尾吊后ERK在大鼠股动脉的相性和紧张性收缩反应中的作用

目的 研究细胞外信号调节激酶（ERK）在尾吊模拟失重后Wistar大鼠股动脉的相性和紧张性收缩反应中的作用.方法 利用离体血管环灌流技术,在无抑制剂存在或给予丝裂原活化蛋白激酶激酶（MEK）抑制剂PD98059后,以Powerlab记录仪检测并记录大鼠股动脉对去甲肾上腺素（NE）的收缩反应,比较两组间收缩反应的曲线下面积（AUC）、相性和紧张性收缩反应.结果 尾吊14 d后,与对照比较,NE诱导的收缩反应的曲线下面积、相性和紧张性收缩反应均下降.PD98059 没有显著影响尾吊后大鼠股动脉收缩反应的AUC,但却显著降低了对照组大鼠股动脉收缩反应的AUC.与对照组比较,PD98059对尾吊后大鼠股动脉的相性和紧张性收缩反应的抑制率明显下降.此外,在对照组,PD98059在血管相性和紧张性收缩反应中的抑制率间差异并无统计学意义（P＞0.05）,但在悬吊组其差异则有统计学意义（P＜0.01）.结论 尾吊模拟失重后,ERK在大鼠股动脉相性和紧张性收缩反应中的作用均下降.尾吊诱发的模拟失重效应可能通过抑制ERK在大鼠股动脉粗、细肌丝调节中的作用从而损伤其收缩反应.     

ERK在NGF诱导PC12 细胞分化中的作用

目的探讨细胞外信号调节激酶(ERK)在NGF。诱导的PC12细胞分化中的作用机制。方法以NGF处理PC12细胞建立分化模型,运用免疫印迹检测不同浓度不同作用时间时NGF对ERK1／2蛋白和磷酸化ERK1／2蛋白水平的影响,并观察MAPK／ERK激酶(MEK)抑制剂U0126对NGF诱导的细胞形态学改变的影响。结果ERK1／2蛋白的磷酸化呈现NGF剂量和时间依赖性。NGF作用细胞5min即可观察到明显的ERK1／2蛋白磷酸化,持续1h左右,2h时降低到初始水平,而细胞形态的改变出现在NCF作用12h以后。倒置相差显微镜观察可见PC12细胞分化的程度与ERK1／2活化持续的时间正相关,U0126可完全即时抑制ERK1／2的活化,而ERK1／2活化的抑制可完全阻断。NGF诱导的PC12细胞分化。结论ERK1／2的活化是PC12细胞发生分化的必需事件,其活化时间的长短对分化具有决定作用。     

PKC和ERK在大鼠心肌缺氧预处理延迟保护机制中的作用

目的:探讨蛋白激酶C(PKC)和细胞外信号调节激酶(ERK)在大鼠缺氧预处理(APC)延迟保护机制中的作用及二者之间的相互关系。方法:建立培养乳鼠心肌细胞缺氧/复氧(A/R)损伤和APC延迟心肌保护模型,用PKC兴奋剂(PMA)模拟预处理延迟保护模型,分别应用PKC和ERK抑制剂干预模型,并在各组相当于预处理后10min取材检测ERK活性。以实验终点检测培养基中乳酸脱氢酶(LDH)活性、细胞存活率、心肌细胞超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量作为心肌细胞损伤的指标。结果:预处理组心肌细胞存活率和SOD含量均显著高于A/R组(P＜0.05),培养液内LDH漏出和心肌细胞内MDA含量显著低于A/R组(P＜0.05),ERK活性显著高于A/R组(P＜0.05),应用PMA激活PKC可以模拟预处理的保护作用;ERK阻滞剂PD98059消除了缺氧和PMA的预处理保护作用,并抑制了预处理后的ERK活性的升高;PKC抑制剂多粘菌素B对APC引起的ERK激活及延迟保护作用无明显影响,但可抑制PMA诱导的保护现象。结论:ERK活化可能是APC延迟保护机制中的必须信号转导途径;PKC活化可以通过激活ERK启动缺氧预处理的延迟保护机制。     

