NN304 [Lys(B29)-tetradecanoyl des(B30) human insulin] is a potentially therapeutic insulin analog designed to exhibit protracted glucose-lowering action. In dogs with infusion rates similar to insulin itself， NN304 exhibits similar glucose uptake (R(d)) stimulation with delayed onset of action. This compartmental modeling study was to determine if NN304 action could be accounted for by the approximately 2% unbound NN304 concentration. NN304 (or human insulin) (n = 6 each) was infused at 10.2 pmol center dot min(-1) center dot kg(-1) under euglycemic clamp conditions in anesthetized dogs. NN304 appearance in lymph， representing interstitial fluid (ISF)， was slow compared with insulin (t(1/2) = 70 +/- 7 vs. 14 +/- 1 min， P < 0.001). R(d) was highly correlated with the ISF concentration for insulin and NN304 (r = 0.86 and 0.93， respectively)， suggesting that slow transendothelial transport (TET) is responsible for sluggish NN304 action. Insulin and NN304 concentration data were fit to a two-compartment (plasma and ISF) model. NN304 plasma elimination and TET were reduced to 10 and 7% of insulin， respectively. Thus， there was reduction of NN304 transport， but not to the degree expected. In ISF， there was no reduction in NN304 elimination. Thus， this acylated insulin analog demonstrates blunted kinetics in plasma， and full efficacy in the compartment of action， ISF.
Mode of transcapillary transport of insulin and insulin analog NN304 in dog hindlimb: evidence for passive diffusion.
A in transcapillary of in skeletal muscle and adipose tissue has been proposed to play a role in the resistance that leads to ， yet the mechanism of transfer across the capillary endothelium from plasma to interstitium continues to be debated. This study examined in vivo the interstitial appearance of in hindlimb using the fatty acid acylated analog Lys()-des-(B30) ， or NN304， as a marker for . If the were a saturable process， then "swamping" the capillary endothelial with native would suppress the subsequent appearance in interstitial fluid of the analog NN304. This analog binds to with an affinity of about 50% of native . Experimental conditions established a physiologic NN304 dose in the absence or presence of pharmacologic and saturating concentrations of regular . Euglycemic clamps were performed in under inhalant anesthesia with deep hindlimb lymphatic sampling， representative of skeletal muscle interstitial fluid (). In group 1 (n = 8)， NN304 alone was infused (3.6 pmol center dot min(-1) center dot kg(-1)) from 60 to 360 min. In group 2 (n = 6)， starting at time 0， was infused at a pharmacologic dose (60 pmol center dot min(-1) center dot kg(-1)) with the addition of NN304 infusion (3.6 pmol center dot min(-1) center dot kg(-1)) from 60 to 360 min. In group 3 (n = 4)， the infusion was increased to a saturating dose (120 pmol center dot min(-1) center dot kg(-1)). Pharmacologic infusion (group 2) established steady-state concentrations of 6，300 plus minus 510 pmol/l in plasma and 5，300 plus minus 540 pmol/l in . Saturating infusion (group 3) achieved steady-state concentrations of 22，000 plus minus 1，800 pmol/l in plasma and 19，000 plus minus 1，500 pmol/l in . Total (bound and unbound) NN304 plasma concentrations rose from a steady state of 1，900 plus minus 110 (group 1) to 2，400 plus minus 200 pmol/l (group 2) and 3，100 plus minus 580 pmol/l (group 3)， consistent with a competition-driven decline in NN304 clearance from plasma as the level increased (P < 0.05 by ANOVA). Steady-state interstitial NN304 concentrations also rose with increasing levels but did not achieve significance in comparison with analog alone (162 plus minus 15 vs. 196 plus minus 22 and 241 plus minus 53 pmol/l for group 1 versus groups 2 and 3， respectively; P = 0.20)， yet the steady-state plasma：ratio for NN304 remained essentially unchanged in the absence and presence of elevated levels (12.6 plus minus 1.2 vs. 12.4 plus minus 0.5 and 13.1 plus minus 1.5 for group 1 versus groups 2 and 3， respectively; P = 0.93). Last， NN304 rate of appearance in interstitial fluid (i.e.， half-time to steady state) was similar between groups; mean half-time of 92 plus minus 4 min (NS between groups). In conclusion， appearance of the analog NN304 in skeletal muscle interstitial fluid was constant whether in the absence or presence of concentrations sufficient to saturate the endothelial . These findings support the hypothesis， provided that the mechanism of and NN304 transcapillary is similar， that transcapillary of in skeletal muscle occurs primarily via a nonsaturable process such as passive diffusion via a paracellular or transcellular route.
