重组改构人TNF-α联合顺铂对人胶质瘤U251细胞的生长抑制作用

目的:观察重组改构人肿瘤坏死因子α(rmhTNF-α)联合顺铂(CDDP)对人胶质瘤细胞生长的抑制作用.方法:人胶质瘤U-251细胞分为对照组(不加药);T组:加入rmhNF-α(20μg/L);C组:加入CDDP(2 mg/L);T C组:加入rmhTNF-α(20μg/L)及CDDP(2 mg/L).MTT法检测细胞生长抑制率,用流式细胞仪检测细胞凋亡率及细胞周期变化情况,用免疫组织化学方法检测细胞PCNA、Bcl-2、P170蛋白的表达情况.结果:T组、C组细胞生长抑制率、细胞凋亡率均高于对照组(P＜0.05),T C组细胞生长抑制率、细胞凋亡率均高于其他各组(P＜0.05).T组PCNA-LI、Bcl-2蛋白表达低于对照组(P＜0.05),T C组PCNA-LI、Bcl-2蛋白表达均明显低于其他各组(P＜0.05),对照组、T组及T C组P170蛋白表达低于C组(P＜0.05).结论:rmhTNF-α能抑制人胶质瘤细胞生长,其机制与诱导细胞凋亡、抑制细胞增殖活性有关,且rmhTNF-α与CDDP联用有协同增效作用.     

Free amino acid pool in the brain of mice homozygous for the gene "dilute lethal"

The neurologic mutant "dilute lethal" (dl) mice, which reveal several neurologic and biochemical disturbances similar to human phenylketonuria, were used to investigate some aspects of amino acid disorder. We have studied the free amino pool in the brain of "dl" mice and of their control littermates as well as phenylalanine and tyrosine levels in brain and liver as a function of age and after phenylalamine overload. The tyrosine level decreased in brain and liver of affected mice whereas the phenylalanine/tyrosine ratio increased as a function of age. The significantly higher phenylalanine level and phenylalanine/tyrosine ratio in the liver of 20-day-old "dl" mice suggest a lower liver phenylalanine hydroxylase activity. After phenylalanine overload, the impairment of phenylalanine metabolism is predominant in the brain of "dl" mice, suggesting a disturbance in phenylalanine hydroxylation. A decrease in the level of several amino acids occurs in the brains of "dl" mice without or after phenylalanine overload; these facts might correspond to a disturbance in the transfer of amino acids to the brain and may lead to impairment in protein synthesis.     

Rapid detection of the hepatitis B virus YMDD mutant using TaqMan-minor groove binder probes

BACKGROUND: TaqMan-minor groove binder (MGB) probes were used in a real-time PCR-based assay for the rapid and accurate detection of hepatitis B virus (HBV) YMDD mutants. METHODS: TaqMan-MGB probes were designed to distinguish between wild-type (YMDD) and mutant (YVDD and YIDD) strains of HBV. The detection limit and sensitivity of the assay were determined using a dilution series of a mixture of wild-type and mutant plasmids. Serum samples collected from four patients with chronic mutant HBV infections during lamivudine therapy were analyzed using this method. RESULTS: The detection limit for YVDD and YIDD was 10 and 50 copies, respectively, whereas the sensitivity was 10% within a mixed virus population. In the clinical samples, mutant strains of HBV could be detected at levels <2.6 log copies/ml of HBV DNA. While 15 of the 21 samples tested by this method were positive for the YMDD mutant, direct sequencing and a reverse hybridization line probe assay (INNO-LiPA HBV DR v2) detected the mutant strain in only 11 and 9 samples, respectively. Moreover, the data for 6 samples analyzed by TA cloning were fully consistent with our TaqMan PCR results. CONCLUSIONS: We successfully established a sensitive and accurate assay for the YMDD mutant of HBV. This method may be useful for monitoring patients treated with lamivudine.     