ERK信号转导途径在哮喘大鼠气道重塑中作用的初步探讨

目的：研究支气管哮喘不同时期细胞外信号调节激酶（External signal regulated kinase,ERK）的磷酸化与c-Fos表达,以探讨ERK信号转导途径在支气管哮喘气道重塑中的作用。方法：复制大鼠哮喘模型,随机分为对照组（包括4周、8周及12周对照组）、哮喘组（包括4周、8周及12周哮喘组）,图像分析软件测定支气管壁厚度（Wat）和平滑肌厚度（Wam）,免疫组化测定肺组织磷酸化的ERK（Phospho-ERK,P-ERK）与c-Fos表达,免疫印迹法测定磷酸化的ERK水平,直线相关分析法显示Wat和Wam与P-ERK的相关性。结果：各哮喘组Wat和Wam,P-ERK和c-Fos的平均吸光度均显著高于相应对照组（P均〈0.01）;各哮喘组磷酸化的ERK水平均显著高于相应对照组（其中A12也与C8组比）（P〈0.01）;Wat、Wam与P-ERK平均吸光度均呈显著正相关性（P〈0.01）。结论：ERK磷酸化水平和c-Fos在哮喘大鼠均增加,ERK信号转导途径在支气管哮喘气道重塑中起重要作用。     

辛伐他汀对大鼠血管平滑肌细胞增殖及bFGF与ERK1/2表达的干预作用

目的:观察辛伐他汀对大鼠血管平滑肌细胞(VSMC)碱性成纤维细胞生长因子(bFGF)与细胞外信号调节激酶1/2(ERK1/2)蛋白表达的影响.方法:体外培养大鼠胸主动脉VSMC,MTT法测定细胞增殖活力;Western blot法检测bFGF与ERK1/2蛋白的表达.结果:与对照组比较,辛伐他汀各浓度组细胞增殖活力均显著降低(P＜0.05).与对照组比较,低、中剂量组bFGF、ERK1/2蛋白表达均显著降低(P＜0.05),高剂量组bFGF、ERK1/2蛋白表达进一步降低(P＜0.01);与低剂量组比较,中剂量组bFGF、ERK1/2蛋白的表达均无统计学意义(P＞0.05),高剂量组则显著降低(P＜0.05);高剂量组bFGF、ERK1/2蛋白的表达又较中剂量组显著降低(P＜0.05),并呈剂量依赖性.结论:辛伐他汀能抑制平滑肌细胞增殖,该作用是通过抑制bFGF及其相应信号通路ERK1/2蛋白的表达来实现的,这可能是辛伐他汀治疗动脉粥样硬化、再狭窄及高血压等心血管疾病的重要机制之一.     

ERK在LPS诱导大鼠雪旺氏细胞TNF-α表达中的作用

目的 研究细胞外信号调节激酶（ERK）对脂多糖（LPS）诱导的大鼠雪旺氏细胞肿瘤坏死因子-α（TNF-α）表达的作用。方法 采用Westernblot检测细胞中ERK激酶的活性；用ERK的特异性抑制剂（PD98059）预处理细胞后，ELISA检测细胞中TNF-α水平；RT-PCR检测细胞中TNF-α mRNA的表达；用免疫荧光双标法检测ERK的表达定位。结果 LPS可显著激活雪旺氏细胞中的ERK信号通路，其激活高峰在LPS刺激后30～45min，60min后ERK磷酸化水平开始逐渐下降；用PD98059预处理细胞后，可显著抑制细胞TNF-α以及TNF-α mRNA的产生；LPS刺激细胞后引起ERK磷酸化，PD98059可抑制该过程。结论 ERK信号通路的激活可能是LPS诱导TNF-α表达的前提。通过阻断细胞内信号转导通路来减少TNF-α及其他细胞因子的产生，为抑制周围神经损伤后的炎症以及免疫反应发生提供了一条新的思路。     

Raf激酶抑制蛋白和p-ERK在糖尿病肾病大鼠肾组织中的表达及意义

 目的:探讨Raf激酶抑制蛋白(Raf kinase inhibitor protein,RKIP)和磷酸化细胞外信号调节激酶(phosphorylated extracellular signal-regulated kinase, p-ERK)在糖尿病肾病大鼠肾组织中的表达及意义。方法:40只Sprague-Dawley （SD）大鼠随机分为2组:正常对照组（N组）和糖尿病肾病组（M组）。苏木精-伊红（hematoxylin-eosin, HE）和过碘酸-雪夫（periodic acid-Schiff,PAS）染色观察肾组织病理改变,免疫组化方法检测各组肾组织RKIP和p-ERK1/2表达,Western blotting 检测各组RKIP和p-ERK1/2的表达。结果:M组大鼠随着病程的延长,血糖升高,出现蛋白尿,肾重增加,肾组织内p-ERK1/2明显增高,RKIP降低,与N组相比有显著差异(P<0.05)。结论: RKIP表达的减少及p-ERK的表达增加可能促进糖尿病肾病的进展。     

