Enzymatic toxicogenation of “Activated” cyclophosphamide by 3′–5′ exonucleases

3'-5' Exonucleases from various sources were found to toxicogenate 4-hydroxycyclophosphamide ("activated" cyclophosphamide) by splitting the oxazaphosphorinane ring and releasing an alkylating moiety and acrolein. Neither cyclophosphamide (CP) nor the deactivated metabolites of CP, 4-keto-CP and carboxyphosphamide nor 4-(S-ethanol)-sulfido-CP were attacked by 3'-5' exonucleases. DNA polymerases with proofreading activity, such as DNA polymerase I from E. coli or DNA polymerase delta from rabbit bone marrow, exhibited a tenfold higher specific activity with "activated" CP than "plain" 3'-5' phosphodiesterases such as snake venom phosphodiesterase or 3',5'cyclic AMP phosphodiesterase from bovine heart tissue. High levels of toxicogenating activity were estimated in peripheric human lymphocytes and tissues of lymphatic origin, suggesting that enzymatic toxicogenation plays a key role with respect to the cytotoxic specificity of "activated" CP.     

副粘病毒Tianjin株基因组3′和5′末端序列测定及分析

目的测定副粘病毒Tianjin株基因组3′和5′末端序列，分析其前导区～级及二级结构与功能的关系。方法从感染病毒的鸡胚尿囊液中提取总RNA，利用cDNA末端快速扩增（RACE）法分别扩增基因组3′和5′端cDNA片段，并克隆至pGM—T载体中，然后分别测定插入片段的核苷酸序列，用DNAStar软件将测得的3′和5′端前导区序列与GenBank中选取的6株仙台病毒代表株相应序列进行相似性比较及序列比对，用RNAdraw软件预测基因组3′和5′端前导区二级结构并进行比较分析。结果副粘病毒Tianjin株基因组3′和5′末端非编码区长度分别为119和142个核苷酸，前导区核苷酸序列与6株仙台病毒基因组的相似性分别在89．1％～96．4％和93．0％～96．5％，二级结构与选取的已知仙台病毒株高度相似而且稳定。结论副粘病毒Tianjin株基因组3′和5′端前导区序列相对保守，可能与病毒复制、转录调控及致病性有关。     

人巨噬细胞中的ph′染色体

已经证明,慢粒白血病的ph′染色体是后天染色体易位引起的,只见于造血细胞和红细胞、粒细胞和巨核细胞的前身。虽然单核细胞和粒细胞可能来自一个共同的干细胞,但在单核—巨噬细胞中从未直接证明ph′染色体的存在。作者用促进巨噬细胞增殖的培养系统研究了3例     

Kartagener′s综合征1例

Kartagener′s综合征又称鼻窦炎支气管扩张内脏转位综合征，是一种罕见疾病，兹将我们所见1例报告如下。患者女，24岁。10年前开始出现心慌、胸闷、气促，活动时明显，感冒后症状加重。近2年来症状明显加重。患者自幼常感冒，鼻塞，常有黄脓状鼻分泌物，时轻时重，并常咳嗽，痰多，常有黄色脓痰，患者系足月顺产，母亲在怀孕时无感冒史。幼年曾患过肺结核，迅速治愈未曾复发过。父母弟妹均健康，发育良好。体检：发育差、身材瘦小，神清自动体位，唇甲无紫绀，浅表淋巴结不肿大，轻度材状指，鼻窦区压痛明显，颈静脉不充盈，胸廓无畸形，肺…     

老年人不稳定型Colles′骨折

Colles?骨折是一种常见的损伤,但关于该骨折治疗方法及效果的报道不尽相同。Sarmiento等人强调患者的功能恢复,认为骨折有某种程度的移位和再错位是可以接受的。其他学者则认为骨折的解剖     

Flow-cytometric enumeration of reticulocytes with the new fluorochrome 1′,3′-diethyl-4,2′-quinolylthiacyanine

Summary Several flow-cytometric methods for reticulocyte enumeration in whole blood have been developed, with different degrees of practical use. Recently, a new fluorochrome, 1,3-diethyl-4,2-quinolylthyacianine (DEQTC) was proposed in a brief report, as an alternative to thiazole orange for reticulocyte counting. We have evaluated the usefulness of this fluorescent stain by assessing the optimal conditions for the flow-cytometric analysis, and by comparing in double-blind assays the quantitative results of this technique with those obtained by manual counting with brilliant cresyl blue. Our results show that flow cytometry with DEQTC is highly correlated to the manual method (r=0.95–0.99), supporting the interest of this particular stain and of flow cytometry for routine laboratory work in hematology.     

