Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery

The function of the human nucleotide excision repair (NER) apparatus is to remove bulky adducts from damaged DNA. In an effort to gain insights into the molecular mechanisms involved in the recognition and excision of bulky lesions, we investigated a series of site specifically modified oligonucleotides containing single, well-defined polycyclic aromatic hydrocarbon (PAH) diol epoxide-adenine adducts. Covalent adducts derived from the bay region PAH, benzo[a]pyrene, are removed by human NER enzymes in vitro. In contrast, the stereochemically analogous N(6)-dA adducts derived from the topologically different fjord region PAH, benzo[c]phenanthrene, are resistant to repair. The evasion of DNA repair may play a role in the observed higher tumorigenicity of the fjord region PAH diol epoxides. We are elucidating the structural and thermodynamic features of these adducts that may underlie their marked distinction in biologic function, employing high-resolution nuclear magnetic resonance studies, measurements of thermal stabilities of the PAH diol epoxide-modified oligonucleotide duplexes, and molecular dynamics simulations with free energy calculations. Our combined findings suggest that differences in the thermodynamic properties and thermal stabilities are associated with differences in distortions to the DNA induced by the lesions. These structural effects correlate with the differential NER susceptibilities and stem from the intrinsically distinct shapes of the fjord and bay region PAH diol epoxide-N(6)-adenine adducts.     

Phosphorylation of the 24p3 protein secreted from mouse uterus in vitro and in vivo

The 24p3 protein is a 25 KDa glycoprotein, having been purified from mouse uterine fluid. Thr⁵⁴, Ser⁸⁸, and Thr¹²⁸/Ser¹²⁹ on the protein molecule were predicted to be the phosphorylation site of casein kinase II, protein kinase C, and cAMP-dependent protein kinase, respectively. Incorporation of phosphate to this protein from [-³²P]-ATP was tested in the solution suitable for the three kinases. Neither casein kinase II nor cAMP-dependent protein kinase reacted to the 24p3 protein; however, protein kinase C demonstrated phosphorylation to this protein. This phosphorylation may be competing with a polypeptide segment: Arg⁷⁹-Tyr-Trp-Ilu-Arg-Thr-Phe-Val-Pro-Ser⁸⁸-Ser-Arg-Ala-Gly-Gln-Phe-Thr-Leu-Gly⁹⁷ in the 24p3 protein molecule. To support this theory, Ser⁸⁸ is a phosphorylation site of protein kinase C on 24p3 protein. The enzyme kinetic parameter, based on the Michaelis-Menten equation, determined Km to be 2.96 M in the phosphorylation of 24p3 protein by the kinase. Both of the phosphorylated and dephosphorylated form of 24p3 protein can enhance the cAMP-dependent protein kinase activity in vitro. In addition, this experiment will show for the first time that serine-phosphorylated 24p3 protein exists in mouse uterine tissue.     

Disruption of the epilepsy KCNQ2 gene results in neural hyperexcitability

Benign familial neonatal convulsion (BFNC) is a common idiopathic epilepsy with autosomal dominant inheritance. Recently, two novel voltage-dependent potassium channel genes, KCNQ2 and KCNQ3, were identified by positional cloning as being responsible for BFNC. Heterotetramers of the products of these genes form M-channels and regulate the threshold of electrical excitability of neurons. We disrupted the mouse KCNQ2 gene via gene targeting to study the relationship between KCNQ2 and epilepsy. Homozygous pups (KCNQ2 -/-) died within a few hours after birth owing to pulmonary atelectasis that was not due to the status of epileptic seizures, although their development was morphologically normal. Heterozygous mice had decreased expression of KCNQ2 and showed hypersensitivity to pentylenetetrazole, an inducer of seizure. These data indicate that the decreased expression of KCNQ2 might cause a hyperexcitability of the CNS, which accounts for the mechanism of BFNC.     

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.     