Biocore 304： Cellular Biology Laboratory-Spring2011ScheduleYour grade will be based on the assignments listed below. Note that the Subcellular Fractionation paper and Signal TransductionPoster are group grades. Check (√) assignments are scored simply adequate or inadequate; papers are graded using the criteriadescribed in theBiocore Writing Manual. Papers & assignments must be handed in on time unless you have contacted your TAaheadof timeto request an extension due to emergency or extenuating circumstances. Otherwise， we will deduct one grade per weekdayit islate from the grade you would have received (e.g.， A->AB for one day late). Note that even an F paper (one week late) counts morethan 0 (not handed in at all) when we total the final grades at the end of the semester. Late reports should be givendirectly to yourTA， Janet Batzli or Michelle Harris (not put in a mailbox or submitted electronically).Lecture topicsWeek/(date)Lab TopicLab Activities/AssignmentsWeightImaging， cellcomposition1 (Jan.18-21)Microscopy：Image FormationRealign messed up microscope–do in labCell observations/diagrams–do in labMacromoleculestructure/function，organelles，bioenergetics2 (Jan.24-28)SubcellularFractionationSubcellular Fractionation pre-labdue in disc.Microscopy assignment(produce hand drawingswith figure legends) due in lab2%3%Enzyme catalysis，membranes &transport， metabolicpathways & role ofATP3 (Jan31-Feb4)Enzyme I：AlkalinePhosphatase &MM KineticsEnzyme pre-lab assignment due in disc.Teams decide final[S]in discSubcell. Fract. Paper(1/pair)due in labGraphs & calculations for Km， Vmax–do in lab2%5%Glycolysis， anaerobicenergy metabolism， fatmetabolism， citric acidcycle4 (Feb.7-11)Enzyme II：ExtendedLiteratureSearch/Bioinformatics/StatisticsComplete BLAST&ClustalW eTeachesandJmoltutorial& turn in answers to questionsatbeginningof discExtended literature search(in disc)Data Analysis pre-lab Part I assignmentduebeginning of labPLAP physicalmodels; Consurf analysis(in lab)Molecular structure/function：E. coliAP mutationactivity(in lab)Introduce t-tests， online stats materials (in lab)Generateenzymequestion， brief rationale，hypothesis， tentative expt. design (1/team)Teams sign upfor formal presentation feedback2%Electron transport &ATP generation，chloroplast &photophosphoryl-ation， photosynthesis5 (Feb.14-18)Enzyme III：ProposalFeedbackPresentation /Pilot StudiesData Analysis pre-lab Part II：focus on yourenzyme protocol and expected data (due in disc).Focus on experimental design with team (in disc)Enzyme proposal feedback presentation (1/team) inlabEnzyme Research Proposaldue 48h afterpresentationEnzyme question/methods/schedule (1/team)2%7%Metabolic integration;DNA & chromosomestructure & replicationEXAM IWednesdayFeb.237：15pm6 (Feb.21-25)Enzyme IV：Final ExperimentFocus on Peer Review： our expectations (in disc.)