睾酮诱导大鼠心肌细胞肥大反应并上调ERK1/2蛋白表达

目的探讨细胞外信号调节激酶(ERK1/2蛋白)在睾酮对大鼠心肌肥大中的作用。方法用差速贴壁法分离及纯化培养新生SD大鼠心肌细胞,以Bradford法测定心肌细胞蛋白质含量,同位素法分析3H-亮氨酸(3H-Leu)掺入,IBAS图像分析心肌细胞表面积,以免疫印迹法检测心肌细胞ERK1/2蛋白表达水平。结果生理浓度的T作用于大鼠心肌细胞24 h后,细胞蛋白质含量3、H-Leu掺入和细胞表面积均增加,其中以10-8mol/L作用最强。雄激素受体拮抗剂氟他胺(Flu)10-5mol/L预处理2 h可抑制T诱导的心肌细胞蛋白质含量的增加,而Flu单独作用对心肌细胞蛋白质含量无影响。ERK1/2信号通路的特异性抑制剂PD98059 50μmol/L预处理2 h,可抑制T诱导的心肌细胞3H-Leu掺入的增加;10-8mol/L T作用24 h使心肌细胞的ERK1/2的蛋白表达显著增加;10-5mol/L Flu预处理2 h可逆转T诱导的心肌细胞ERK1/2蛋白表达的增加。结论生理浓度的T可以诱导心肌细胞肥大反应,该作用可能由ERK1/2信号通路介导。T通过雄激素受体(AR)上调ERK1/2蛋白表达。     

不同标本制备法对小鼠小肠组织中ERK1/2和pERK1/2免疫组织化学定位的影响

目的:本研究针对丝裂原活化蛋白激酶(MAPK)家族成员细胞外信号调节激酶(ERK1/2),以及磷酸化的ERK1/2(pERK1/2),探讨不给任何刺激时,用不同标本制备法检测ERK1/2在小鼠小肠组织中的免疫组织化学定位。方法:分别应用灌流固定脱水法(PF-DH),浸泡固定脱水法(IM-DH),快速冻结-冻结置换法(QF-FS)制备的标本,通过免疫组织化学显色检测ERK1/2和pERK1/2在肠上皮中的定位。结果:应用PF-DH法,ERK1/2位于几乎所有上皮细胞的核中,而pERK1/2在上皮细胞中很难检测到。应用IM-DH法,ERK1/2位于所有上皮细胞的胞质和核内,pERK1/2仅位于小肠绒毛顶部的上皮细胞内,而不存在于肠隐窝区域的上皮细胞中。应用QF-FS法制备的标本中,ERK1/2定位于所有上皮细胞的胞质中,而pERK1/2主要存在于肠隐窝部位和肠绒毛顶部的上皮细胞中。结论:本研究结果提示,用不同的标本制备方法会影响ERK1/2的免疫组织化学定位。通过比较不同标本制备方法取材的小鼠小肠组织中的ERK1/2和pERK1/2的免疫组织化学定位,得出QF-FS法可能为最接近活体状态的小鼠小肠组织中的ERK1/2和pERK1/2定位的方法。     

