptor-mediated Ca(2+) mobilization was not affected by the Anx7(+/-) mutation, suggesting that the mechanism underlying the observed differences in electrical and secretory responsiveness does not involve intracellular Ca(2+) stores. Our results provide evidence that both IP3 receptors and ryanodine receptors play important roles in regulating β-cell membrane potential and insulin secretion, and that the Anx7(+/-) mutation is associated with alterations in the signaling pathways related to these receptors.     

An intravascular chemoattractant lectin inhibits neutrophil migration

KM+, a lectin purified from Artocarpus integrifolia seeds, is an attractant for neutrophils, and has properties similar to fMLP, IL-8 and MNCF. The endogenous lectin MNCF, inhibits carrageenan-induced neutrophil migration when intravenously administered in rats. In an attempt to mimic the activity of MNCF with KM+, we determined the effect of intravenous (iv) injection of KM+ (5 g) on neutrophil migration to the peritoneal cavity of Wistar rats induced by KM+ (50 g, intraperitoneal, ip), fMLP (5 ng, ip) and carrageenan (300 g, ip). Initially we evaluated the effect of the time interval between intravenous and intraperitoneal administration of KM+. The intervals ranged from 20 to 120 min and progressively stronger inhibition was observed with increasing time intervals up to a maximum of 60 min, with effect decreasing thereafter. With injections at the optimum interval of 60 min, we observed that KM+ inhibited KM+- and carrageenan-induced neutrophil migration by 72%, and fMLP-induced migration by 56%. White cell counts for Wistar rats that only received KM+iv, performed at 0 to 120 min intervals after injection, revealed early neutropenia lasting 60 min, followed by a marked increase in circulating neutrophils that reached a maximum of twice the initial levels within 90 min and after 120 min returned to levels near to that observed before intravenous administration of KM+. These results indicate that when KM+ is present in the intravascular space, it produces an inhibitory effect on neutrophil migration similar to that caused by the intravenous administration of other chemoattractants, regardless of whether they act through a mechanism independent of carbohydrate recognition, as does IL-8, or are dependent on carbohydrate recognition, like MNCF.     

4周电针干预对大鼠白色脂肪“棕色化”的影响及其机制*

目的: 以有氧运动作为参照,观察4周电针干预对大鼠白色脂肪“棕色化”的影响,探究其可能的分子机制。方法: 将24只8周龄雄性SD大鼠随机分为安静对照组(Sed组)、有氧运动组(Exe组)、电针组(ElA组),每组8只,每组分别干预4周。Exe组采用65%最大摄氧量强度跑台运动,1 h/d,每周6 d。ElA组采用电针刺激“足三里”和“天枢”穴,20 min/d,每周6 d,每周记录大鼠体重。4周干预结束后,心尖和腹主动脉取血处死,分离双侧腓肠肌、肾周和附睾周围白色脂肪组织并称湿重,检测大鼠体脂含量和血清Irisin的水平,检测大鼠腓肠肌中AMPKα、p-AMPKα、PGC-1α、FNDC5和脂肪组织中UCP1蛋白的表达,检测大鼠棕色脂肪组织脂滴的形态。结果: ①与Sed组相比,从第2周开始,Exe、ElA组大鼠体重的增长明显降低,4周后Exe、ElA组大鼠体重和体脂含量明显降低(P＜0.01),而Exe、ElA组之间未见显著差异(P＞0.05)。②4周干预后,ElA组和Exe组大鼠白色脂肪湿重均明显降低(P＜0.05或P＜0.01)。③Exe和ElA组大鼠的血清Irisin水平明显升高(P＜0.05),Exe、ElA组腓肠肌p-AMPKα/AMPKα、PGC-1α、FNDC5蛋白表达和白色脂肪、棕色脂肪组织中UCP1蛋白表达均明显升高(P＜0.05)。④Exe、ElA组大鼠白色和棕色脂肪组织中脂滴面积明显降低(P＜0.01)。结论: 4周的电针干预,可有效控制大鼠体重,诱导白色脂肪“棕色化”,具有与4周有氧运动近似的减脂效果,其可能是通过AMPKα-PGC-1α-FNDC5-Irisin信号通路,释放Irisin,进而与脂肪组织“crosstalk”,上调UCP1的表达,诱导白色脂肪组织“棕色化”。     

KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection.