Structural insights into the effects of 2′-5′ linkages on the RNA duplex

The mixture of 2′-5′ and 3′-5′ linkages generated during the nonenzymatic replication of RNA has long been regarded as a central problem for the origin of the RNA world. However, we recently observed that both a ribozyme and an RNA aptamer retain considerable functionality in the presence of prebiotically plausible levels of linkage heterogeneity. To better understand the RNA structure and function in the presence of backbone linkage heterogeneity, we obtained high-resolution X-ray crystal structures of a native 10-mer RNA duplex (1.32 Å) and two variants: one containing one 2′-5′ linkage per strand (1.55 Å) and one containing three such linkages per strand (1.20 Å). We found that RNA duplexes adjust their local structures to accommodate the perturbation caused by 2′-5′ linkages, with the flanking nucleotides buffering the disruptive effects of the isomeric linkage and resulting in a minimally altered global structure. Although most 2′-linked sugars were in the expected 2′-endo conformation, some were partially or fully in the 3′-endo conformation, suggesting that the energy difference between these conformations was relatively small. Our structural and molecular dynamic studies also provide insight into the diminished thermal and chemical stability of the duplex state associated with the presence of 2′-5′ linkages. Our results contribute to the view that a low level of 2′-5′ substitution would not have been fatal in an early RNA world and may in contrast have been helpful for both the emergence of nonenzymatic RNA replication and the early evolution of functional RNAs.The capacity of RNA to act as both a carrier of genetic information and as a catalyst has led many to investigate its potential role as the first biopolymer (1–4). An early stage involving nonenzymatic replication simplifies RNA-first scenarios, but known nonenzymatic copying reactions generate a mixture of 3′-5′ and 2′-5′ backbone linkages because of the similar nucleophilicity and orientation of the 2′ and 3′ hydroxyl groups on ribose (Fig. 1). Although regioselectivity for the 3′-5′ linkage can be improved by using different metal ions or activated monomers, it reaches, at most, ∼90% (5–11). This lack of regiospecificity has been regarded as a central problem for the emergence of the RNA world, because the resulting backbone heterogeneity was expected to disrupt the folding, molecular recognition, and catalytic properties of functional RNAs. However, we recently observed that functional nucleic acid molecules can still evolve in the presence of nonheritable mixed DNA/RNA backbone heterogeneity (12), and known functional RNAs retain catalytic and ligand binding behavior in the presence of 2′-5′/3′-5′ backbone linkage heterogeneity (13).Open in a separate windowFig. 1.Template-directed chemical incorporation of an activated monomer at the 3′ end of an RNA primer. (LG, leaving group: in contemporary biochemistry, LG = pyrophosphate, in model prebiotic reactions, LG = 2-methylimidazole). Extant enzyme polymerases produce homogeneous 3′-5′–linked RNA (left product), whereas known model prebiotic reactions produce both 3′-5′ and 2′-5′ (right product) linked RNA.The well-known duplex-destabilizing property of 2′-5′ linkages can enable thermal strand separation of long RNA duplexes in the presence of the high Mg²⁺ concentrations required for known prebiotic copying reactions (13–16). However, the mechanism responsible for this destabilization has not yet been satisfactorily elucidated, although a very preliminary modeling study has suggested that the reduced base overlap between adjacent intrastrand bases caused by the 2′-5′ linkage might be one of the reasons for the decreased T_m (17). In addition, the diminished chemical stability of this linkage in the duplex state has been suggested as a potential proofreading mechanism for linkage heterogeneity in prebiotic RNA synthesis (5). These observations, coupled with the fact that strands containing these linkages can still template RNA primer extension (18), suggest that, far from being a problem, 2′-5′ backbone linkages may have been an essential feature of early (pre)-RNA.Given the potential importance of mixed RNA backbone isomers in early evolution, we sought to elucidate the structural origins of the properties of mixed-backbone RNA duplexes. Although NMR structures of a homogeneously 2′-5′-linked DNA and RNA duplex as well as X-ray crystal structures of 2′-5′-linked dinucleotides have been reported (19–26), no crystallographic data are available on mixed-backbone RNA. Here we report high-resolution crystal structures of three RNA 10mer duplexes of the same self-complementary sequence, the first being native RNA, the second containing two, and the third containing six 2′-5′ linkages. These data, along with accompanying molecular dynamics simulations, provide clear structural insights into the origins of the above phenomena, as well as explaining how RNA duplexes adjust their overall and local structures to accommodate mixed regioisomers. Additionally, both duplexes containing 2′-5′ linkages crystallized more readily than the native RNA duplex, most likely due to additional interhelical interactions mediated by the surface exposed 3′-hydroxyl of the 2′-linked sugars, suggesting that the incorporation of 2′-5′ linkages into RNA structures may facilitate duplex packing and RNA crystallographic analysis.     

