Structural Studies on the Reaction of Isopenicillin N Synthase with a Sterically Demanding Depsipeptide Substrate Analogue

Isopenicillin N synthase (IPNS) is a nonheme iron(II)‐dependent oxidase that catalyses the central step in penicillin biosynthesis, conversion of the tripeptide δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐valine (ACV) to isopenicillin N (IPN). This report describes mechanistic studies using the analogue δ‐(L ‐α‐aminoadipoyl)‐(3S‐methyl)‐L ‐cysteine D ‐α‐hydroxyisovaleryl ester (A^SmCOV), designed to intercept the catalytic cycle at an early stage. A^SmCOV incorporates two modifications from the natural substrate: the second and third residues are joined by an ester, so this analogue lacks the key amide of ACV and cannot form a β‐lactam; and the cysteinyl residue is substituted at its β‐carbon, bearing a (3S)‐methyl group. It was anticipated that this methyl group will impinge directly on the site in which the co‐substrate dioxygen binds. The novel depsipeptide A^SmCOV was prepared in 13 steps and crystallised with IPNS anaerobically. The 1.65 Å structure of the IPNS–Fe^II–A^SmCOV complex reveals that the additional β‐methyl group is not oriented directly into the oxygen binding site, but does increase steric demand in the active site and increases disorder in the position of the isovaleryl side chain. Crystals of IPNS–Fe^II–A^SmCOV were incubated with high‐pressure oxygen gas, driving substrate turnover to a single product, an ene‐thiol/C‐hydroxylated depsipeptide. A mechanism is proposed for the reaction of A^SmCOV with IPNS, linking this result to previous crystallographic studies with related depsipeptides and solution‐phase experiments with cysteine‐methylated tripeptides. This result demonstrates that a (3S)‐methyl group at the substrate cysteinyl β‐carbon is not in itself a block to IPNS activity as previously proposed, and sheds further light on the steric complexities of IPNS catalysis.     

将数字印刷新策略转变为利润

正如我们在美国所说的,再没有什么比变化更持续.近段时期这种说法更是得到了验证,因为似乎我们的世界每天都有许多方面在发生变化.除了政治局势和气候状况的变化,印刷业发生的变化也比任何时候都来得迅猛.不久前,我们以很高的价格销售了高质量的印刷品,建立了业务.然后我们通过个性化直销市场的销售赚了些钱,业务包括一对一的促销信件、个性化网址(PURLs)和更多的互动工具将印刷材料连接到高度相关的互动营销渠道上.然后,我们的世界又改变了,因为全球消费者开始越来越多地使用社会媒体渠道.     

Isolation of natural arachidonic acid as its methyl ester

Summary Fresh beef suprarenal glands were ground and extracted thoroughly with alcohol and then with ethyl ether. After removal of solvent the total lipid residue was saponified, and the fatty acids were recovered by extraction. The less unsaturated acids were removed by crystallization from acetone at −40°C. At this stage the filtrate contained essentially all the arachidonic acid originally present in the glands and also unsaponifiable matter. After the unsaponifiable material was removed, the arachidonic acid content of the concentrate was about 25%. These unsaturated acids were converted to their methyl esters and fractionated on a silicic acid adsorption column. The progress of the adsorption fractionation was followed by spectrophotometric examination and determination of iodine values of the eluted fractions. Methyl arachidonate of 90% purity was obtained by this means. It was further purified by fractional distillation in vacuo. The final product had an iodine value of 316.1; theory, 318.8. The purity of this preparation was further established by spectrophotometric examination, by saponification equivalents, mean molecular weight, and by x-ray diffraction patterns and melting points of a completely hydrogenated portion. Evidence of acids of greater unsaturation than arachidonic acid in suprarenal lipids was also clearly established by spectrophotometric examination. A fraction was obtained which was estimated to contain about 80–85% of a C₂₀ acid with five double bonds. Presented at the 41st Annual Meeting of the American Oil Chemists’ Society, Atlanta, Ga., May 1–3, 1950. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Endosomally Localized RGLG-Type E3 RING-Finger Ligases Modulate Sorting of Ubiquitylation-Mimic PIN2

Intracellular sorting and the abundance of sessile plant plasma membrane proteins are imperative for sensing and responding to environmental inputs. A key determinant for inducing adjustments in protein localization and hence functionality is their reversible covalent modification by the small protein modifier ubiquitin, which is for example responsible for guiding proteins from the plasma membrane to endosomal compartments. This mode of membrane protein sorting control requires the catalytic activity of E3 ubiquitin ligases, amongst which members of the RING DOMAIN LIGASE (RGLG) family have been implicated in the formation of lysine 63-linked polyubiquitin chains, serving as a prime signal for endocytic vacuolar cargo sorting. Nevertheless, except from some indirect implications for such RGLG activity, no further evidence for their role in plasma membrane protein sorting has been provided so far. Here, by employing RGLG1 reporter proteins combined with assessment of plasma membrane protein localization in a rglg1 rglg2 loss-of-function mutant, we demonstrate a role for RGLGs in cargo trafficking between plasma membrane and endosomal compartments. Specifically, our findings unveil a requirement for RGLG1 association with endosomal sorting compartments for fundamental aspects of plant morphogenesis, underlining a vital importance for ubiquitylation-controlled intracellular sorting processes.     