–peer review practice exerciseEnzyme proposal returned with commentsTeams collect data (open lab)Transcription & RNAprocessing， translation& genetic code，mutation & DNArepair7 (Feb28-Mar4)MolecularGenetics I： IntrotoC. elegans，heat shockproteins， andRNAi / GFP tostudy geneexpressionPeer reviewconferenceFinal Enzyme papers indisc.Start to introduce Worm unit in disc， observewildtype wormsFinal Enzyme paper (13%)+peer review (2%)+author’s response + GEA formsdue in lab orlectureView Podcast on wormmorph(Part 1)& life-cycle，RNAi， GFP(Part 2)beforelab15%In-classstations：C. elegansgene expressionworksheetDevelop study question and tentative biorationaleBacterial genetics，transposable elements8 (Mar7-11)MolecularGenetics II：Developingexperiments ongene expressioninC. elegansWorm stress/gene expression+ statistics(ANOVA， Chi-square)Prelabdue in disc.Worm proposal feedback presentationsin labMaterials and schedule sheet-detailing how， when，and who will perform each step of experimentMid-semester evaluations3%Spring Recess March 14-18Genetic engineering，prokaryotic generegulation9 (Mar21-25)MolecularGenetics III：WormexperimentsWorm proposal mini-poster peer review indiscussionWorm research proposalposterdue in labDo worm experiments-establish treatments， pilotstudies，and RNAi set up， gather data8%Prokaryotic &eukaryotic generegulation， genomics10(Mar.28-Apr1)MolecularGenetics IV：DataAnalysisand PosterDevelopmentBiocore 324 preview in lab (in disc.)Worm data collectionand poster developmentWorm data analysis(chi-square)： what do your dataindicate?Worm proposal posters returned with commentsProtein trafficking，cellsignalingEXAM IITuesdayApr.57：15pm11(Apr.4-8)SignalTransduction I：Intro to YeastSystemDo genetic transformations in discussion，introduction to cell shape andB-gal assayGenerate tentative question for Sign. Trans project(1/team) due inlabC. elegansmini-posters + GEAduein Fri. lecture15%G-linked receptors，second messengers，growth factors &receptor tyrosinekinases12(Apr.11-15)SignalTransduction II：PresentProposals (disc& lab);Signal Transduct. prelabdue in disc.Signal Transduction feedback presentation (1/team)in labIntro to 2-way ANOVA during presentationsMaterials & schedule for signal transduction project(1/team) due in lab3%Ras signaling，hormones， cytoskeletalproteins13(Apr.18-22)SignalTransduction III：Pilot Studies &Your ExperimentPeer reviewers meet to discuss signal transductionproposal papers (in disc)Signal Transduction Proposal Paper (13%) +peer review (2%)+ author’s response–dueWedlecture orlabData collection I in lab15%Cell movement， celladhesion， cell cycle14(Apr.25-29)SignalTransduction IV：Data AnalysisGenetics Concept Assessment in discData collection II in labCell cycle & apoptosis，cancer15(May2-6)SignalTransduction V：Poster Presen.Practice presentation & group poster peer reviewduringdiscussionSignal