起始密码下游序列与16srRNA3′端序列的匹配性对基因表达效率的影响

目的 :研究起始密码下游序列与 16srRNA 3′端 +146 9——— +1482局部序列间的匹配关系对基因表达效率的影响。方法 :我们一方面对人IL_2、人IL_8、人IL_6 ,人GM_CSF、人G_CSF、人IL_1ra、人IL_9等全长cDNA序列及 5′末端局部序列与 16srRNA 3′端 +146 9——— +1482序列进行匹配性进行分析 ,寻找序列匹配性与表达效率间的关系 ;另一方面 ,对人IL_6、人GM_CSFcDNA 5′端序列进行沉默突变 ,增强其与 16srRNA 3′端 +146 9——— +1482序列间的匹配性 ,并克隆入pKpL4表达型质粒载体 ,通过大肠杆菌进行表达 ,实验验证序列匹配性与基因表达效率的关系。结果 :分析发现起始密码下游局部序列与 16srRNA 3′端 +146 9——— +1482核酸匹配性越好 ,则目的蛋白表达效率越高 ;目的基因编码区内与 16srRNA 3′ +146 9——— +1482间的最大匹配区域越靠近 5′末端 ,目的蛋白表达效率越高。通过对人IL_6、人GM_CSFcDNA 5′末端序列进行沉默突变 ,改善其与 16srRNA 3′端 +146 9——— +1482序列的匹配性后 ,目的蛋白的表达量均呈不同程度地提高。结论 :提高起始密码下游序列与 16srRNA 3′端 +146 9——— +1482局部序列间的匹配性 ,可增强目的基因的表达效率。     

Enhancement of the response to purinergic agonists in P2Y1 transfected 1321N1 cells by antagonists suramin and PPADS

1. We have previously shown that both suramin and pyridoxal-phosphate-6-azophenyl-2′, 4′ disulphonic acid (PPADS) act as antagonists at transfected P2Y₁ receptors. Here we show that under certain experimental conditions these two P2 antagonists can enhance the response to agonists acting at these receptors.
2. The expression of either P2Y₁ or P2Y₂ receptors in 1321N1 human astrocytoma cells results, on a change of medium, in an elevation of basal (no added agonist) accumulation of [³H]-inositol(poly)phosphates([³H]-InsP_x) compared to cells not expressing these receptors. This elevation is much greater in P2Y₁ transfectants than in P2Y₂ transfectants.
3. Both PPADS and suramin reduced this basal level of [³H]-InsP_x accumulation in the P2Y₁ expressing cells.
4. When a protocol was used which required changing the culture medium, antagonists were added at a concentration which reduced the basal accumulation by about 50%, there was a significant stimulation in response to increasing concentrations of 2-methylthioadenosine 5′-triphosphate (2MeSATP), in the absence of antagonists there was no significant effect of the agonist.
5. However, when 2MeSATP was added in the absence of a change of medium and with no antagonist present, there was a several fold increase in [³H]-InsP_x accumulation. These results show that a release of endogenous agonist activity (possibly ATP/ADP) from the P2Y₁ expressing cells can create conditions in which a response to an agonist such as 2MeSATP can only be seen in the presence of a competitive antagonist.

     

Differential roles of neurokinin 1 and neurokinin 2 receptors in the development and maintenance of heat hyperalgesia induced by acute inflammation

1. Following induction of acute inflammation by intraarticular injection of kaolin and carrageenan into the knee joint in rats, there was a significant decrease in the withdrawal latency to radiant heat applied to the paw (i.e. heat hyperalgesia), an increased joint circumference and increased joint temperature.
2. A neurokinin₁ (NK₁) receptor antagonist (CP-99,994, 10 mM) had no effect on the paw withdrawal latency when it was administered spinally through a microdialysis fibre before the induction of inflammation. Pretreatment with a NK₂ receptor antagonist (SR48968, 1 mM) administered spinally through the microdialysis fibre prevented the heat hyperalgesia from developing in the early stages of the inflammation.
3. Post-treatment through the microdialysis fibre with the NK₁ receptor antagonist (0.0110 mM) was effective in reversing the heat hyperalgesia. In contrast, post-treatment spinally with the NK₂ receptor antagonist (0.011 mM) had no effect on the heat hyperalgesia. The inactive stereoisomers of the NK₁ receptor antagonist, CP100,263, or the NK₂ receptor antagonist, SR48965, administered at the same doses, had no effect on the joint inflammation or the heat hyperalgesia.
4. Pretreatment systemically with the NK₁ receptor antagonist (30 mg kg⁻¹) had no effect on the heat hyperalgesia or pain-related behaviour ratings where 0 is none and 5 is non weight bearing and complete avoidance of limb contact. Pretreatment with a NK₂ receptor antagonist (10 mg kg⁻¹) systemically prevented the heat hyperalgesia and pain-related behaviour ratings from developing in the early stages of the inflammation. The inactive stereoisomers of NK₁ receptor antagonist, CP100,263, or the NK₂ receptor antagonist, SR48965, administered at the same doses, had no effect on the joint inflammation or the heat hyperalgesia.
5. Post-treatment systemically with either the NK₁ (0.130 mg kg⁻¹) or the NK₂ (0.110 mg kg⁻¹) receptor antagonist resulted in a dose-dependent reversal of the heat hyperalgesia. Pain-related behaviour ratings were reduced by post-treatment only with the NK₁ receptor antagonist. The inactive stereoisomers of the NK₁ receptor antagonist, CP100,263, or the NK₂ receptor antagonist, SR48965, administered at the same doses, had no effect on the behavioural responses.
6. Direct pretreatment of the knee joint with either the NK₁ (30 mg) or the NK₂ (10 mg) receptor antagonist prevented the heat hyperalgesia from developing without affecting joint swelling. The inactive stereoisomers of the NK₁ receptor antagonist, CP100,263, or the NK₂ receptor antagonist, SR48965, administered at the same doses, had no effect on the joint inflammation or the heat hyperalgesia.
7. There appears to be a differential role for the spinal tachykinin receptors in the development and maintenance of the heat hyperalgesia associated with acute joint inflammation. The NK₂ receptors appear to be activated early in the development of the heat hyperalgesia and NK₁ receptors are involved in the maintenance of the heat hyperalgesia.
8. Peripherally, both NK₁ and NK₂ receptors are involved in the development of heat hyperalgesia and pain-related behaviour ratings induced by acute inflammation.

