Validating Excised Rodent Lungs for Functional Hyperpolarized Xenon-129 MRI

Ex vivo rodent lung models are explored for physiological measurements of respiratory function with hyperpolarized (hp) ¹²⁹Xe MRI. It is shown that excised lung models allow for simplification of the technical challenges involved and provide valuable physiological insights that are not feasible using in vivo MRI protocols. A custom designed breathing apparatus enables MR images of gas distribution on increasing ventilation volumes of actively inhaled hp ¹²⁹Xe. Straightforward hp ¹²⁹Xe MRI protocols provide residual lung volume (RV) data and permit for spatially resolved tracking of small hp ¹²⁹Xe probe volumes during the inhalation cycle. Hp ¹²⁹Xe MRI of lung function in the excised organ demonstrates the persistence of post mortem airway responsiveness to intravenous methacholine challenges. The presented methodology enables physiology of lung function in health and disease without additional regulatory approval requirements and reduces the technical and logistical challenges with hp gas MRI experiments. The post mortem lung functional data can augment histological measurements and should be of interest for drug development studies.     

Hyperpolarized 129Xe MR Imaging of Alveolar Gas Uptake in Humans

Background

One of the central physiological functions of the lungs is to transfer inhaled gases from the alveoli to pulmonary capillary blood. However, current measures of alveolar gas uptake provide only global information and thus lack the sensitivity and specificity needed to account for regional variations in gas exchange.

Methods and Principal Findings

Here we exploit the solubility, high magnetic resonance (MR) signal intensity, and large chemical shift of hyperpolarized (HP) ¹²⁹Xe to probe the regional uptake of alveolar gases by directly imaging HP ¹²⁹Xe dissolved in the gas exchange tissues and pulmonary capillary blood of human subjects. The resulting single breath-hold, three-dimensional MR images are optimized using millisecond repetition times and high flip angle radio-frequency pulses, because the dissolved HP ¹²⁹Xe magnetization is rapidly replenished by diffusive exchange with alveolar ¹²⁹Xe. The dissolved HP ¹²⁹Xe MR images display significant, directional heterogeneity, with increased signal intensity observed from the gravity-dependent portions of the lungs.

Conclusions

The features observed in dissolved-phase ¹²⁹Xe MR images are consistent with gravity-dependent lung deformation, which produces increased ventilation, reduced alveolar size (i.e., higher surface-to-volume ratios), higher tissue densities, and increased perfusion in the dependent portions of the lungs. Thus, these results suggest that dissolved HP ¹²⁹Xe imaging reports on pulmonary function at a fundamental level.     

Depolarization Laplace Transform Analysis of Exchangeable Hyperpolarized 129Xe for Detecting Ordering Phases and Cholesterol Content of Biomembrane Models

We present a highly sensitive nuclear-magnetic resonance technique to study membrane dynamics that combines the temporary encapsulation of spin-hyperpolarized xenon (¹²⁹Xe) atoms in cryptophane-A-monoacid (CrA_ma) and their indirect detection through chemical exchange saturation transfer. Radiofrequency-labeled Xe@CrA_ma complexes exhibit characteristic differences in chemical exchange saturation transfer-driven depolarization when interacting with binary membrane models composed of different molecular ratios of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). The method is also applied to mixtures of cholesterol and POPC. The existence of domains that fluctuate in cluster size in DPPC/POPC models at a high (75–98%) DPPC content induces up to a fivefold increase in spin depolarization time τ at 297 K. In POPC/cholesterol model membranes, the parameter τ depends linearly on the cholesterol content at 310 K and allows us to determine the cholesterol content with an accuracy of at least 5%.     

Differential Response of Neural Cells to Trauma-Induced Free Radical Production In Vitro

CNS trauma has been associated with an increase in free radical production, but the cellular sources of this increase or the mechanism involved in the production of free radicals are not known. We, therefore, investigated the effects of trauma on free radical production in cultured neurons, astrocytes and BV-2 microglial cells. Free radicals were measured with the fluorescent dye DCFDA following in vitro trauma. At 30 and 60 min following trauma, there was a 132% and 64% increase, respectively, in free radical production in neurons when compared to controls. In astrocytes, there was a 94% and 133% increase at 30 and 60 min, respectively. Microglial cells, however, displayed no significant increase in free radicals at 30, 60 or 120 min following trauma. Since trauma can induce the mitochondrial permeability transition (MPT), a process associated with mitochondrial dysfunction, we further investigated whether cyclosporin A (CsA), an agent known to block the MPT, could prevent free radical formation following trauma. In neurons CsA did not block free radical production at 30 min but blocked it by 90% at 60 min. In contrast, in astrocytes CsA completely blocked free radical production at 30 min but did not block it at 60 min. Our results indicate that a differential sensitivity to trauma-induced free radical production exists in neural cells; that the MPT may be involved in the production of free radical post-trauma; and that the CsA-sensitive phase of free radical production is different in neurons and astrocytes.     