transduction Posters FormalPresentations (1/team)+ GEAformsFinal course evaluation15%Team work， class participation and checkassignments3%Grade BreakdownPercentageLetter Grade90-100A80-89B70-79CAssignments， due dates， and individual assignment weight in percent are detailed above.Biocore 304 is aComm-B course and therefore is writing intensive. Grades on assignmentswill not be curved. For those few individuals that are on the borderline at the end of thesemester， we will assign intermediate grades (AB and BC) based on our evaluation of yourparticipation (in both lab AND discussion)， teamwork and your effort.60-69DPercentageLetter Grade90%A80%B70%C60%DPercentageLetter Grade90-100A80-89B70-79C60-69D
摘 要： 目的评价304rim中波高能紫外线联合复方甘草酸苷片治疗斑秃的临床疗效和安全性。方法将入选的84例斑秃患者随机分为两组，各42例。全部患者予304nm中波高能紫外线照射，2次／周.治疗组还予口服复方甘草酸苷片50mg，对照组还予口服胱氨酸片100mg，均为3＆／d。治疗8周后评价临床疗效。结果治疗组有效率为73.81％，对照组有效率为52.38％，两组比较差异有统计学意义（P〈0.05）。二者均无严重的不良反应。结论304nm中波高能紫外线联合复方甘草酸苷片治疗斑秃疗效好.且无明显不良反应。
Berberine Inhibiting Staphylococcus Aureus-Induced Apoptosis in ECV-304 Cells via Regulating Bcl-2,Bax and Caspase-3 Expressions
Objective： To study the protection of berberine against ECV-304 apoptosis induced by Staphylococcus aureus( S.aureus) via regulating Bcl-2，Bax and Caspase-3 expressions and to discuss the mechanism that berberine inhibits apoptosis of ECV-304 cells induced by S. aureus. Methods： ECV-304 was pretreated with 128 μg / m L BBR for 2 h，and then S aureus( 1∶100) was added. The expression levels of apoptosis related genes were detected with semi-quantity RT-PCR and Western-blot. Results： Pretreatment of BBR helped to survive in S aureus-induced ECV-304. BBR significantly increased the expression levels of Bcl-2 and decreased that of Bax and Caspase-3 after staphylococcus aureus infection. Conclusion： Berberine inhibits staphylococcus aureus-induced apoptosis in ECV-304 cells via regulating Bcl-2，Bax and Caspase-3 expressions. This study provides evidence for clinical treatment.
研究新型紫杉烷类化合物 NPB304逆转肿瘤多药耐药的作用。MTT法测定化疗药物的IC50，Western blotting方法分析P-糖蛋白(P-gp)的表达，分别通过罗丹明123(Rh123)蓄积实验和分析试剂盒测定化合物对P-gp功能及P-gp ATPase活性的影响，采用分子对接预测化合物与P-gp结合能力的强弱，用MDCKⅡ和MDR1-MDCKⅡ细胞模型分析NPB304的跨膜转运。在 KBV细胞中，NPB304具有多药耐药逆转作用;在MCF-7/paclitaxel细胞中，NPB304协同P-gp抑制剂维拉帕米增强逆转耐药的活 性，10μmol·L-1维拉帕米与paclitaxel合用时逆转倍数为56.5倍，合用NPB304增加耐药逆转倍数;NPB304与维拉帕米合用时 协同增加Rh123在耐药细胞中的蓄积，NPB304(0~1μmol·L-1)增强维拉帕米激活P-gp ATPase活性的作用;NPB304与P-gp的TM区存在疏水相互作用，与TM区A链的结合力较强;NPB304在较低浓度 (0~1.5μmol·L-1)时激活P-gp ATPase，发挥抑制P-gp功能的作用，但不具有明显的P-gp底物特征。NPB304通过抑制P-gp的功能活性发挥自身及协同维拉帕米逆转耐药的 作用。
目的 了解胆道感染时胆汁中致病菌菌群及耐药变化趋势.方法将2006年1月-2009年12月培养阳性的269例胆汁标本，共304株病原菌及药敏结果进行统 计分析.结果 304株细菌中包含了52种细菌，其中革兰阴性杆菌占59.5%，革兰阳性球菌占40.5%，排列前5位的细菌依次为：大肠埃希菌20.4%、粪肠球菌 16.8%、肺炎克雷伯菌11.2%、铜绿假单胞菌8.9%、阴沟肠杆菌7.9%;细菌对青霉素类、头孢类、喹诺酮类、大环内酯类抗菌药物敏感性普遍下 降，对亚胺培南、美罗培南、万古霉素、替考拉宁仍保持相当高的敏感率.结论胆道感染菌种在增加，菌群在变化，对常用的抗菌药物耐药率在增加;治疗胆道感染 应动态监测菌群和药物敏感性的变化及合理应用抗菌药物.