     

The NK1 receptor and its participation in the synergistic enhancement of corneal epithelial migration by substance P and insulin-like growth factor-1

1. We have previously shown that substance P (SP) and insulin-like growth factor-1 (IGF-1) act synergistically to enhance the migration of rabbit corneal epithelial cells in an organ culture model. The present study was designed to identify the epithelial cell SP receptor that participates in this synergistic effect.
2. Rabbit corneal blocks were incubated for 24 h, then the length of the path of epithelial migration was measured. Reagents tried in the TC-199 culture medium, in the presence or absence of IGF-1, were: SP, agonists of tachykinin receptors NK₁, NK₂ or NK₃ and antagonists of tachykinin receptors NK₁ or NK₂.
3. The binding characteristics of SP receptors were examined in rabbit cultured corneal epithelial cells by binding assays with [¹²⁵I]-SP in the presence or absence of excess unlabelled SP or ligands of NK₁, NK₂ or NK₃ receptors.
4. As was demonstrated previously, SP and IGF-1 stimulated epithelial migration when they were added to the culture medium together, but individually they had no effect. NK₁ agonists had the same synergistic effect with IGF-1 as did SP, but the NK₂ and NK₃ agonists did not. Furthermore, the NK₁ antagonist abolished the synergistic effect of SP and IGF-1, but the NK₂ antagonist had no effect.
5. SP bound specifically to rabbit cultured corneal epithelial cells. The binding affinity was 0.44 nM and there were 2.43×10⁴ binding sites per cell. The NK₁ ligand competed, in a dose-dependent fashion, with the binding of SP to corneal epithelial cells, but neither the NK₂ nor NK₃ ligand affected binding.
6. We conclude that the SP receptor in rabbit corneal epithelial cells is NK₁ and that this receptor participates in the synergistic enhancement of corneal epithelial migration by SP and IGF-1. The precise mechanism(s) of this interaction requires more study. These findings imply that both neural and humoral factors are essential for the maintenance and healing of corneal epithelium.

     

Amyloid-P-component-like immunoreactivity in β/A4-immunoreactive deposits in Alzheimer-type dementia brains

An immunohistochemical study using the mirror-image technique was performed in order to establish whether amyloid P component is involved in the mechanism of deposition of amyloid fibrils in senile plaques (SPs) in Alzheimer-type dementia (ATD). Ninety percent of /A4 protein-immunoreactive SPs were also stained by the anti-amyloid P component immunchistochemistry, and this applied to all of the diffuse, primitive and classical types of /A4 deposits. These findings may suggest an involvement of amyloid P component in the formation of amyloid fibrils in senile plaques in ATD brains.     

