Isolation and mapping of plasmids containing the Salmonella typhimurium origin of DNA replication.

A purified EcoRI restriction endonuclease fragment that determines resistance to kanamycin and is incapable of self-replication was used to select autonomously replicating fragments from an EcoRI digest of a Salmonella typhimurium F' plasmid containing the chromosomal region believed to include the S. typhimurium origin of DNA replication. Both the F factor and S. typhimurium chromosome replication origins were cloned by this procedure. The EcoRI fragmentment containing the S. typhimurium origin of replication is 19.4 kilobase pairs long and includes functional asp+ and uncB+ genes. Restriction endonuclease analysis of deletions obtained from the S. typhimurium origin plasmid indicated that the replication origin (ori region) is contained within a 3.3-kilobase pair region. Comparison with Escherichia coli origin plasmids shows colinearity of gene arrangement on the chromosomes in this region and suggests that some, but not all, regions of the nucleotide sequence in the origin region may be conserved (identical) in these two bacterial species.     

Conduction characteristics at the crista terminalis during onset of pulmonary vein atrial fibrillation

INTRODUCTION: Focal atrial fibrillation (AF) may initiate with an irregular rapid burst of atrial ectopic (AE) activity from a pulmonary vein (PV) focus, but how AF is maintained it is not known. The crista terminalis (CT) is an important line of block in atrial flutter (AFL), but its role in AF has not been determined. The aim of this study was to examine the conduction properties of the CT during onset of AF. METHODS AND RESULTS: In 10 patients (mean age 38 +/- 8 years), we analyzed conduction across the CT during onset of focal AF from an arrhythmogenic PV and during pacing from the same PV at cycle lengths of 700 and 300 ms. A 20-pole catheter was positioned on the CT using intracardiac echocardiography. In 10 control patients with no history of AF, we analyzed conduction across the CT during pacing from the distal coronary sinus at 700 and 300 ms. In all 10 AF patients, AF was initiated with 1 to 9 AE beats (median 5) from a PV. During sinus rhythm, there were no split components (SC) recorded on the CT. During PV AE activity, discrete SC were recorded on the CT in all patients over 6.3 +/- 0.9 bipoles (3.7 +/- 0.3 cm). Maximal splitting of SC was 66 +/- 31 ms (37-139). There was an inverse relationship between AE coupling intervals and the degree of splitting between SC in all patients. Degeneration to AF was preceded by progressive decrement across the CT. SC were recorded during PV pacing at 700 and 300 ms (maximal distance between SC of 24 +/- 3 ms and 43 +/- 5 ms, respectively, P < 0.001). Maximum SC at CT in controls was 13 +/- 8 ms at 700 ms (P = 0.06 vs AF patients) and 16 +/- 9 ms at 300 ms (P < 0.01 vs AF patients). CONCLUSION: (1) These observations provide evidence of anisotropic, decremental conduction across the CT during onset of focal AF and during pacing from the same PV. A line of functional conduction block develops along this anatomic structure (CT). Whether this line of block acts as an initiator of AF or simply contributes passively to nonuniform fibrillatory conduction is unknown. (2) In some patients with focal AF, development of conduction block along the CT may provide a substrate for typical AFL.     

First Psychometric Properties of the Dutch Interview for Diagnostic Assessment of Autism Spectrum Disorder in Adult Males Without Intellectual Disability

Journal of Autism and Developmental Disorders - For autism spectrum disorder (ASD) in adults there are several diagnostic instruments available with a need for consideration of the psychometric...     

A Protein Kinase A–Independent Pathway Controlling Aquaporin 2 Trafficking as a Possible Cause for the Syndrome of Inappropriate Antidiuresis Associated with Polycystic Kidney Disease 1 Haploinsufficiency