The Free Space of the Xylem Translocation Pathway of the Tomato Stem

In this paper a method is presented for measurement of the apparentfree space (AFS) directly involved in the xylem vessel translocation. The AFS of xylem vessel bundles was measured by perfusing tomatointernodes with solutions of a non-absorbed (inulin-carboxylicacid), a weakly absorbed (-aminoisobutyric acid), and a stronglyabsorbed compound (L--alanine). The xylem cross-section actually involved in the longitudinalflow of these substances plus the area which is in rapid (10to 15 min), diffusional, reversible equilibrium with the flowarea was found to amount to 2?5 times the cross-section of thexylem vessels. Since all apoplasmic compartments of the xylemconstitute only 70% of the AFS of the xylem vessels, 30% ofthe AFS seems to be situated outside the xylem.     

产生L-异亮氨酸的黄色短杆菌的代谢途径分析

目的:代谢工程要解决的主要问题是改变某些途径中的碳架物质流量或改变碳架物质流在不同途径中的流量分布,其目标就是修饰初级代谢,将碳架物质流导入目的产物的载流途径,以获得产物的最大转化率。方法:利用途径分析方法对黄色短杆菌生产L-异亮氨酸的途径进行了分析。结果:建立了9种基础模型,确定L-异亮氨酸理论最高摩尔产率是1;确定了黄色短杆菌生产L-异亮氨酸的最佳途径的通量分布,并以此为依据进行发酵溶氧控制优化,溶氧分阶段控制发酵生产L-异亮氨酸比溶氧恒定控制方式发酵的产率提高了8.2%。结论:根据途径分析结果,通过改变发酵过程有关参数,可使目的产物产率得到提高。     

Correlations Between Free Radical Production and Viability of Lyophilized Bacteria

When Serratia marcescens cultures were treated with dilute solutions of phenol or hydrogen peroxide before drying or by lyophilization at suboptimal pH, the log of the number of cells surviving lyophilization was correlated with subsequent free radical production by the dried cells. Since the rate of free radical production and rate of death were similarly affected by temperature, the log of the number of cells surviving after 6 days was inversely related to the free radical concentration at that time. Free radicals were produced in proportion to the log of oxygen pressure, and viability was inversely related to oxygen tension; again, free radical concentration appeared to be correlated with the death of organisms.     

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD⁺]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD⁺]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD⁺]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD⁺]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD⁺]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.     

TDP-l-Megosamine Biosynthesis Pathway Elucidation and Megalomicin A Production in Escherichia coli

