Role of the 2 adenine (g.11293_11294insAA) insertion polymorphism in the 3' untranslated region of the factor VII (FVII) gene: molecular characterization of a patient with severe FVII deficiency

Polymorphic variants in the gene encoding factor VII (F7) affect the plasma levels of this coagulation protein and modify the clinical phenotype of FVII deficiency in some patients. In this study we report the in vitro functional analysis of a novel polymorphic variant located in the 3' untranslated region of F7: g.11293_11294insAA. To determine whether this variant regulates FVII expression, we initially compared an expression vector containing FVII cDNA with g.11293_11294insAA with the FVII wild-type (WT) construct. The kinetics of mRNA production showed that the insertion decreases the steady-state FVII mRNA levels. To assess whether the insertion influences the phenotype of FVII-deficient patients, we evaluated its effect on the expression of FVII in a patient with severe FVII deficiency (undetectable FVII activity and antigen) carrying two additional homozygous missense variations (p.Arg277Cys and p.Arg353Gln). The two substitutions alone reduced the expression of FVII activity and antigen in vitro, but with the insertion polymorphism in our expression vector the patient's phenotype of undetectable plasma FVII was recapitulated. The insertion polymorphism in the 3' untranslated region of F7 is another modifier of FVII expression that might explain the poor genotype-phenotype correlation in some FVII-deficient patients.     

Effects of selective α1 and α2 adrenoceptor active drugs on 86Rb+ efflux from pieces of rat parotid gland

Minute pieces of rat parotid gland were used in studies of adrenergic regulation of K+ efflux using ⁸⁶Rb+ as a probe for K+. Noradrenaline induced a concentration-dependent Rb+ efflux, whereas the β₁-selective agonist prenalterol was without effect. On the other hand, the β₂-selective drug, terbutaline, at high concentrations displayed a small enhancement of Rb+ -secretion. The selective α₁-adrenoceptor drug, phenylephrine, was as potent as noradrenaline, whereas the α₂-agonist clonidine had only a small effect. The noradrenaline-induced Rb+-efflux was effectively inhibited in the presence of prazosin, an α₁-blocker, whereas the α₂-antagonist, yohimbine, was roughly 50 times less potent. The results suggest that catecholamine-induced K+-secretion from the rat parotid gland is mediated via activation of post-synaptic α-adrenoceptors of the α₁-subtype.     

Isolation and characterization of a cDNA coding for a novel human 17.3K myelin basic protein (MBP) variant

Human fetal spinal cord poly A (+) mRNA was found to direct the synthesis of three major myelin basic protein (MBP) variants with molecular weights of 17K, 18.5K, and 21.5K when translated in reticulocyte lysates. In order to investigate the structural relationships between these MBP variants and their corresponding mouse variants, human fetal spinal cord and mouse brain cDNA libraries were constructed and screened for MBP cDNAs. A number of MBP cDNA clones were isolated and characterized. One of these, PP535 contained the entire coding region of the mouse 14K MBP; and another mouse cDNA clone, PP1.85, was almost full-length and coded for either the 21.5K MBP or the 18.5K MBP. A human clone (KK36), 1,173 nucleotides in length, contained the entire coding region of an MBP variant with a molecular weight of 17,342. The structure of this clone within its coding region is significantly different from the corresponding mouse 17K MBP cDNA. It is missing two sequences found in the mouse 17K MBP cDNA (exons 2 and 5); and it contains a sequence (exon 6) that is missing from the mouse 17K MBP cDNA. Thus, this human 17.3K cDNA codes for a "17K" human MBP variant that is quite different from the corresponding mouse variant and is identical to the human 18.5K MBP except for a deletion of a peptide consisting of 11 amino acids that includes the single tryptophan residue of the 18.5K MBP. An analysis of the structure of this 17.3K human MBP cDNA suggests that the major pathway for splicing the primary human MBP gene product may be different from that in the mouse.     

Second dose of the BNT162b2 mRNA vaccine: Value of timely administration but questionable necessity among the seropositive

This study monitored titers of neutralizing IgG against the receptor-binding domain of the SARS-CoV-2 S1 subunit 14 days post-injection of each dose of the BNT162b2 mRNA Covid-19 vaccine in 401 Greek healthcare workers aged 20–67. After the first dose, titers varied upon age and history of infection, being lower in the 50+ age group and significantly higher among the seropositive. After the second dose, immunogenicity was significantly boosted in the age 50+ and SARS-CoV-2-naïve individuals, indicating the effectuality of its timely administration, yet questioning its value among the seropositive.     