IL-13及其变异体对气道平滑肌细胞信号传导的影响

目的探讨野生型人IL-13(whIL-13)及其变异体(mhIL-13,Arg130Gln)对培养的气道平滑肌细胞(AM-SCs)信号传导的影响。方法利用不同浓度的whIL-13及mhIL-13分别对AMSCs进行刺激,收集细胞,流式细胞仪检测STAT-6磷酸化情况,从而判断这两种蛋白分子在信号传导上的差异。结果whIL-13及mhIL-13引起的STAT-6磷酸化随浓度增加而增强。mhIL-13在较低浓度(0.3 ng/ml)即可引起STAT-6磷酸化,而whIL-13则需要更高浓度(1 ng/ml);相同浓度时STAT-6磷酸化情况在两种蛋白分子间具有显著性差异;mhIL-13作用强度大约为whIL-13分子的3-5倍。结论mhIL-13分子致气道平滑肌细胞STAT-6磷酸化能力明显强于whIL-13,低浓度mhIL-13即可在体外培养的AMSCs中引起信号传导。     

RNAi suppression of rice endogenous storage proteins enhances the production of rice-based Botulinum neutrotoxin type A vaccine

Mucosal vaccines based on rice (MucoRice) offer a highly practical and cost-effective strategy for vaccinating large populations against mucosal infections. However, the limitation of low expression and yield of vaccine antigens with high molecular weight remains to be overcome. Here, we introduced RNAi technology to advance the MucoRice system by co-introducing antisense sequences specific for genes encoding endogenous rice storage proteins to minimize storage protein production and allow more space for the accumulation of vaccine antigen in rice seed. When we used RNAi suppression of a combination of major rice endogenous storage proteins, 13 kDa prolamin and glutelin A in a T-DNA vector, we could highly express a vaccine comprising the 45 kDa C-terminal half of the heavy chain of botulinum type A neurotoxin (BoHc), at an average of 100 μg per seed (MucoRice-BoHc). The MucoRice-Hc was water soluble, and was expressed in the cytoplasm but not in protein body I or II of rice seeds. Thus, our adaptation of the RNAi system improved the yield of a vaccine antigen with a high molecular weight. When the mucosal immunogenicity of the purified MucoRice-BoHc was examined, the vaccine induced protective immunity against a challenge with botulinum type A neurotoxin in mice. These findings demonstrate the efficiency and utility of the advanced MucoRice system as an innovative vaccine production system for generating highly immunogenic mucosal vaccines of high-molecular-weight antigens.     

Studies in the diabetic mutant mouse: III. Physiological factors associated with alterations in beta cell proliferation

Summary Beta cell replication was studied in normal (C 57 BL/Ks) and diabetic mutant (C 57 BL/Ks-db/db) mice following thymidine-³H administration. The specific activity of DNA of isolated islets (DPM/g islet DNA) was used as an index of proliferative activity and correlated with labeling determined by radioautography. Although thymidine-³H incorporation in islets of prehyperglycemic 5 to 6 week old mutants was limited, it was significantly greater than that in normal mice. With the elevation of blood glucose values, incorporation rose sharply, reaching a maximum level above 130 mg glucose/100 ml blood. Sustained, severe hyperglycemia subsequently correlated with a decline in islet DNA synthesis. Food restriction early in the syndrome reduced hyperglycemia and resulted in low incorporation of label. Animals refed ad lib for periods of 1, 2, or 3 weeks showed significant increases in labeling, with maximal values after 1 week of refeeding. Electron microscopic radioautographs of the islets revealed labeled beta cells but no labeled alpha cells, suggesting that proliferative activity is predominantly restricted to the beta cell population.USPHS Research Career Development Awardee, Grant K4-AM-7394.     