In vivo reconstitution of the TDP-l-megosamine pathway from the megalomicin gene cluster of Micromonospora megalomicea was accomplished by the heterologous expression of its biosynthetic genes in Escherichia coli. Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-l-megosamine from TDP-4-keto-6-deoxy-d-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of l-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis in vivo of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.Most of the deoxy sugars found in natural products belong to the 6-deoxyhexose (6DOH) family (21). Since many of these 6DOHs are essential for the bioactivity of natural compounds, extensive efforts have been made to investigate the relevant genetics, enzymology, and mechanistic features of the biosynthetic pathways leading to these sugars. The amino sugar l-megosamine is found within a family of macrolide compounds produced by the actinomycete Micromonospora megalomicea, named megalomicins A (MegA) (structure 1), B, C1, and C2 (Fig. ​(Fig.11 A) (27). These compounds consist of a 14-membered macrolactone ring carrying three deoxy sugar residues, l-mycarose, d-desosamine, and l-megosamine. The megalomicin congeners differ from each other in the specific acetyl or propionyl groups attached at the 3′′′ or 4′′′ hydroxyls of the mycarose moiety. These macrolides were originally discovered as antibacterial agents which inhibit protein synthesis through selective binding to the bacterial 50S ribosomal subunit in a mode similar to that of erythromycins and other macrolides (25). Due to the similarities with erythromycin in terms of structure, antibacterial activity, and pharmacological properties, megalomicins did not receive much attention until antiviral and antiparasitic activities of these compounds were reported (1, 3). These studies demonstrated that megalomicins interfere with protein trafficking, resulting in an anomalous protein glycosylation (4, 5) that affects the maturation of enveloped viruses, including herpes simplex virus, Semliki Forest virus, vesicular stomatitis virus, and more importantly the human immunodeficiency virus type 1 (HIV-1) (1, 22). In HIV replication, inhibition of gp160 protein processing to gp120 and gp41 resulted in noninfectious virions (22). In addition, megalomicins also showed antiparasitic activity against the epimastigote stage of Trypanosoma cruzi, Leishmania spp., and Plasmodium falciparum, although in this case the mechanism of action still remains unclear (3).Open in a separate windowFIG. 1.(A) Structures of megalomicins and erythromycins. (B) Genetic organization of the meg gene cluster from M. megalomicea. A 12-kb fragment, including putative l-megosamine biosynthesis genes, is indicated.The main structural difference between megalomicins and erythromycins is the presence of the l-megosamine sugar moiety at C-6 (Fig. ​(Fig.1A).1A). Since erythromycin does not exhibit antiparasitic and antiviral activities, the presence of this additional amino sugar in megalomicins could be associated with the differential properties of these compounds (1, 3). Due to the potential pharmacological relevance of megalomicins and the lack of a detailed characterization of the l-megosamine biosynthetic pathway from the megalomicin (meg) gene cluster, an in-depth metabolic route study was deemed warranted.Analysis of the overall organization of the meg gene cluster revealed that l-megosamine biosynthesis genes are grouped together within this gene cluster (Fig. ​(Fig.1B)1B) (25). This was demonstrated by the heterologous expression of a 12-kb DNA fragment that included the putative megosamine biosynthesis genes in the erythromycin producer strain Saccharopolyspora erythraea, which allowed the production of megalomicins in this host (25). Six biosynthetic steps were proposed for the biosynthesis of TDP-l-megosamine (l-Meg) (structure 2) from the intermediate TDP-4-keto-6-deoxy-d-glucose (TKDG) (structure 3). Neither the biosynthesis pathway nor the enzymes involved in each catalytic step have been confirmed.Herein, the investigation focused on the biosynthesis of l-Meg from M. megalomicea by the heterologous expression of meg genes in Escherichia coli. The sequence of enzymatic reactions implicated in this pathway was confirmed by analyzing the TDP-sugar intermediates generated from the expression of operons containing different sets of genes. This methodology allowed the validation of a new pathway for the biosynthesis of l-Meg from the precursor TKDG through the use of five enzymatic steps. Bioconversion experiments furthermore demonstrated that the attachment of l-megosamine to the macrolide intermediate required both a specific glycosyltransferase and a helper protein.     

Failure to thrive-an analysis of 83 cases

The case histories of 83 children admitted to the hospital with a diagnosis only of failure to thrive were examined. In twenty-six cases there was evidence of maternal deprivation as a factor. Forty patients were found to have significant organic diseases as a possible or probable cause or contributing influence.Twenty-six were found to have some degree of mental retardation, either documented or suspected, but in nearly all of them there were associated factors presumably responsible, at least in part, for failure to thrive.Several children had birth weight less than 2,500 grams, but no child was thought to grow poorly because of prematurity alone. Congenital anomalies such as cleft palate and other problems leading to feeding difficulties were not unusual.In any case of persistent failure of an infant to gain adequately in weight and length, in which the cause is not evident, the child should be admitted to a hospital to determine response in a new environment. Also an adequate social history should be sought and siblings more closely evaluated; and careful study should be made of the renal, gastro-intestinal, cardiac, pulmonary and central nervous systems, even if there are no symptoms or signs referable to these systems.     