急性冷热应激对大鼠脊髓和脊神经节ERK信号转导的影响

ERK是细胞内MAPK信号转导系统的重要蛋白激酶,参与多种刺激的应激反应。本实验建立冷热应激模型,应用免疫组织化学和Western blot方法,研究冷热应激刺激对大鼠脊髓和脊神经节ERK的激活作用。结果显示:(1)正常脊髓非磷酸化ERK1(pan-ERK1)阳性细胞分布于RexedIII-X层,而I-II层没有免疫反应,胞浆着色,没有核膜断裂;磷酸化ERK1/2(p-ERK1/2)阳性细胞见于灰质Rexed各层(I-X),散在分布。免疫产物位于胞浆核周区,少见于细胞核,核膜有断裂。脊神经节p-ERK1/2免疫反应见于大、中、小型神经元,定位于细胞核;没有pan-ERK1免疫反应。(2)20min热应激后,RexedII层p-ERK1/2阳性细胞数明显增多,核周着色区增大,核膜常有断裂,脊神经节阳性细胞数没有明显变化;10min热应激和体温(38℃)应激后,脊髓和脊神经节阳性细胞数和核着色没有显著变化。(3)20min冷应激后,脊髓II层p-ERK1/2阳性细胞数轻度减少,核周着色区变窄,核膜有断裂,脊神经节阳性细胞数没有变化;10min冷应激后阳性细胞数没有明显变化。(4)Western blot显示,热应激和冷应激分别提高和降低了胸段脊髓p-ERK1/2的蛋白表达水平。本研究证实,ERK存在于脊髓灰质各层和脊神经节各型神经元,热应激提高了ERK激活水平而冷应激则相反,提示MAPK信号转导激活具有应激类型和应激强度的依赖性,参与了感觉神经元的功能调节。     

Fictive motor activity in rat after 14 days of hindlimb unloading

The goal of the present study was to examine the effects of chronic hindlimb unloading on fictive motor patterns which can be developed in hindlimb nerves of adult rats. The animals were divided into two groups. The first group was submitted to hindlimb unloading for 2 weeks by tail suspension. The second group served as controls. After this initial phase, the animals of both groups were acutely decorticated, paralysed and electroneurographic efferent activity was recorded from hindlimb muscle nerves under conditions of "fictive locomotion" in order to evaluate variations in central locomotor command. Fictive rhythmic motor episodes were either spontaneous or evoked by electrical stimulation of the mesencephalic locomotor region. Only the second ones were recognised as locomotor-like activities. The motor pattern was not fundamentally affected by unloading except that, after the unloading period, extensor muscle nerves were significantly more frequently activated and their burst durations were increased compared to activity in control animals, despite the fact that the phasic sensory afferent inputs were suppressed. This suggests that unloading induces plastic modifications of the central networks of neurons implicated in the locomotor command. The origin of this extensor hyperactivity is discussed. It is proposed that it could be the consequence of either changes in motoneuronal properties or of an increase in afferent input to motoneurones. Electronic Publication     

Upregulation of HSP72 in reloading rat soleus muscle after prolonged hindlimb unloading

To clarify the changes of heat shock protein (HSP) 72 in the rat soleus muscle after hind-limb unloading (HU) and during reloading, 7-week-old male Wistar rats were hind-limb-suspended for 9 weeks, thereafter ambulatory recovery was permitted for 8 weeks. The body and absolute soleus weights were significantly lower in the HU than in the age-matched control group after HU and during reloading. The soleus weight relative to body weight was also significantly lower in the HU than in the age-matched control group at the end of 9 weeks of suspension, but returned to the control level after 2 weeks of reloading. The HSP72 content decreased to 38% of the control level after HU and conversely increased to 165 and 175% of the control level after 2 and 4 weeks of reloading, respectively. The HSP72 content returned to the control level after 8 weeks of reloading. Thus our results showed that the expression of HSP72 was downregulated by HU and upregulated temporally over the level of the control during the reloading period, and they suggested that these down- and up-regulations of HSP72 may be related to many factors including mechanical stress or load applied to the muscle.     

Functional recovery of the plantarflexor muscle group after hindlimb unloading in the rat

Research into skeletal muscles response to hindlimb unloading (HU) of the rodent has focused on that of the markedly affected slow-twitch anti-gravity muscles (e.g., soleus). However, the ability of the animal to locomote following HU should be best determined by the in vivo functional properties of the muscle groups involved and, to our knowledge, this has not been investigated. Our objective was to determine how the in vivo functional properties of the rat ankle plantarflexor group change after 28 days of HU and during a subsequent 28-day recovery. Rats (n=48) were unloaded for 28 days after which they were either tested immediately or allowed to recover for 7, 14, or 28 days before being tested. Control rats (n=61) were tested at comparable times. In vivo functional properties of the ankle plantarflexors were assessed under anesthesia using an isokinetic dynamometer and included determination of the isometric torque-frequency relationship, the concentric torque-ankle angular velocity relationship, and fatigability. Immediately after HU, plantarflexor muscle weight was reduced by 24% but isometric torque production was reduced by 7–9% only at 100 Hz and concentric torque production was not significantly affected. However, after 7 days of recovery, in vivo function was more adversely affected; isometric and concentric torques were reduced by 12–33% and 16–36%, respectively, relative to control levels. In vivo plantarflexor function was recovered by 14 days. In conclusion, 28 days of HU has minor adverse effects on the in vivo function of the rat ankle plantarflexors. During the first week of recovery from HU, injury apparently occurs to the plantarflexors resulting in a transient impairment of functional capacity.     