Molecular Mechanisms of Polymyxin B-Membrane Interactions: Direct Correlation Between Surface Charge Density and Self-Promoted Transport

We have studied the interaction of the polycationic peptide antibiotic polymyxin B (PMB) with asymmetric planar bilayer membranes via electrical measurements. The bilayers were of different compositions, including those of the lipid matrices of the outer membranes of various species of Gram-negative bacteria. One leaflet, representing the bacterial inner leaflet, consisted of a phospholipid mixture (PL; phosphatidylethanolamine, -glycerol, and diphosphatidylglycerol in a molar ratio of 81:17:2). The other (outer) leaflet consisted either of lipopolysaccharide (LPS) from deep rough mutants of PMB-sensitive (Escherichia coli F515) or -resistant strains (Proteus mirabilis R45), glycosphingolipid (GSL-1) from Sphingomonas paucimobilis IAM 12576, or phospholipids (phosphatidylglycerol, diphytanoylphosphatidylcholine). In all membrane systems, the addition of PMB to the outer leaflet led to the induction of current fluctuations due to transient membrane lesions. The minimal PMB concentration required for the induction of the lesions and their size correlated with the charge of the lipid molecules. In the membrane system resembling the lipid matrix of a PMB-sensitive strain (F515 LPS/PL), the diameters of the lesions were large enough (d= 2.4 nm ± 8%) to allow PMB molecules to permeate (self-promoted transport), but in all other systems they were too small. A comparison of these phenomena with membrane effects induced by detergents (dodecyltriphenylphosphonium bromide, dodecyltrimethylammonium bromide, sodiumdodecylsulfate) revealed a detergent-like mechanism of the PMB-membrane interaction. Received: 16 September 1997/Revised: 25 November 1997     

两种高危型HPV检测技术诊断宫颈病变的临床价值比较

目的:比较酶切信号放大法(Cervista)与导流杂交基因芯片技术(Hybri Ma)检测高危型人乳头状瘤病毒(HR-HPV)诊断宫颈上皮内瘤变2级或2级以上(CIN2+)的临床价值。方法:随机选择288例2012年3月至2013年1月在哈尔滨医科大学附属第一医院妇科门诊进行新柏氏液基细胞学检查的年龄在20~65岁的宫颈细胞学检测未明确意义的不典型鳞状细胞(ASCUS)的患者,采用Cervista技术与Hybri Ma技术进行高危型HPV检测,并对入组的患者行阴道镜下宫颈活组织检查。以病理学诊断结果为金标准,比较Cervista技术与Hybri Ma技术诊断宫颈上皮内瘤变2级或2级以上(CIN2+)的敏感度、特异度及ROC曲线。结果:在入组的288例患者中,Cervista技术和Hybri Ma技术检出高危型HPV的阳性率分别为49.31%和51.39%(P0.05),其诊断CIN2+的敏感度分别为95.65%和91.30%(P0.05),特异度分别为59.50%和56.20%(P0.05),阳性预计值分别为30.99%和28.38%(P0.05),阴性预计值分别为98.63%和97.14%(P0.05)。两组ROC曲线下面积分别为0.776和0.738(P0.05)。结论:Cervista技术与Hybri Ma技术诊断CIN2+的临床价值相当。     

腹腔镜辅助下阴式子宫切除术与阴式子宫全切除术治疗子宫良性病变的临床比较

目的:探究阴式子宫全切除术与腹腔镜辅助下阴式子宫切除术治疗子宫良性病变的临床效果。方法:选择2011年10月~2013年9月我院收治的子宫良性病变的患者120例,其中行腹腔镜辅助下阴式子宫切除术80例(研究组)和行阴式子全宫切除术40例(对照组),观察并分析两组的临床指标及手术并发症。结果:研究组住院费用明显高于对照组(P0.05),手术时间、术中出血量及住院时间均低于对照组(P0.05);研究组无邻近脏器损伤,对照组输尿管损伤2例(5.00%),研究组患者出现手术损伤率明显低于对照组,差异存在统计学差异(x2=3.968,P=0.046);对两组患者术后随访1年,两组患者均无出现再手术情况;两组患者术后阴道残端均愈合较好,未出现阴道残端漏,患者无大小便困难及其他不适。结论:腹腔镜辅助下阴式子宫切除术治疗子宫良性病变疗效优于阴式子宫全切术,其手术时间、术中出血量及住院时间均较低,创伤小,更有利于患者恢复健康,在临床上值得应用推广。     

