Adipocyte differentiation of 3T3-L1 cells involves augmented expression of a 43-kDa plasma membrane fatty acid-binding protein.

A previously described 43-kDa plasma membrane fatty acid-binding protein (FABPPM) was not observed by immunohistochemical methods in proliferating 3T3-L1 fibroblasts. However, it was detectable in plasma membranes by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated adipocytes. These observations were confirmed by extraction of plasma membrane proteins and subsequent immunoblotting. Kinetics of initial [3H]oleate uptake by both fibroblasts and adipocytes consisted of the sum of a saturable and a non-saturable component. During differentiation the saturable component increased progressively. Vmax increased from 3 to 25 to 110 pmol.s-1.mg cell protein-1 between the fibroblast, the 4-day, and 8 day adipocyte stages; Km was 24 nM in fibroblasts and approximately 55 nM in both 4- and 8-day differentiated adipocytes. By contrast, the rate constant for nonsaturable oleate influx decreased progressively from 0.026 to 0.010 ml.s-1.mg protein-1 between the fibroblast and 8 day adipocyte stages. In 8-day adipocytes saturable oleate uptake was inhibited by up to 55% by antibodies against rat liver FABPPM; these antibodies had no effect on uptake of 2-deoxyglucose or the medium chain fatty acid octanoate. They also had no effect on oleate uptake by fibroblasts. These studies support the hypothesis that FABPPM is a component of a saturable transport mechanism for long chain fatty acids.     

Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with ^•NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged ^•NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief ^•NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief ^•NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of ^•NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of ^•NO.     

Synthesis,Biological Evaluation,and Docking Study of Ring-Opening Amide Analogs of Matrine as Antitumor Agents

In this article, we designed and synthesized two series of matrine analogs with ring-opening in the lactam portion of the molecule. Our in vitro cytotoxicity study showed that analog N-(3-bromophenyl)-4-[(1R,3aS,10aR,10bS)-decahydro-1H,4H-pyrido[3,2,1-ij][1,6]naphthyridin-1-yl]butanamide ( B11 ) with a meta-bromide on the phenyl ring displayed the best antiproliferative activity. Moreover, B11 induced cell cycle arrest in G1 phase and cell apoptosis in a dose-dependent manner in A549 cells. Molecular modeling revealed that B11 achieved a higher docking score compared to its precursor tert-butyl (1R,3aS,10aR,10bS)-1-[4-(3-bromoanilino)-4-oxobutyl]octahydro-1H,4H-pyrido[3,2,1-ij][1,6]naphthyridine-2(3H)-carboxylate ( A11 , an analog of B11 with a Boc group) and parent compound matrine, possibly because B11 formed a hydrogen bond with SER91 and a halogen bond with GLN320 on the binding site of annexin A2. Overall, we discovered the potential anticancer lead compound B11 , which can be used for further study both in vitro and in vivo.     

Quantitative structure-activity relationships of structurally similar odorless and odoriferous benzenoid musks

To reveal the difference of molecular property between structurallysimilar odorless and odoriferous musk compounds, 10 pairs ofbenzenoids (monocyclic-, dicyclic- and tricyclic-) were examined.Molecular structures of all compounds were optimized by MNDO(modified neglect of diatomic differential overlap) consideringconformation. Parameters effective in discriminating two groups,group A of 10 odorless compounds and group B of 10 musk odorcompounds, were searched from 34 candidate parameters by adaptiveleast squares. The best three parameters found were log P value(octanol/water partition coefficient), the longest side lengthof hexahedron circumscribing a molecule, and the parameter whichexpresses structural hindrance to the functional group whena molecule approaches the receptor site. The two groups of compoundswere completely discriminated using these three parameters.     

The Tumor Suppressor pVHL Down-regulates Never-in-Mitosis A-related Kinase 8 via Hypoxia-inducible Factors to Maintain Cilia in Human Renal Cancer Cells

NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.     

Prevalence of Post-Stroke Cognitive Impairment in China: A Community-Based,Cross-Sectional Study

International hospital-based studies have indicated a high risk of cognitive impairment after stroke, evidence from community-based studies in China is scarce. To determine the prevalence of post-stroke cognitive impairment (PSCI) and its subtypes in stroke survivors residing in selected rural and urban Chinese communities, we conducted a community-based, cross-sectional study in 599 patients accounting for 48% of all stroke survivors registered in the 4 communities, who had suffered confirmed strokes and had undergone cognitive assessments via the Montreal Cognitive Assessment (MoCA), Mini-Mental State Examination (MMSE), and Hachinski Ischemia Scale (HIS). Detection of PSCI was based on scores in these neuropsychological scales. Factors potentially impacting on occurrence of PSCI were explored by comparing demographic characteristics, stroke features, and cardiovascular risk factors between patients with and without PSCI. The overall prevalence of PSCI was 80.97% (95%CI: 77.82%-84.11%), while that of non-dementia PSCI (PSCI-ND) and post-stroke vascular dementia (PSD) was 48.91% (95%CI: 44.91%-52.92%) and 32.05% (95%CI: 28.32%-35.79%), respectively. Prior stroke and complications during the acute phase were independent risk factors for PSCI. The risk of recurrent stroke survivors having PSCI was 2.7 times higher than for first-episode survivors, and it was 3 times higher for those with complications during the acute phase than for those without. The higher prevalence of PSCI in this study compared with previous Chinese studies was possibly due to the combined effects of including rural stroke survivors, a longer period from stroke onset, and different assessment methods. There is an urgent need to recognize and prevent PSCI in stroke patients, especially those with recurrent stroke and complications during the acute phase.     

Mechanisms of HIV-1 evasion to the antiviral activity of chemokine CXCL12 indicate potential links with pathogenesis

HIV-1 infects CD4 T lymphocytes (CD4TL) through binding the chemokine receptors CCR5 or CXCR4. CXCR4-using viruses are considered more pathogenic, linked to accelerated depletion of CD4TL and progression to AIDS. However, counterexamples to this paradigm are common, suggesting heterogeneity in the virulence of CXCR4-using viruses. Here, we investigated the role of the CXCR4 chemokine CXCL12 as a driving force behind virus virulence. In vitro, CXCL12 prevents HIV-1 from binding CXCR4 and entering CD4TL, but its role in HIV-1 transmission and propagation remains speculative. Through analysis of thirty envelope glycoproteins (Envs) from patients at different stages of infection, mostly treatment-naïve, we first interrogated whether sensitivity of viruses to inhibition by CXCL12 varies over time in infection. Results show that Envs resistant (RES) to CXCL12 are frequent in patients experiencing low CD4TL levels, most often late in infection, only rarely at the time of primary infection. Sensitivity assays to soluble CD4 or broadly neutralizing antibodies further showed that RES Envs adopt a more closed conformation with distinct antigenicity, compared to CXCL12-sensitive (SENS) Envs. At the level of the host cell, our results suggest that resistance is not due to improved fusion or binding to CD4, but owes to viruses using particular CXCR4 molecules weakly accessible to CXCL12. We finally asked whether the low CD4TL levels in patients are related to increased pathogenicity of RES viruses. Resistance actually provides viruses with an enhanced capacity to enter naive CD4TL when surrounded by CXCL12, which mirrors their situation in lymphoid organs, and to deplete bystander activated effector memory cells. Therefore, RES viruses seem more likely to deregulate CD4TL homeostasis. This work improves our understanding of the pathophysiology and the transmission of HIV-1 and suggests that RES viruses’ receptors could represent new therapeutic targets to help prevent CD4TL depletion in HIV+ patients on cART.