Synthetic Pathway for Production of Five-Carbon Alcohols from Isopentenyl Diphosphate

Synthetic biological pathways could enhance the development of novel processes to produce chemicals from renewable resources. On the basis of models that describe the evolution of metabolic pathways and enzymes in nature, we developed a framework to rationally identify enzymes able to catalyze reactions on new substrates that overcomes one of the major bottlenecks in the assembly of a synthetic biological pathway. We verified the framework by implementing a pathway with two novel enzymatic reactions to convert isopentenyl diphosphate into 3-methyl-3-butenol, 3-methyl-2-butenol, and 3-methylbutanol. To overcome competition with native pathways that share the same substrate, we engineered two bifunctional enzymes that redirect metabolic flux toward the synthetic pathway. Taken together, our work demonstrates a new approach to the engineering of novel synthetic pathways in the cell.     

G蛋白偶联受体介导游离脂肪酸的信号通路及生理功能

游离脂肪酸（free fatty acid,FFA）是动物一种重要能量来源,同时它还是一种重要的信号分子,其生理功能和作用机制长期以来倍受关注. 最近研究表明,细胞膜存在FFA的特定孤儿型G蛋白偶联膜受体家族.中长链游离脂肪酸是GPR40和GPR120的配基,而短链游离脂肪酸则是GPR41和GPR43的配基. 该受体家族可以介导游离脂肪酸,通过ERK、PI3K-Akt和MAPK信号通路,在维持机体内的葡萄糖稳态、脂肪形成、白细胞功能和细胞增殖等生理过程中发挥重要作用. 本文就游离脂肪酸G蛋白偶联受体的结构、分布、配体选择性、下游信号通路,及其介导FFA生理功能的最新研究进展进行简要综述.     

Suppression of FAM83D Inhibits Glioma Proliferation,Invasion and Migration by Regulating the AKT/mTOR Signaling Pathway

ObjectiveTo explore the mechanism by which the family with sequence similarity 83, member D (FAM83D)-mediated AKT/mTOR signaling pathway activation affects the proliferation and metastasis of glioma cells.MethodsFAM83D protein expression in glioma cells and tissues was detected by western blotting. Glioma U87 and U251 cells were selected and divided into the Mock, siNC, siFAM83D, FAM83D, MK2206 and FAM83D + MK2206 groups. Cell proliferation was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and clone formation assays, while invasion and migration were evaluated by Transwell assays and wound healing tests. The protein expression of members of the AKT/mTOR pathway was determined via western blotting. Xenograft models were also established in nude mice to observe the in vivo effect of FAM83D on the growth of glioma.ResultsFAM83D was upregulated in glioma patients, especially in those with Stage III-IV. In addition, cells treated with siFAM83D had significant downregulation of p-AKT/AKT and p-mTOR/mTOR, with decreased proliferation and colony numbers, as well as decreased invasion and migration compared to the Mock group. However, FAM83D overexpression could activate the Akt/mTOR pathway and promote the proliferation, invasion and migration of glioma cells. Moreover, treatment with MK2206, an inhibitor of AKT, reversed the promoting effect of FAM83D on the growth of glioma cells. The in vivo experiments demonstrated that silencing FAM83D could inhibit the in vivo growth of glioma cellsConclusionFAM83D was upregulated in glioma and silencing FAM83D suppressed the proliferation, invasion and migration of glioma cells via inhibition of the AKT/mTOR pathway.     

Regulation of Alternative Pathway Activation and C3a Production by Adipose Cells

Adipose tissue is a major source of adpisin/factor D of the alternative pathway of complement. Adipose tissue also expresses the two other complement components which are involved in the first activation step of the alternative pathway, factor B and C3, and this step is activated in adipose tissue, producing C3a/Acylation Stimulating Protein (C3a/ASP), a stimulator of triglyceride synthesis. Complement activation is a highly regulated process, however, nothing is known about regulation of complement activation in adipose tissue. To gain insight into the nature of adipose complement activation and its regulation, we have now examined the expression of several complement activation regulatory genes, and analyzed the production of C3a/ASP in lean vs. obese, adipsin-deficient mice. We found that undifferentiated preadipocytes expressed the mRNAs encoding the negative regulatory proteins Crry and factor H, but expression of both genes was decreased upon differentiation. The positive regulator properdin, as well as Crry and factor H, were found in adipose tissue. None of these genes was regulated in murine genetic obesity. To investigate the relative levels of complement activation in lean vs. adipsin-deficient obese mice, we developed a radioimmunoassay for measurement of murine C3a/ASP in plasma. We report that there was no significant difference in the level of C3a in lean vs. obese plasma; however, we found a positive correlation between C3a and plasma triglyceride levels in normal lean mice.