The tumor multidrug resistance reversal effect of NPB304， a novel taxane， was studied. MTT assay was used to determine the IC50 of chemotherapy drugs. Western blotting assay was applied to analyze the expression of P-glycoprotein(P-gp). The effect of compounds on the P-gp function and P-gp ATPase activity was determined by rhodamine 123(Rh123) accumulation assay and analysis kit， respectively. Molecular docking was employed to predict the binding force between compounds and P-gp. Transmembrane transport of NPB304 was analyzed using MDCKⅡ and MDR1-MDCKⅡ cell model. NPB304 displayed multidrug resistance reversal effect on KBV cells and MCF-7/paclitaxel cells， NPB304 collaborative with P-glycoprotein(P-gp) inhibitors verapamil enhanced the reversal activity， specifically， 10 μmol·L-1 verapamil in combination with paclitaxel reversed resistance by 56.5-fold， while combined with NPB304 increased the reversal fold; NPB304 synergistically increased Rh123 accumulation in the resistant cells when combined with verapamil， and NPB304 at 0-1 μmol·L-1 enhanced the ATPase activity activated by verapamil was observed. NPB304 existed the hydrophobic interactions with the TM regions of P-gp， and the binding force between NPB304 and the A chain of the TM region was stronger. P-gp ATPase activity assay demonstrated NPB304 at lower concentrations(0-1.5 μmol·L-1) could activate the P-gp ATPase， playing a role on inhibition of P-gp function. However， NPB304 did not have an obvious feature of P-gp substrate. NPB304 exerted itself and synergy with verapamil activity on reversing tumor resistance via inhibiting the P-gp function.
采用计算机检索和人工检索相结 合方法，对1994～2011年糖尿病相关文献进行收集整理，共收集合格文献304篇，其中中医治疗糖尿病140篇，中西医结合治疗糖尿病164篇。运用 统计学方法对收集文献进行分析，中医治疗糖尿病出现频率较多的证型依次为血瘀证(44.29%)、气阴两虚证(39.29%)、阴虚燥热证 (25.00%)、肾阴虚证(11.43%)、脾气虚弱证(11.43%)、阴阳两虚证(9.29%)、肾气亏虚证(7.86%)、阴虚火旺证 (7.14%)、痰湿证(5.00%)，中西医结合治疗糖尿病出现频率较多的证型依次是血瘀证(53.05%)、气阴两虚证(31.10%)、阴虚燥热证 (17.07%)、阴阳两虚证(10.37%)、气虚证(8.54%)、脾肾两虚证(7.93%)、痰湿证(5.49%)，初步总结出糖尿病证候分布情 况，即血瘀证、气阴两虚证、阴虚燥热证为糖尿病的主要证型，治疗应益气养阴、清热润燥、活血化瘀为主。由于现代糖尿病血瘀证明显增多，在发病中所占的比例 最高，其预防应注意调整身心状态、饮食清淡、保精养津。
Objective To study the effect Panax Notoginseng Saponins(PNS) on proliferation of ECV304 cells.Methods The proliferation of ECV304 cells were evaluated by MTT assay.The expression levels of VEGF and KDR mRNA in ECV304 cells were detected by real-time quantitative polymerase chain reaction.The expression levels of ERK1/2 and pErk1/2 were detected by Western blot.Results Compared with the control group，PNS(100，200，400，800mg/L) promoted the proliferation of ECV304 cells in a dose-dependent manner obviously(P0.05).The proliferation rates of ECV304 cells induced by 48h PNS were 112.44%±5.64%，117.20%±11.05%，134.34%±0.40% and 141.45%±5.49 %，respectively.Compared with the control group，the relative quantity of VEGF expression and KDR mRNA expression in ECV304 cells treated with PNS were increased significantly(P0.05).Western blot results showed that PNS increased the expression of proliferation-related protein pERK1/2.Conclusion The PNS can promote the proliferation of ECV304 cells，the mechanism of which may be related with up-regulating the expression of VEGF，KDR和pERK1/2.