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNaV1.7

Improper function of voltage-gated sodium channels (Na_Vs), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target Na_Vs hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of Na_V subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of Na_V through a systematic structure–activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human Na_V1.7 (hNa_V1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective Na_V inhibitors.Modulation of action potentials in electrically excitable cells is controlled by tight regulation of ion channel expression and distribution. Voltage-gated sodium ion channels (Na_Vs) constitute one such family of essential membrane proteins, encoded in 10 unique genes (Na_V1.1–Na_V1.9, Na_x) and further processed through RNA splicing, editing, and posttranslational modification. Sodium channels are comprised of a large (∼260 kDa) pore-forming α-subunit coexpressed with ancillary β-subunits. Misregulation and/or mutation of Na_Vs have been ascribed to a number of human diseases including neuropathic pain, epilepsy, and cardiac arrhythmias. A desire to understand the role of individual Na_V subtypes in normal and aberrant signaling motivates the development of small-molecule probes for regulating the function of specific channel isoforms (1–4).Nature has provided a collection of small-molecule toxins, including (+)-saxitoxin (STX, 1) and (−)-tetrodotoxin (TTX), which bind to a subset of mammalian Na_V isoforms with nanomolar affinity (5–7). Guanidinium toxins inhibit Na⁺ influx through Na_Vs by occluding the outer pore above the ion selectivity filter (site 1). This proposed mechanism for toxin block follows from a large body of electrophysiological and site-directed mutagenesis studies (Fig. 1A and refs. 8–10). The detailed view of toxin binding, however, is unsupported by structural biology, as no high-resolution structure of a eukaryotic Na_V has been solved to date (11–16). Na_V homology models, constructed based on X-ray analyses of prokaryotic Na⁺ and K⁺ voltage-gated channels, do not sufficiently account for experimental structure–activity relationship (SAR) data (6, 17–20), and the molecular details underlying distinct differences in toxin potencies toward individual Na_V subtypes remain undefined (5, 6, 21–23). The lack of structural information motivates a comprehensive, systematic study of toxin–protein interactions.Open in a separate windowFig. 1.(A) Schematic drawing of 1 bound in the Na_V outer pore as suggested by previous electrophysiology and mutagenesis experiments. Each of the four domains (I, orange; II, red; III, gray; and IV, teal) is represented by a separate panel. (B) Schematic representation of double-mutant cycle analysis and mathematical definition of coupling energy (ΔΔE_Ω). X¹ = IC₅₀(WT⋅STX)/IC₅₀(MutNa_V⋅STX), X² = IC₅₀(WT⋅MeSTX)/IC₅₀(MutNa_V⋅MeSTX), Y¹ = IC₅₀(MutNa_V⋅STX)/IC₅₀(MutNa_V⋅MeSTX), and Y² = IC₅₀(WT⋅STX)/IC₅₀(WT⋅MeSTX).Double-mutant cycle analysis has proven an invaluable experimental method for assessing protein–protein, protein–peptide, and protein–small-molecule interactions in the absence of crystallographic data (Fig. 1B and Fig. S1 and refs. 9, 10, and 24–31). Herein, we describe mutant cycle analysis with Na_Vs using STX and synthetically modified forms thereof. Our results are suggestive of a toxin–Na_V binding pose distinct from previously published views. Our studies have resulted in the identification of a natural variant of STX that is potent against the STX-resistant human Na_V1.7 isoform (hNa_V1.7). Structural insights gained from these studies provide a foundation for engineering guanidinium toxins with Na_V isoform selectivity.Open in a separate windowFig. S1.Mutant cycle analysis definition and examples. (A) Schematic of a single mutant cycle with mathematical expressions for coupling energy ΔΔE_Ω. R is the ideal gas constant and T is temperature. Each IC₅₀ is the half maximal inhibition concentration determined by whole-cell voltage-clamp electrophysiology. When the separation between IC₅₀ values for the reference compound and the modified compound is different with a mutant than with the WT protein, a nonzero value for ΔΔE_Ω is obtained (B), but when the separation is the same (C), ΔΔE_Ω is equal to 0. In B, the difference in the relative affinity of 1 and 4 with Y401A is smaller than the difference with the WT channel, indicating a positive coupling (ΔΔE_Ω > 0). In C, the relative affinities of 1 and 8 against WT rNa_V1.4 and Y401A are similar, and ΔΔE_Ω ∼0 kcal/mol.     