TURP联合经尿道膀胱颈切开术治疗小体积前列腺增生所致膀胱出口梗阻的疗效分析

目的:探讨经尿道前列腺电切术(TURP)联合经尿道膀胱颈切开术(TUIBN)治疗小体积前列腺增生(BPH)所致膀胱出口梗阻的疗效。方法:选择2009年1月~2013年12月我院收治的小体积BPH患者,其中单纯经尿道前列腺电切术(TURP组)48例,经尿道前列腺电切术联合经尿道膀胱颈切开术(TURP+TUIBN组)48例。比较两组的术前、术后国际前列腺症状评分(IPSS)、残余尿量(PVR)、最大尿流率(Qmax)等,以及术后并发症的发生情况。结果:TURP+TUIBN组术中出血量较TURP组明显增多(P0.05),两组手术时间、组织切除质量比较,差异均无统计学意义(P0.05);与TURP组比较,TURP+TUIBN组术后6个月IPSS评分、PVR明显下降,Qmax、膀胱压力明显上升(P0.05);TURP+TUIBN组并发症发生率为4.2%,显著低于TURP组16.7%(P0.05)。结论:TURP+TUIBN治疗小体积前BPH所致膀胱出口梗阻,可彻底切除增生腺体,消除小体积BPH的各种梗阻因素,减少术后膀胱颈挛缩的发生。     

Oral mucosal lesions in denture wearers

doi:10.1111/j.1741‐2358.2009.00289.x
Oral mucosal lesions in denture wearers Objective: To determine the prevalence of oral mucosal lesions (OMLs) and denture‐related mucosal lesions (DMLs) in denture wearers and to co‐relate the prevalence with age, gender, type of denture and any systemic conditions. Materials and methods: Dental records of 380 denture wearers were retrospectively reviewed for OMLs and DMLs. Results: We found 45% of the denture wearers had DMLs and 60.8% had OMLs not related to denture wearing. Although the prevalence of DMLs was higher in complete denture wearers than in partial denture wearers (49% vs. 42.2%), this difference was not significant. The most common DMLs were traumatic ulcer (19.5%) and denture‐induced stomatitis (18.1%). When analysed by type, traumatic ulcer, denture hyperplasia, frictional keratosis and candidiasis were more common in complete denture wearers, whereas denture‐induced stomatitis was more common in partial denture wearers. Frictional keratosis was more common in men than in women. The prevalence of OMLs not related to denture wearing was higher in complete denture wearers than in partial denture wearers, and the most common OML was fissured tongue (27.6%). No association between DMLs and systemic conditions or xerostomic drugs was noted. Conclusion: No differences in the prevalence of DMLs in association with denture type were found. The prevalence of OMLs not related to denture wearing was higher in complete denture wearers than in partial denture wearers. This difference was affected by age, and the data were similar to the findings observed in the elderly.     

Hormonal concentrations in bitches with primary uterine inertia

Normal labor is accompanied by sequential changes in blood concentrations of prostaglandin F2α (measured as 15-ketodihydro-PGF2α = PGFM), progesterone, estradiol, oxytocin, vasopressin, and of elevated cortisol levels. The aim of this study was to investigate hormone concentrations in dogs diagnosed with primary uterine inertia before and during treatment by cesarian section. The hypothesis was the dogs would have abnormally low plasma concentrations in one or several of the hormones involved in parturition. The study comprised seven bitches with total primary uterine inertia (dystocia group) treated with cesarian section and six healthy bitches (control group) subjected to planned cesarean section. Blood samples were taken before anesthesia, before surgery started, on delivery of the first puppy and on delivery of the last puppy. The progesterone:PGFM ratio in plasma was higher in the dystocia group than in the control group, but the serum estradiol concentration did not differ between groups. The plasma concentrations of oxytocin and vasopressin increased in both groups when the first puppies were delivered, but both hormones were more elevated in the control group than in the dystocia group on delivery of the last puppies. The plasma cortisol concentration increased to the same level in both groups. In conclusion, the ratio between progesterone and PGFM was higher and the oxytocin and vasopressin concentrations lower in the dystocia dogs than in the control dogs. The findings indicate that these hormones are involved in the pathophysiology of total primary uterine inertia in bitches.