Multi-walled carbon nanotubes induce COX-2 and iNOS expression via MAP Kinase-dependent and -independent mechanisms in mouse RAW264.7 macrophages

ABSTRACT: BACKGROUND: Carbon nanotubes (CNTs) are engineered graphene cylinders with numerous applications in engineering, electronics and medicine. However, CNTs cause inflammation and fibrosis in the rodent lung, suggesting a potential human health risk. We hypothesized that multi-walled CNTs (MWCNTs) induce two key inflammatory enzymes in macrophages, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), through activation of extracellular signal-regulated kinases (ERK1,2). METHODS: RAW264.7 macrophages were exposed to MWCNTs or carbon black nanoparticles (CBNPs) over a range of doses and time course. Uptake and subcellular localization of MWCNTs was visualized by transmission electron microscopy (TEM). Protein levels of COX-2, iNOS, and ERK1,2 (total ERK and phosphorylated ERK) were measured by Western blot analysis. Prostaglandin-E2 (PGE2) and nitric oxide (NO) levels in cell supernatants were measured by ELISA and Greiss assay, respectively. RESULTS: MWCNTs, but not CBNPs, induced COX-2 and iNOS in a time- and dose-dependent manner. COX-2 and iNOS induction by MWCNTs correlated with increased PGE2 and NO production, respectively. MWCNTs caused ERK1,2 activation and inhibition of ERK1,2 (U0126) blocked MWCNT induction of COX-2 and PGE2 production, but did not reduce the induction of iNOS. Inhibition of iNOS (L-NAME) did not affect ERK1,2 activation, nor did L-NAME significantly decrease COX-2 induction by MWCNT. Nickel nanoparticles (NiNPs), which are present in MWCNTs as a residual catalyst, also induced COX-2 via ERK-1,2. However, a comparison of COX-2 induction by MWCNTs containing 4.5 and 1.8% Ni did not show a significant difference in ability to induce COX-2, indicating that characteristics of MWCNTs in addition to Ni content contribute to COX-2 induction. CONCLUSION: This study identifies COX-2 and subsequent PGE2 production, along with iNOS induction and NO production, as inflammatory mediators involved in the macrophage response to MWCNTs. Furthermore, our work demonstrates that COX-2 induction by MWCNTs in RAW264.7 macrophages is ERK1,2-dependent, while iNOS induction by MWCNTs is ERK1,2-independent. Our data also suggest contributory physicochemical factors other than residual Ni catalyst play a role in COX-2 induction to MWCNT.     

Role of the Renin–Angiotensin–Aldosterone and Kinin–Kallikrein Systems in the Cardiovascular Complications of COVID-19 and Long COVID

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the virus responsible for the COVID-19 pandemic. Patients may present as asymptomatic or demonstrate mild to severe and life-threatening symptoms. Although COVID-19 has a respiratory focus, there are major cardiovascular complications (CVCs) associated with infection. The reported CVCs include myocarditis, heart failure, arrhythmias, thromboembolism and blood pressure abnormalities. These occur, in part, because of dysregulation of the Renin–Angiotensin–Aldosterone System (RAAS) and Kinin–Kallikrein System (KKS). A major route by which SARS-CoV-2 gains cellular entry is via the docking of the viral spike (S) protein to the membrane-bound angiotensin converting enzyme 2 (ACE2). The roles of ACE2 within the cardiovascular and immune systems are vital to ensure homeostasis. The key routes for the development of CVCs and the recently described long COVID have been hypothesised as the direct consequences of the viral S protein/ACE2 axis, downregulation of ACE2 and the resulting damage inflicted by the immune response. Here, we review the impact of COVID-19 on the cardiovascular system, the mechanisms by which dysregulation of the RAAS and KKS can occur following virus infection and the future implications for pharmacological therapies.     

Modeling and inference of multisubject fMRI data.

This article reviews four commonly used approaches to group modeling in fMRI. The methods differ in their computational intensity (FSL with its two-level estimation including MCM being the most intense) and assumptions (SPM2 with its assumption of spatially homogeneous covariance V/sub g/ being most restrictive). This study also distinguishes fixed-effects models from mixed-effects models and motivates the importance of a mixed-effects model for group fMRI analysis. The sections following that describe single-subject modeling and show a general method for estimating the group model.     

Cognitive Status, Information Structure, and Pronominal Reference to Clausally Introduced Entities

This paper investigates reference to clausally introduced entities and proposes an explanation for why these are more readily available to immediate subsequent reference with a demonstrative pronoun than with the personal pronoun,it. New evidence is provided supporting proposals that such entities are typically activated, but not brought into focus, upon their introduction into a discourse. The study also provides further insight into the role of information structure, lexical semantics, presuppositional contexts, and syntactic structure in bringing an entity into focus of attention.     

Long-term follow-up of patients with acute myocarditis by magnetic resonance imaging

Magnetic resonance imaging (MRI) reveals cardiac signal intensity changes in patients with acute myocarditis; however, the natural history of these changes and their relationship to individual outcomes are unknown. The relationship of MRI findings to long-term outcome was studied by serial MRI studies in 16 patients with acute myocarditis who were followed for 30±4 (SE) months. Myocardial contrast enhancement was monitored using contrast-enhanced T1-weighted fast spin-echo images. Left ventricular ejection fraction was measured with gradient-echo sequences. Clinical symptoms were scored. The results were compared to a control group of 26 age-matched, healthy volunteers. Myocardial contrast enhancement, which was markedly increased in the early course of the disease, decreased at 4 weeks and remained within the normal range in most patients after 30 months. Contrast enhancement 4 weeks after onset of symptoms was predictive for the functional and clinical long-term outcome. Contrast-enhanced MRI may be a useful, noninvasive tool for long-term follow-up of patients with acute myocarditis. Furthermore, relatively early MRI findings may predict longer-term outcomes. Electronic Publication