Diabetic neuropathy in db/db mice develops independently of changes in ATPase and aldose reductase

Summary ATPase activity was investigated in sciatic and optic nerves of female mutant diabetic C57Bl/Ks (db/db) mice and age-matched control mice (db/m and m/m). Nerves from animals aged 50, 70, 125, 180 and 280 days were assayed in vitro for ATPase activity in the presence or absence of ouabain: the ouabain-sensitive fraction contained Na⁺,K⁺-ATPase. Enzymatic activity was compared within and between age-matched groups. No significant difference in Na⁺,K⁺-ATPase activity was detected between the diabetic and control mice, whether expressed as mol P_i/h^–1 formed per gramme wet weight or per nerve (protein content). The activity decreased by about 25% in both the sciatic and optic nerves of the oldest animals. These results were strikingly similar in all groups, regardless of the type of nerve examined, confirming that the development of neuropathy in this animal model is unrelated to the postulated derangement of Na⁺,K⁺-ATPase activity. Among possible explanations, a lack of polyol pathway activation was investigated by staining the sciatic nerves of animals from all groups with the peroxidase-antiperoxidase procedure using a polyclonal antiserum raised against the enzyme aldose reductase. Histological sections of all nerves were consistently negative, suggesting that these animals actually lack the enzyme involved in activating the self-perpetuating metabolic cycle leading to deranged nerve function. The db/db mouse appears to present particular biochemical changes which merit attention with a view to clarifying the pathogenesis of diabetic neuropathy.     

Molecular investigations of a novel iduronate-2-sulfatase mutant in a Chinese patient

BACKGROUND: Molecular investigations of iduronate-2-sulfatase (IDS) mutants for the X-linked lysosomal storage disease mucopolysaccharidosis type II (MPS II, Hunter disease), commonly depends on transient expression studies to verify a single nucleotide change to be pathogenic. In 2 severely affected patients, IDS missense mutations, c.1016T>C (novel) and c.1016T>G (known) were identified predicting the substitution of an ambivalent cyclic proline and a hydrophilic arginine respectively for the hydrophobic leucine at residue 339. We hypothesized that residue Leu339 may be functionally critical. METHODS: We performed a study for the 2 mutations by in-situ mutagenesis, in vitro expression, and functional analysis. RESULTS: Transient expression revealed that both the missense variants had stable mRNA but their residual enzyme activities remained <2.5% of normal level. The effect of the missense mutations on protein expression was detected by Western blot analysis. Both the missense mutations synthesized the precursor form but had reduced mature form of IDS. CONCLUSION: The novel mutation p.L339P is a disease-causing mutation affecting maturation of the protein.     

牙龈卟啉单胞菌唾液酸酶基因突变株黏附与侵入KB细胞能力研究

目的    研究PG0352基因缺失对牙龈卟啉单胞菌（P. gingivalis）黏附和侵入人口腔上皮癌KB细胞能力的影响。方法    本研究于2014年7—12月在中国医科大学口腔医学院中心实验室完成。实验组：将P. gingivalis W83菌株（W83菌株组）和PG0352基因突变株（PG0352突变株组）分别以100∶1的比例与KB细胞共培养2 h,制备P. gingivalis与KB细胞的黏附和侵入模型;对照组：单纯KB细胞培养。透射电镜观察KB细胞表面及细胞内是否有P. gingivalis存在;用PBS清除未黏附于KB细胞的细菌,裂解细胞后涂板检测P. gingivalis黏附和侵入KB细胞情况,抗生素保护法检测P. gingivalis侵入KB细胞情况。结果    透射电镜结果发现,W83菌株组和PG0352突变株组KB细胞内均有细菌侵入;通过涂板后菌落计数发现P. gingivalis W83菌株和PG0352基因突变株对KB细胞的黏附率分别为（15.559 ± 2.020）%和（9.309 ± 1.750）%,侵入率分别为（0.651 ± 0.287）%和（1.517 ± 0.233）%,两者差异均有统计学意义（均P < 0.05）。结论    P. gingivalis W83菌株和PG0352基因突变株均能够黏附和侵入KB细胞;与W83菌株相比,PG0352基因突变株黏附上皮细胞的能力较弱而侵入能力较强。