The outcome and severity of some diseases correlate with the dominance of either the T helper 1 (Th1) or Th2 immune response, which is stimulated by IL-12 or IL-4, respectively. In the present study we demonstrate that gamma interferon (IFN-gamma) secretion by murine spleen cells stimulated with KM(+), a mannose-binding lectin from Artocarpus integrifolia, is due to IL-12 induction, because (1) macrophages from several sources (including cell lines) produced IL-12 p40 in response to KM(+), and (2) lectin-free supernatants from J774 cell line cultures stimulated with KM(+) induced the secretion of IFN-gamma by spleen cell cultures, an effect blocked by the supernatant pretreatment with anti-IL-12 antibody. The known pattern of susceptibility of BALB/c mice to infection with Leishmania major, attributed to high levels of IL-4 production leading to a Th2 nonprotective immune response, was modified by administration of KM(+). Draining lymph node cells from these immunized BALB/c mice (in contrast to cells from animals immunized only with soluble leishmanial antigen [SLA]) secreted high levels of IFN-gamma and low levels of IL-4, which characterized a Th1 rather than a Th2 response pattern. The footpad thickness of BALB/c mice immunized with SLA plus KM(+) and challenged with L. major was similar to that of uninfected mice. This beneficial effect against leishmanial infection was blocked by pretreatment of these mice with anti-IL-12 antibody. These observations indicate that KM(+) induces IL-12 p40 in vivo and has a protective effect against L. major infection.     

Sibutramine alters the central mechanisms regulating the defended body weight in diet-induced obese rats

Chronic administration of sibutramine lowers body weight, presumably by altering brain monoamine metabolism. Here the effect of sibutramine on sympathoadrenal function (24-h urine norepinephrine and epinephrine levels) and arcuate nucleus (ARC) neuropeptide Y (NPY) and proopiomelanocortin (POMC) expression was assessed in diet-induced obese rats fed a low-fat diet. Chronic (10 wk) sibutramine [5 mg. kg(-1). day(-1) ip; rats fed ad libitum and injected with sibutramine (AS)] lowered body weight by 15% but only transiently (3-4 wk) reduced intake compared with vehicle-treated controls [rats fed chow ad libitum and injected with vehicle daily (AV)]. Other rats food restricted (RS) to 90% of the weight of AS rats and then given sibutramine restored their body weights to the level of AS rats when allowed libitum food intake. After reequilibration, RS rats were again energy restricted to reduce their weight to 90% of AS rats, and additional vehicle-treated rats (RV) were restricted to keep their body weights at the level of AS rats for 3 wk more. Terminally, total adipose depot weights and leptin levels paralleled body weights (AV > AS = RV > RS), although AS rats had heavier abdominal and lighter peripheral depots than RV rats of comparable body weights. Sibutramine treatment increased sympathetic activity, attenuated the increased ARC NPY, and decreased POMC mRNA levels induced by energy restriction in RV rats. Thus sibutramine lowered the defended body weight in association with compensatory changes in those central pathways involved in energy homeostasis.     

Effects of Neonatal Treatments on Pain and Audiogenic Sensitivity and Brain Content of Monoamines in Adult Rats

In adult Wistar, KM, and Wag/Rij rats, the threshold of pain sensitivity (tail-flick test) and audiogenic sensitivity were estimated after neonatal administration of Semax (analog of ACTG_4–10 fragment) or after placebo (administration of saline for the control of the effect of neonatal pain stimulation). These neonatal treatments did no affect the rates of sensomotor development at an early age (Fox tests), i.e., did not affect directly the physiological activity of rat pups at the age of up to 21 days. In all control rats injected with saline (pain stimulation), the latencies of audiogenic fits increased reliably, while their degree decreased. Administration of Semax raised these parameters to the level of those in intact animals, i.e., increased the sensitivity to sound. Neonatal administration (per os) of caffeine to KM rats increased reliably the latency of audiogenic fits. The thresholds of pain sensitivity in the rats of all strains were significantly lower saline injected animals than in the intact control, just as the level of dopamine in the hippocampus of KM rats. These data are interpreted as an evidence of changes in the development of some brain systems in response to neonatal treatments.     