Human vascular smooth muscle cells express a constitutive nitric oxide synthase that insulin rapidly activates, thus increasing guanosine 3′ : 5′-cyclic monophosphate and adenosine 3′ : 5′-cyclic monophosphate concentrations

Aims/hypothesis. Insulin incubation of human vascular smooth muscle cells (hVSMC) for 120 min increases both guanosine 3′ : 5′-cyclic monophosphate (cGMP) and adenosine 3′ : 5′-cyclic monophosphate (cAMP) and these effects are blocked by inhibiting nitric oxide synthase (NOS). These data suggest that insulin activates a constitutive Ca²⁺-dependent NOS (cNOS), not described at yet in hVSMC. To test this hypothesis, we evaluated in hVSMC: i) the kinetics of the insulin-induced enhancement of the two cyclic nucleotides; ii) the ability of nitric oxide (NO) to increase both cyclic nucleotides; iii) NO involvement in the short-term influence of insulin on both cyclic nucleotides; iv) the ability of insulin to increase NO production in a few minutes; v) the presence of a cNOS activity; vi) the expression of mRNA for cNOS. Methods. In hVSMC incubated with insulin, NO donors and the Ca²⁺ ionophore ionomycin, we measured cAMP and cGMP (RIA); in hVSMC incubated with insulin and ionomycin we measured NO, evaluated as l-(³H)-citrulline production from l-(³H)-arginine; by northern blot hybridization, we measured the expression of cNOS mRNA. Results. i) By incubating hVSMC with 2 nmol/l insulin for 0–240 min, we observed an increase of both cGMP and cAMP (ANOVA: p = 0.0001). Cyclic GMP rose from 0.74 ± 0.01 to 2.62 ± 0.10 pmol/10⁶ cells at 30 min (p = 0.0001); cAMP rose from 0.9 ± 0.09 to 11.65 ± 0.74 pmol/10⁶ cells at 15 min (p = 0.0001). ii) Sodium nitroprusside (100 μmol/l) and glyceryltrinitrate (100 μmol/l) increased both cGMP and cAMP (p = 0.0001). iii) The effects of insulin on cyclic nucleotides were blocked by NOS inhibition. iv) An increase of NO was observed by incubating hVSMC for 5 min with 2 nmol/l insulin (p = 0.0001). v) Ionomycin (1 μmol/l) enhanced NO production (p = 0.0001) and increased both cyclic nucleotides (p = 0.0001). vi) hVSMC expressed mRNA of cNOS. Conclusion/interpretation. Human VSMC express cNOS, which is rapidly activated by insulin with a consequent increase of both cGMP and cAMP, suggesting that insulin-induced vasodilation in vivo is not entirely endothelium-mediated. [Diabetologia (1999) 42: 831–839] Received: 30 November 1998 and in revised form: 25 January 1999     

Tietze′s综合征52例分析

Tietze′s综合征即非特异性肋软骨炎,是指不明原因所引起的上部胸肋软骨及周围的软组织的疼痛性非化脓性肿痛的一组病症.是一种非化脓性肋软骨肿大,以青少年和女性多发[1].1984年-2009年共诊治52例Tietze′s综合征患者,现报道如下.     

Polymorphisms of Thymidylate Synthase in the 5′- and 3′-Untranslated Regions and Gastric Cancer

Studies investigating the association of polymorphisms in the 5′-untranslated regions (5′UTR) and 3′-untranslated regions (3′UTR) of thymidylate synthase with gastric cancer susceptibility and sensitivity to fluoropyrimidine-based chemotherapy report conflicting results. The objective of this study was to quantitatively summarize the evidence for such a relationship. This meta-analysis included ten studies, which included 1,730 gastric cancer cases and 1,843 controls. The combined results based on all studies showed that there was no significant difference in genotype distribution of 5′UTR or 3′UTR between gastric cancer and noncancer patients. When stratifying for race, we found that: (1) among Asians, patients with gastric cancer had significantly higher frequency of 2R/2R of 5′UTR than did noncancer patients, and (2) among Caucasians, patients with gastric cancer had significantly lower frequency of ins6/ins6 and higher frequency of ins6/del6 of 3′UTR than did noncancer patients. No significantly different response rate or survival of gastric cancer with fluoropyrimidine-based chemotherapy were observed with genotype distribution of 5′UTR or 3′UTR among Caucasians or Asians. This meta-analysis suggests that polymorphisms in the 5′UTR and 3′UTR of thymidylate synthase may be associated with gastric cancer susceptibility, but are not correlated with sensitivity of gastric cancer to fluoropyrimidine-based chemotherapy.     