Renal water reabsorption is controlled by arginine vasopressin (AVP), which binds to V2 receptors, resulting in protein kinase A (PKA) activation, phosphorylation of aquaporin 2 (AQP2) at serine 256, and translocation of AQP2 to the plasma membrane. However, AVP also causes dephosphorylation of AQP2 at S261. Recent studies showed that cyclin-dependent kinases (cdks) can phosphorylate AQP2 peptides at S261 in vitro. We investigated the possible role of cdks in the phosphorylation of AQP2 and identified a new PKA-independent pathway regulating AQP2 trafficking. In ex vivo kidney slices and MDCK-AQP2 cells, R-roscovitine, a specific inhibitor of cdks, increased pS256 levels and decreased pS261 levels. The changes in AQP2 phosphorylation status were paralleled by increases in cell surface expression of AQP2 and osmotic water permeability in the absence of forskolin stimulation. R-Roscovitine did not alter cAMP-dependent PKA activity but specifically reduced protein phosphatase 2A (PP2A) expression and activity in MDCK cells. Notably, we found reduced PP2A expression and activity and reduced pS261 levels in Pkd1^+/− mice displaying a syndrome of inappropriate antidiuresis with high levels of pS256, despite unchanged AVP and cAMP. Similar to previous findings in Pkd1^+/− mice, R-roscovitine treatment caused a significant decrease in intracellular calcium in MDCK cells. Our data indicate that reduced activity of PP2A, secondary to reduced intracellular Ca²⁺ levels, promotes AQP2 trafficking independent of the AVP–PKA axis. This pathway may be relevant for explaining pathologic states characterized by inappropriate AVP secretion and positive water balance.In most mammals, regulation of water balance is critically dependent on water intake and excretion, which is under control of the antidiuretic hormone arginine vasopressin (AVP). In the kidney, AVP binds to the V2 vasopressin (V2R) receptor, activating the cAMP/protein kinase A (PKA) signal transduction cascade, promoting the fusion of intracellular vesicles containing aquaporin 2 (AQP2) to the apical plasma membrane, and increasing luminal permeability.^1–3 This translocation is accompanied by AVP-dependent phosphorylation of AQP2 at serine-256 (pS256).Mice in which S256 could not be phosphorylated (AQP2-S256L) develop polyuria and hydronephrosis because of a defect in AQP2 trafficking to the plasma membrane.⁴ Interestingly, it connects to polycystic kidney disease (PKD). Mutations in polycystin-1 (Pkd1^+/−) gene cause PKD, whereas PKD1 haplo-insufficient mice (Pkd1^+/−), showing an inappropriate antidiuresis, display significantly higher levels of pS256 compared with wild-type (WT) littermates; the prominent expression at the apical plasma membrane of collecting duct principal cells, despite normal V2R expression and normal cAMP levels, is associated with unchanged AVP expression in the brain, despite chronic hypo-osmolality.⁵These observations underscore the crucial role of AQP2 phosphorylation at S256 in controlling the cellular distribution and fate of AQP2.^1,6,7 As for many proteins, the function and the trafficking of AQP2 are modulated by a balance of reversible phosphorylation and dephosphorylation. Preventing dephosphorylation of AQP2 with okadaic acid, inhibitor of phosphatase 1 (PP1), inhibitor of phosphatase 2A (PP2A), and inhibitor of phosphatase 2B (PP2B) significantly increased AQP2-pS256.⁸ Proteomic analysis of inner medulla collecting duct identified PP2A as a phosphoprotein isolated from inner medullary collecting duct samples treated with either calyculin-A, a specific PP2A inhibitor, or vasopressin,⁹ suggesting the possible participation of this phosphatase in cellular events triggered by physiologic stimulus, such as vasopressin in renal collecting duct cells.The complexity of AQP2 regulation was further increased by phosphoproteomics studies showing that, other than S256, vasopressin modulates the phosphorylation status of three other sites within the C terminus (S261, S264, and S269). Although vasopressin increases S264 and S269 phosphorylation, it decreases S261 phosphorylation.^9–12 Regarding the potential kinases responsible for the phosphorylation of these sites, c-Jun N-terminal kinase, p38, and cyclin-dependent kinases (cdks) cdk1 and cdk5 can phosphorylate AQP2 peptides at S261 in vitro.^13,14 Here, in the attempt to investigate the potential involvement of cdks in AQP2 regulation, we discovered a new PKA-independent signal transduction pathway regulating AQP2 phosphorylation and localization. We found that selective inhibition of cdks with R-roscovitine is associated with a decrease of intracellular Ca²⁺ levels and a significant downregulation of the phosphatase PP2A activity, resulting in an increase of AQP2 phosphorylation at S256 and targeting to the apical membrane. Physiologically, this novel regulatory mechanism might be of clinical interest, because it better elucidates the molecular bases of pathologic states characterized by disturbances in water balance.     

Development of a catadioptric endoscope objective with forward and side views

Autofluorescence endoscopy is a promising functional imaging technique to improve screening of pre-cancerous or early cancer lesions in the gastrointestinal (GI) tract. Tissue autofluorescence signal is weak compared to white light reflectance imaging. Conventional forward-viewing endoscopes are inefficient in the collection of light from objects of interest along on the GI luminal wall. A key component of a complete autofluorescence endoscope is the light collection module. In this paper, we report the design, optimization, prototype development, and testing of an endoscope objective that is capable of acquiring simultaneous forward and radial views. The radial-view optical design was optimized for a balance between image quality and light collection. Modulation transfer function (MTF), entrance pupil radius, manufacturability, and field-of-view were parameters used in the lens optimization. In comparison with the typical forward-viewing endoscopes, our nonsequential ray trace simulations suggest the proposed radial-view design is more practical in the light collection. To validate the proposed simulation methods, a 3:1 scaled-up prototype was fabricated. Contrast measurements were taken with the prototype, and then compared with the simulated MTF.     

甘草酸对大鼠体内芍药苷药代动力学的影响

目的:建立一种简单、快速的HPLC方法用于研究在大鼠体内甘草酸对芍药苷药代动力学特性的影响.方法:采用反相高效液相色谱及内标法测定大鼠血浆中芍药苷浓度,实验动物分为对照组和实验组,对照组单独给予芍药苷,实验组给予芍药苷和甘草酸,尾静脉注射给药,应用WinNonlin软件以非房室模型计算药代动力学参数.结果:Auc(min·mg·L~(-1)),V_d(mL·kg~(-1))、CL(mL·min~(-1)·kg~(-1))对照组分别为166.81±26.94,394.33±29.52,18.40±3.12,实验组分别为235.44±46.48,266.63±48.43,13.16±2.59.结论:静注甘草酸可以增加芍药苷的AUC,降低CL,减小V_d(P<0.05),对其他药代动力学参数没有影响,推测可能是影响了其代谢和排泄.