Effects of the antiestrogens,MER-25 and CI 628, on rat and hamster lordosis

Antiestrogens were used to test the hypothesis that estrogen exerts a “maintenance,” as well as a “priming,” effect on rat and hamster sexual receptivity as it apparently does for guinea pigs. MER-25 (75 or 150 mg/kg) significantly reduced rat LQ when given ?2 hr or 8 hr after EB injection. MER-25 given at 34 hr (2 hr prior to P) failed to diminish rat LQ. With hamsters, MER-25 in large doses (750 mg/kg) given either at ?2 hr or 34 hr reduced lordosis duration to 40% of controls, but this effect was confounded by severe illness among the MER-25 injected animals. Lower doses failed to block behavior, but still produced some toxicity. CI 628 (50 mg/kg) greatly reduced hamster lordosis duration and increased lordosis latency when given 0 hr, but not 34 hr, after EB. The results are consistent with similar previous work on rats and do not support the concept of estrogen “maintenance” in either rats or hamsters.     

Amphiregulin and hepatocyte-derived extracellular matrix regulate proliferation and autocrine growth factor expression in colon cancer cell lines of varying liver-colonizing capability

We studied the effect of two members of the epidermal growth factor (EGF) family--amphiregulin and heparin-binding EGF-like growth factor (HB-EGF)-on cell proliferation, growth factor and growth factor receptor expression, and cell differentiation in two human colon cell lines of varying liver-colonizing potential. The effect of amphiregulin and HB-EGF was assessed both in cells grown on plastic, as well as on cells grown on hepatocyte-derived extracellular matrix (ECM). We found that both colon cell lines were sensitive to HB-EGF stimulation of cell proliferation. Amphiregulin inhibited cell proliferation in KM12 cells and stimulated the strongly metastatic cell line KM12SM to a slight extent. When the cells were cultured on hepatocyte-derived ECM, amphiregulin inhibited the weakly metastatic KM12 and stimulated the growth of KM12SM. HB-EGF synergistically acted with hepatocyte-derived ECM to enhance cell proliferation in both colon cell lines. Expression of ligands of the EGF family, such as transforming growth factor-alpha (TGF-alpha) and amphiregulin, was decreased in both cell lines when cultured on ECM. Hepatocyte-derived ECM decreased expression of cripto in KM12 and increased it in KM12SM cells. Neither cripto nor TGF-alpha mRNA levels was affected by growing the cells in the presence of amphiregulin. However, amphiregulin increased expression of its own mRNA in the weakly metastatic KM12 and decreased it in the strongly metastatic KM12SM when the cells were cultured on plastic. Amphiregulin and HB-EGF stimulated expression of erb-B2 in both cell lines cultured on plastic. Surprisingly, when the cells were grown on hepatocyte-derived ECM, amphiregulin inhibited erb-B2 expression in both cell lines. We observed no effect of amphiregulin on cell differentiation as assessed by alkaline phosphatase expression. Our studies demonstrate one mechanism that could play a role in site-specific metastasis. We found an inhibitory response to an autocrine growth factor in the context of hepatocyte-derived ECM in a weakly metastatic cell and a stimulatory effect of the same growth factor when strongly metastatic cells were cultured on the same ECM.     

Perturbations of the stress-induced GLUT4 localization pathway in slow-twitch muscles of obese Zucker rats

Past studies have suggested that the stress-induced GLUT4 localization pathway is damaged in fast-twitch muscles (white muscles) of obese subjects. In this study, we used obese rodents in an attempt to determine whether the stress-induced GLUT4 localization pathway is abnormal in slow-twitch muscles (red muscles), which are responsible for most daily activities. Protein expression levels of the intracellular stress sensor AMP-activated protein kinase (AMPK), its upstream kinase LKB1, its downstream protein AS160 and the glucose transporter protein 4 (GLUT4) in the red gastrocnemius muscle were measured under either resting or stress conditions (1 h of swimming or 14% hypoxia) in both lean and obese Zucker rats (n = 7 for each group). At rest, obese rats displayed higher fasting plasma insulin levels and increased muscle AMPK and AS160 phosphorylation levels compared with lean controls. No significant difference was found in the protein levels of LKB1, total GLUT4, or membrane GLUT4 between the obese and lean control groups. After one hour of swimming, AMPK and AS160 phosphorylation levels and the amount of GLUT4 translocated to the plasma membrane were significantly elevated in lean rats but remained unchanged in obese rats relative to their resting conditions. One hour 14% hypoxia did not cause significant changes in the LKB1-AMPK-AS160-GLUT4 pathway in either lean or obese rats. This study demonstrated that the AMPK-AS160-GLUT4 pathway was altered at basal levels and after exercise stimulation in the slow-twitch muscle of obese Zucker rats.