P0.1 is a useful parameter in setting the level of pressure support ventilation

Objective The purpose of this study was to investigate whether changes in breathing pattern, neuromuscular drive (P0.1), and the work involved in breathing might help to set the individual appropriate level of pressure support ventilation (PSV) in patients with acute respiratory failure (ARF) requiring ventilatory assistance.Design: A prospective, interventional study.Setting An 8-bed multidisciplinary intensive care unit (ICU).Patients Ten patients with ARF due to adult respiratory distress syndrome (ARDS), sepsis or airway infection were included in the study. Chronic obstructive pulmonary disease (COPD) patients with acute exacerbation were excluded. None of these patients was in the weaning process.Interventions We found a level of pressure support able to generate a condition of near-relaxation in each patient, as evidenced by work of breathing (WOB) values close to 0 J/l. This level was called PS 100 and baseline physiological measurements, namely, breathing pattern, P 0.1 and WOB were obtained. Pressure support was then reduced to 85%, 70% and 50% of the initial value and the same set of measurements was obtained.Measurements and results Flow ( ) was measured by a flow sensor (Varflex) positioned between the Y-piece of the breathing circuit and the endotracheal tube. Tidal volume was obtained by numerical integration of the flow signal. Airway pressure (P_aw) was sampled through a catheter attached to the flow sensor. Esophageal pressure (P_es) was measured with a nasogastric tube incorporating an esophageal balloon. The esophageal balloon and flow and pressure sensors were connected to a portable monitor (CP 100 Bicore) that provided realtime display of flow, volume, P_aw and P_es tracings and loops of P_es/V, P_aw/V and /V relationships. The breathing pattern was analyzed from the flow signal. Patient work of breathing (WOB) was calculated by integration of the area of the P_es/V loop. Respiratory drive (P 0.1) was measured at the esophageal pressure change during the first 100 ms of a breath, by the quasiocclusion technique. When pressure support was reduced, we found that the respiration rate significantly increased from PS 100 to PS85, but varied negligibly with lower pressure support levels. Tidal volume behaved in a similar way, decreasing significantly from PS 100 to PS85, but hardly changing at PS 70 and PS 50. In contrast, WOB and P 0.1 increased progressively with decreasing pressure support levels. The changes in WOB were significant at each stage in the trial, whereas P 0.1 increased significantly from PS 100 at other stages. Linear regression analysis revealed a highly positive, significant correlation between WOB and P 0.1 at decreasing PSV levels (r=0.87), whereas the correlation between WOB and ventilatory frequency was less significant (r=0.53). No other correlation was found.Conclusions During pressure support ventilation, P 0.1 may be a more sensitive parameter than the assessment of breathing pattern in setting the optimal level of pressure support in individual patients. Although P 0.1 was measured with an esophageal balloon in the present study, non-invasive techniques can also be used.     

Glutamatergic regulation of striatal peptide gene expression in rats

Summary The mRNA levels encoding enkephalin and substance P were measured in the rat striatum following cortical ablation, blockade of N-methyl-D-aspartate (NMDA) receptors or inhibition of glutamate release by lamotrigine. Unilateral ablation of the cerebral cortex resulted in a decrease of substance P mRNA levels particularly in the rostral dorsolateral and dorsomedial striatum ipsilateral to the lesion. There was a similar trend for a reduction in levels of enkephalin mRNA. Continuous, intrastriatal infusion of the competitive NMDA receptor antagonist, 3-((±)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, (CPP, 0.12 and 1.2g/day) decreased both enkephalin mRNA and substance P mRNA in dose-dependent manner evenly throughout the striatum adjacent to the infusion site. Following subchronic administration of the presumed glutamate release inhibitor, lamotrigine (5 and 20 mg/kg IP) there was no significant alterations in either enkephalin mRNA or substance P mRNA levels in the striatum. Both enkephalin mRNA and substance P mRNA expression in the rat striatum appear tonically stimulated through postsynaptic NMDA receptor mediated mechanisms. This contrasts with differential dopaminergic modulation of peptides in striatal output neurons.     

原位PCR和免疫组化检测垂体腺癌P16基因

目的研究人垂体腺瘤P16基因的改变,同时探索原位PCR技术的适宜条件和结果确认,以及用于基因缺失检测的可行性。方法原位PCR和免疫组化技术。结界绝大部分间质细胞为P16蛋白免疫组化阴性,而一部分垂体肿瘤细胞呈P16阳性;针对P16基因的原位PCR信号则可见于垂体肿瘤细胞和间质细胞等各种细胞,即P16组化阴性的细胞同样表明有P16基因存在。结论原位PCR可以是P16基因研究的一种有效手段,提示垂体腺癌的P16基因改变形式可能主要是表达过度而不是基因缺失。     

p16在肝细胞癌组织内变异的研究

应用分子杂交及免疫组化方法检测５６例ＨＣＣ组织内的抑癌基因ｐ１６。结果显示,ＨＣＣ标本中ｐ１６蛋白在癌细胞内表达的阳性率为３７５％（２１／５６）,而ｐ１６ＤＮＡ斑点杂交的阳性率为５５３６％（３１／５６）。Ｓｏｕｔｈｅｒｎ转膜杂交证实,１２５％（７／５６）ＨＣＣ标本存在ｐ１６的甲基化变异,４４６４％（２５／５６）ｐ１６基因缺失。提示ＨＣＣ发生、发展过程中存在ｐ１６基因的缺失和甲基化变异     

聚合酶链反应检测细菌16S rRNA基因

根据细菌１６ＳｒＲＮＡ基因的高度保守性,设计合成所有细菌、革兰氏阳性细菌及革兰氏阴性细菌的共同引物,采用聚合酶链反应检测已知细菌１３株,三对引物分别扩增的阳性率为１００％,倍比稀释法能检出细菌的最低浓度为４ＣＦＵ·ｍｌ－１,同时检测临床样本４０份,阳性率为６７５％（２７／４０）,同期细菌培养阳性率为４５％（１８／４０）,二者比较差异有显著性（Ｐ＜０．０５）。结果提示聚合酶链反应检测细菌１６ＳｒＲＮＡ基因具有高度的特异性和敏感性。