Cellular adaptations in soleus muscle during recovery after hindlimb unloading

Aim: We used a model of chronic unloading followed by reloading to examine the apoptotic responses associated with soleus muscle atrophy and subsequent recovery. Methods: Male Wistar rats were subjected to hindlimb unloading (HU) for 2 weeks and subsequent reloading for 0, 3, 7 and 14 days. One‐half of the HU‐reloaded rats were administered cyclosporine A (CsA), a calcineurin (CaN) inhibitor. Results: There was fibre atrophy (73%) and a decrease in slow type I fibre/myosin heavy chain (MyHC) composition in the soleus muscle after 2 weeks of HU. Fibre size and type I MyHC composition recovered to near the age‐matched control levels by recovery day 14 in non‐treated, but not in CsA‐treated, rats. Myonuclear number was lower and the number of apoptotic nuclei higher in 2‐week HU than control rats. These values returned to control levels after 7 and 14 days of recovery, respectively, in both HU‐recovery groups. After 2 weeks of HU, the levels of heat shock proteins (Hsp) 60 and 72, mitochondrial cytochrome c oxidase subunit IV (Cox IV), and peroxisome proliferator‐activated receptor gamma coactivator 1 (PGC‐1) proteins were lower than control. The levels of all of these proteins gradually increased to or above the control levels during cage recovery in both groups. Conclusion: Our results indicate that apoptotic mechanisms are involved in the modulation of myonuclear number during chronic unloading and subsequent reloading. Furthermore, it appears that CaN is related to fibre size and phenotype adaptations, but not to apoptotic responses.     

Adaptation of rat lateral gastrocnemius muscle motor units during hindlimb unloading

Contractile and fatigue-resistance properties of 71 lateral gastrocnemius muscle (LG) motor units (MU) following 14 days of hindlimb unloading (HU) were compared to those of 60?LG MU from control rats. The MU properties were assessed from isolated and stimulated individual motor axons. The MU were classified using standard criteria (shape of unfused tetani and fatigue resistance). The HU did not affect LG MU composition, but diminished the maximal tetanic tension (P ₀) of all MU types: P ₀ was significantly reduced by about 40% for the slow and fast-resistant MU, and by 18% for the fast-fatigable ones. The speed-related properties of fast-resistant MU became more similar to those of slower MU. The fatigue properties of MU were evaluated during a 5-min exercise test, using two fatigue indexes, FI₂ and FI₅, which expressed the relative capacity of MU to generate tension after 2 and 5?min, respectively. Results showed that 14 days of HU did not change the fatigue sensitivity of the LG MU. However, when FI₅ was compared to FI₂, a greater decrease was observed after HU than in control conditions for the fast-resistant and fast-intermediate MU. It was concluded that a prolonged fatigue test may show changes in metabolic properties of muscle fibres during 14 days of HU. Specific adaptations of LG MU as well as comparisons with those of the soleus muscle under the same conditions are discussed.     

Analysis by two-dimensional Blue Native/SDS-PAGE of membrane protein alterations in rat soleus muscle after hindlimb unloading

Muscle atrophy occurring in several pathophysiological conditions determines decreases in muscle protein synthesis, increases in the rate of proteolysis and changes in muscle fiber composition. To determine the effect of muscle atrophy induced by hindlimb unloading (HU) on membrane proteins from rat soleus, a proteomic approach based on two-dimensional Blue Native/SDS-PAGE was performed. Proteomic analysis of normal and HU soleus muscle demonstrates statistically significant changes in the relative level of 36 proteins. Among the proteins identified by mass spectrometry, most are involved in pathways associated with muscle fuel utilization, indicating a shift in metabolism from oxidative to glycolytic. Moreover, immunoblotting analysis revealed an increase in aquaporin-4 (AQP4) water channel and an alteration of proteins belonging to the dystrophin–glycoprotein complex (DGC). AQP4 and DGC are regulated in soleus muscle subjected to simulated microgravity in response to compensatory mechanisms induced by muscle atrophy, and they parallel the slow-to-fast twitch conversion that occurs in soleus fibers during HU. In conclusion, the alterations of soleus muscle membrane proteome may play a pivotal role in the mechanisms involved in disuse-induced muscle atrophy.     