Activated cyclophosphamide: An enzyme-mechanism-based suicide inactivator of DNA polymerase/3′–5′ exonuclease

Summary DNA polymerase I fromE. coli can toxify activated cyclophosphamide (CP) by means of the 3–5 exonuclease activity associated with the enzyme. Acrolein and an alkylating moiety are released in the process. Preincubation of DNA polymerase I with activated CP for 15–60 min leads to an increasing inhibition of DNA polymerase activity, which can be prevented when preincubation of DNA polymerase I with activated CP is carried out in the presence of 5 AMP, a competitive inhibitor of the 3–5 exonuclease subsite of the enzyme. This demonstrates that toxification of activated CP by the 3–5 exonuclease subsite of DNA polymerase is a prerequisite for the inhibition of DNA polymerase activity. The kinetics and the degree of DNA polymerase inhibition suggest that the alkylating moiety rather than acrolein released from activated CP during toxification is responsible for the inhibition of DNA polymerase. DNA polymerase with associated 3–5 exonuclease activity has also been isolated from eukaryotic cells, and toxification of activated CP by such an enzyme (DNA polymerase from rabbit bone marrow) has been shown previously. Thus we suggest that toxification of activated CP by DNA polymerases/3-5 exonucleases present mainly in proliferating cells might lead to the specific alkylation of macromolecules involved in the cell proliferation process, such as the DNA polymerase subsite of these enzymes and probably also the DNA bound to the enzymes. The relatively high cancerotoxic selectivity and cytotoxic specificity of activated CP could be based on this specific enzyme-mediated alkylation.Supported by the Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg     

慢性乙型肝炎2′，5′—寡腺苷酸合成酶活性与干扰素疗效的关系

探讨慢性乙型病毒性肝炎患者血液中单个核细胞的2′,5′-寡腺苷酸合成酶（2′,5′-OAS）活性与干扰素疗效的关系。为临床预测干扰疗效,避免盲目使用干扰素提供依据。将17例HBeAg、HBV-DNA双阳性的慢乙肝患者干扰素治前、治疗一周及治疗三月后进行2′,5′-OAS检测。结果示11例治前2′,5′-OAS活性较低,治疗一周有明显升高,其中8例HBeAg、HBV-DNA双项转阴,3例HBeAg单项转阴,其余6例治疗前2′,5′-OAS活性较高,治后1周升高幅度不大,双项均无转阴,提示2′,5′-OAS的水平可能是临床预测干扰素疗效的指标之一。     

微P′型持久性房性心动过速

慢性房性心动过速是房性心动过速(房速)的少见类型。文献报告慢性房速又分2个亚型,即持久性和慢性反复性房速,并且认为均多见于无器质性心脏病的青少年和儿童。本文报告3例与上述2个亚型不同的持久性房速。这3例的临床表现和心电图(ECG)特征有许多相似之处。临床和心电图资料例1 患者女性,53岁。心悸、气短、浮肿6年余。近3年反复住院4次,末次住院为1980年3月。全身明显浮肿,发绀,严重颈静脉怒张,肝大平脐。X线心影呈普大型,心胸比例0.78。临床诊断:原发性扩张型心肌病,全心扩大,心功能Ⅳ级。住院治疗半年,心衰症状无明显改善,自动出院。2月后死亡。图1A(见第170页,后同)中Ⅰ、Ⅱ、aVF导联的第3个QRS波为室性早搏。各导联P′波电压极小,难以辨认。图1B(不连续记录)中由于室率较慢     

Graves′病的介入栓塞治疗

Graves′病的介入栓塞治疗目前在国内外应用刚刚起步,但已取得了显著效果,使一些久治不愈或有某些禁忌症的甲亢患者病情得到缓解,开辟了治疗Graves′病的一条新途径。传统上,Graves′病有三种治疗方法:抗甲状腺药物治疗,放射性~(131)I治疗和手术治疗。每一种治疗各有优缺点。抗甲状腺药物治疗疗程长,治愈率低,易复发,可引起药物过敏和粒细胞缺少,限制了其应用范围。~(131)I治疗方便快捷,但甲状腺功能减退发生率高,需甲状腺素片终身替代治疗,在儿童、青少年和生育期妇女,甲减可影响生