用非同位素原位杂交组化在光镜和电镜水平观察前阿黑皮素mRNA在垂体的分布

用地高辛标记反意前阿黑皮素(pro-opiomelanocortin,POMC)cRNA探针原位杂交组化在光镜和电镜水平观察了POMC mRNA在大鼠垂体的分布,并比较了碱性磷酸酶(AKP)显色系统和辣根过氧化酶(HRP)显色系统在光镜水平上的敏感性.结果:POMC mRNA广泛地分布于垂体的中间叶和前叶.中间叶全部细胞均为POMC mRNA阳性.前叶中除前叶的腹侧缘和前叶与中间叶交界处阳性细胞较少外,都有较多的POMC mRNA阳性细胞分布.AKP显色系统比HRP显色系统敏感.在电镜水平,POMC mRNA主要分布于粗面内质网,少数分泌颗粒可能呈阳性反应,胞核未见阳性反应沉淀.文内还就阳性分泌颗粒在调节细胞合成功能方面可能起的作用进行了讨论.     

A Study of Rb Gene in Oral Squamous Cell Carcinoma

Thefirsttumorsuppressorgenemolecularlyisolatedistheretinoblastoma(Rb).ThedeletionofRbplaysanessentialroleinRetinoblastoma[1].TheosteosarcomaisalsoassociatedwiththedoublelossoftheRbgene[2].AbnormalitiesinthestructureandexpressionoftheRbgenehadbeenidentifiedinsmallcellcanceroflung[3]andbreastcancers[4].ThesefindingssuggestthattheRbgeneisapleiotropicsuppressorgene.Oralsquamouscellcarcinomaisthecommonmalignanttumor,whichaccountsfor80%ofthetotalofmalignanttumorsinoralmaxillofacialregion.Theprog…     

间歇性气囊挤压大鼠腿部对挤压部位和远端骨骼肌一氧化氮合酶mRNA表达的影响

目的:为了进一步了解间歇性气囊挤压法(Intermittent pneumatic compression, IPC)挤压大鼠腿部与一氧化氮(NO)的关系.方法:检测了大鼠骨骼肌中3种一氧化氮合酶(NOS)同工酶:神经型NOS(nNOS);诱导型NOS(iNOS)和内皮细胞型NOS(eNOS)mRNA在IPC作用后的表达变化.25只SD大鼠被随机分为3个模拟实验组和4个IPC实验组.每只鼠取右侧胫前肌(AT)和提睾肌(CM)作为正常对照.IPC组挤压0.5,1,和5h,及挤压5h加等待4h,模拟实验组除不挤压外,其他操作均与实验组相同,然后分离左侧AT和CM作为处理后样品.所有样品应用RT-PCR进行NOS mRNA测定.以样品中看家基因2,3-二羟基丙醛-3-磷酸脱氢酶(GAPHD)cDNA为内参,与NOS cDNA 共同扩增.PCR产物电泳条带密度用NIH图像分析软件定量,并以与正常对照的对比值作为变化比率.结果:在IPC作用0.5、1和5h后,eNOS mRNA显著上升,在AT中分别达到正常对照的1.2,1.8和2.6倍;在CM中分别达到1.2,1.8和2.7倍,而其他NOS,除5hIPC组的nNOS外,总体表现下调.在IPC作用1h加等待4h组中,eNOS mRNA回复至正常对照水平.结论:该结果证实了IPC产生的机械压力至少部分增加了血管壁的剪切压,使内皮细胞增加了NO产物的释放量,导致了挤压部位及远端肌肉的血管扩张和改善了微循环.     

Functional expression of the renal organic cation transporter and P-glycoprotein inXenopus laevis oocytes

The hypothesis that P-glycoprotein (P-gp) mediates the renal secretion of organic cations was tested by functional expression of mRNAs in theXenopus laevis oocyte system. Efflux of 2-deoxytubercidin (dTub), a substrate for the renal organic cation transporter (OCT) but not for P-gp, was enhanced by injection of renal mRNA but not by injection of mRNA from P-gp-overexpressing cells (MDCK cells transduced with the cDNA for humanMDR1). The functional capacity of the MDCK-MDR mRNA was established by its ability to reduce, the steady-state uptake of a classical P-gp substrate, vinblastine. Thus, these data indicate OCT and P-gp to be distinct entities. TheXenopus oocyte system provides a functional approach to further characterize the OCT.Work supported by NIH Grant RO1DK41606 from the Institute for Digestive Diseases and Kidney, and NIH Cancer Center Core Grant, CA 16672 from the National Cancer Institute     

CARD In Situ Hybridization: Sights and Signals

During the last decade, several strategies have been developed to improve the detection sensitivity ofin situ hybridization (ISH) by amplification of either target nucleic acid sequences prior to ISH (e.g.,in situ PCR), or the detection signals after the hybridization procedures (signal amplification). Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, summarize applications as well as possible limitations of CARD ISH, and discuss some future directions ofin situ nucleic acid detection using this amplification strategy.