Effect of hindlimb unloading on interlimb coordination during treadmill locomotion in the rat

Effects of hindlimb unloading on interlimb coordination were examined in adult rats walking on a treadmill at moderate speed. In the first group of animals, the electromyographic activity (EMG) of soleus muscle of both hindlimbs was recorded after 7 and 14 days of unloading. In the second group, the EMG was recorded daily until the 14th day of unloading. The general organization of locomotion was preserved in the two groups whatever the duration of the unloading. The step cycles of the two hindlimbs were always strictly alternating. However, the locomotor pattern was very irregular. A lateral instability was observed. It was accompanied by an abduction of the hindlimbs, and frequent hyperextensions of the ankle when walking. The EMG analysis showed an increase in step cycle duration and in coactivation duration of the soleus muscles (i.e. in the double stance duration). In the rats recorded daily, mean EMG was dramatically reduced the 1st day of unloading, suggesting a decrease in the neural drive. Taken together, these data indicate that 14 days of hindlimb unloading can alter the neuromuscular pattern during locomotion. It is proposed that these changes are related to changes in the peripheral sensory information.     

Effect of hindlimb unloading on locomotor strategy during treadmill locomotion in the rat

Electromyographic activity (EMG) was recorded from the soleus muscles of adult rats during treadmill locomotion after 7 and 14 days of hindlimb unloading, and after 7 days of recovery. Observation of the rats indicated that treadmill locomotion was disrupted after unloading since the animals had some difficulty in moving. Soleus muscle EMG analysis was performed. Onset and offset of bursts of activity were determined, and the relationships between step duration and cycle duration were analysed. Our main results were as follows: firstly, mean cycle duration was increased after 14 days of hindlimb unloading when walking at low speed. At high speed, no difference was observed. Secondly, linear regression analysis indicated that the relationships between step duration and cycle duration were altered after 7 days of unloading. Thirdly, adaptation occurred, since the normal slope and correlation coefficient were restored after 14 days of unloading. Fourthly, when speed increased, no variation of mean EMG was demonstrated after hindlimb unloading whereas an increase occurred in normal rats. Fifthly, video analysis showed that the rats presented frequent hyperextension of the hindlimb after unloading. These abnormal steps were more numerous when walking at low speed. These data would indicate that a transitory disruption of the soleus muscle motor pattern occurred after 7 days of unloading. This disruption depended on the treadmill belt speed. Possible origins of these modifications are discusssed.     

Contractile properties of rat soleus motor units following 14 days of hindlimb unloading

The purpose of this study was to compare the isometric contractile properties of rat soleus motor units after 14 days of hindlimb unloading (HU) to those under control conditions. The motor units (MU) were classified using two mechanical criteria: the presence or not of a sag during unfused tetani and the value of the twitch time-to-peak (TTP). Under control conditions, the soleus muscle was composed of 85% of slow-type (sag −, TTP > 20 ms) and 15% of fast-type (sag +, TTP < 20 ms) units. Following HU, these two populations were still present and results showed: (1) large decreases in their maximal tetanic tensions (of −67% and −60% for slow- and fast-type, respectively), and (2) changes in their relative proportions, i.e. a decrease in the percentage of slow-type units and a twofold increase in the percentage of fast-type units were observed. These latter changes might be the consequence of a complete transformation of slow-towards fast-type units. A third population appeared in the HU solei, 26% of the samples, combining the presence of a sag and speed-related properties between those of slow- and fast-type units. These slow-intermediate units might come from slow units partially transformed into a faster type during HU. Thus the present study showed that unloading conditions induced a reorganisation of the soleus motor unit profile. The complete or partial transformation of the motor units could be related to the changes in the electromyographical activity of the unloaded soleus. Received: 30 June 1995/Received after revision: 12 January 1996/Accepted: 22 January 1996